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the neuroendocrine tumors ( nets ) are a heterogeneous group of neoplasms that share some biological characteristics , that have been addressed as a common entity . in 62%82% of the cases , the nets are located in the digestive system , being designated as gastroenteropancreatic neuroendocrine tumors ( gep - nets ) . the latest figures from the uk , sweden , and switzerland suggested that their incidence is 2 - 3/100000 with a higher prevalence in females . also data from the surveillance , epidemiology , and end results ( seer ) program database report an increased incidence between 1973 and 2004 of 1.09 to 5,25/100000 [ 3 , 4 ] . nets are classically characterized by their ability to secrete hormones and/or vasoactive peptides , which results in many of their clinical manifestations , ganging up on specific hormonal syndromes . the most frequent syndrome is the carcinoid syndrome , which results from the secretion of serotonin and includes symptoms such as skin flushing , severe diarrhea , abdominal cramping , and electrolyte abnormalities [ 4 , 5 ] . their clinical course is variable , having been considered traditionally with an indolent natural history [ 1 , 4 ] . all of them have the potential of metastatic dissemination and , therefore , are currently recognized as malignant neoplasms . the prognosis and the approach of nets the tumors with grade 3 , mitotic count exceeding 20/10 high - powered fields and/or with ki-67 proliferative index exceeding 20% represent aggressive malignant disease with rapid clinical course and a low survival . the liver is the most important place of metastatic disease . more than 75% of patients with nets of the small intestine and 30%85% of patients with pancreatic nets have liver metastases at the time of diagnosis or during the course of their disease . additionally 5%10% of patients with nets of unknown primary location have liver metastases at the time of the diagnosis [ 6 , 8 ] . patients with liver metastases have a worsening of their morbidity and mortality , as compared to patients without these lesions . according to touzios et al . survival rate to 5 years of patients with untreated liver metastasis is about 13%54% , against 75%99% in patients without liver metastases [ 6 , 9 ] . this revision aims to address the theme of gep - nets with metastatic liver , their clinical significance , prognosis , and its current therapeutic approach with a special focus on medical treatment . patients with nets often develop liver metastases ( nlms ) affecting significantly their morbidity and mortality . about 75%80% of patients present with hepatic metastasis at the time of diagnosis ( synchronous ) , while 20%25% the presence of liver metastases is closely related to the appearance / aggravation of symptoms . the presence of hepatic metastases leads to less metabolization of these peptides leading to an increase of their circulating levels . sometimes , only then its distribution , in the way it modifies therapeutic management , reflects the aggressiveness of the tumor . type i metastases are lesions only confined to one hepatic lobe or limited to two adjacent segments , corresponding to about 20%25% of cases . type ii metastases include the presence of dominant injury with small satellites contralateral ( 10%15% of cases ) . type iii metastases correspond to diffuse multifocal , being the most prevalent being ( 60%70% cases ) . because of the possibility of surgical resection , type i is associated with a favorable outcome . type iii metastases have a worse prognosis regarding the greater liver involvement and the lack of surgical approach . in this group of patients however , the complete excision of secondary lesions is only an option for a small number of patients ( 10%20% ) . this differs from the normal vascularization system which is derived from the venous system port . the handling of hepatic artery prevents the supply of nutrients and oxygen to the tumor cells with its consequent destruction . for that reason , several techniques have been developed by exploiting duality in the liver vasculature in order to control the disease process . these modalities are include the ablative techniques , the hepatic artery embolization , chemoembolization , and radioembolization ( selective internal radiation therapy ) . patients with predominant metastatic liver disease are those who benefit the most from these therapies . curative intention depends on the stage and presentation of the disease [ 13 , 14 ] . even with hepatic involvement , surgical approach , when feasible , is the best treatment option , with proven benefits in terms of overall survival and quality of life improvement [ 1 , 6 , 12 ] . patients treated with surgical resection of the primary tumor and liver metastases had a survival at 5 years of more than 60% , reaching 80% in some studies , with minimum mortality ( less than 5% ) and admissible morbidity ( less than 30% ) [ 6 , 12 ] . patients with liver metastasis that are not candidates to surgical treatment ( 80%90% of cases ) , still benefit from primary tumor resection . this approach can be combined with other liver - direct therapies increasing patients ' outcome [ 12 , 13 , 15 ] . in these cases , where primary tumor was resected therefore , the palliation of the symptoms is also a criterion for surgery [ 5 , 6 ] . regarding the criteria for selecting patients with nlms , there is no consensus , since the several studies developed included a small number of patients and different surgical procedures . however , when the complete excision of a significant number of liver metastasis is possible , surgery must always be considered . in a recent retrospective study , 74 cases were analyzed and the 5-year survival rate was more than 60% in all patients who underwent surgical resection of liver metastases . in 65% of these patients , excision was incomplete , and there was no worsening of the prognosis [ 6 , 12 ] . however , the identification of other predictive variables in patients ' selection for surgery was needed . therefore , clinical studies were conducted , and patients were selected based on tumor grade . the analyses of the results from these studies showed that tumors with high grade had a worse postsurgical outcome when compared to low grade tumors . high grade net tumors are good candidates to chemotherapy and do not seem to beneficiate from surgery . translating these results into clinical practice , in 2008 , the european neuroendocrine tumor society ( enets ) issued general guidelines for surgical resection based on the three types of liver involvement . for patients with hepatic involvement of type i surgical resection is the first therapeutic option , while in patients with the involvement of type iii it is totally excluded . in order to give the best possible treatment to patients with net tumors this evaluation should take into account the following aspects : performance status , localization of the primary tumor , tumor grade , possibility of primary tumor resection , presence and localization of metastases , possibility of complete resection of metastases , presence of extrahepatic disease ( extrahepatic disease should be excluded based on adequate image methods ) , and the presence or absence of carcinoid heart disease [ 6 , 17 ] . in patients with carcinoid heart disease , symptoms control and clinical stabilization should be achieved before aggressive surgery is considered . also , in patients with carcinoid syndrome , even in those under treatment with somatostatin analogues , intra- and postoperative carcinoid crisis must be prevented , using these same somatostatin analogues . preoperative administration of octreotide ( 300 micrograms subcutaneously ) can reduce the incidence of carcinoid crisis and is recommended for patients with a history of carcinoid syndrome who require surgical procedures . calcium and catecholamines may provoke the release of mediators from the tumor and worsen , rather than ameliorate , the syndrome . during a carcinoid crisis , the blood pressure should be supported by the infusion of plasma and octreotide ( 300 micrograms iv ) given immediately . a continuous iv drip of octreotide may be needed . in patients with high risk of carcinoid crisis , an iv octreotide drip may be initiated preoperatively [ 1 , 1823 ] . the presence of abdominal lymph node involvement , including local recurrence , is not an absolute contraindication for surgery , if the excision of lymph nodes and liver metastases and/or recurrent lesions could be done simultaneously or in planned staggered phases . extensive lymphadenectomy and a careful exploration of the entire abdominal cavity should be carried out during surgical procedure . the use of intraoperative echography seems to be of additional value as it allows to define the extent of known lesions and to detect small additional lesions . it is usually recommended in cases where more than 90% of the tumor can be excised or in young patients [ 6 , 12 , 20 , 25 , 26 ] . symptomatic patients also benefit from cytoreductive surgery , as it seems to improve the effectiveness of medical therapy . liver transplantation is a therapeutic approach with an intention to cure , to prolong survival or control of carcinoid symptoms . liver transplantation is only indicated in cases of metastatic disease if the primary tumor is an net . in 1998 , lehnert analyzed a total of 103 net patients with liver transplant . the tumor histology and its primary location did not affect the survival in this study , unlike the extent of surgery and the patient 's age [ 12 , 27 ] . however , in a more recent clinical trial , primary location in the duodenum or pancreas was associated with worse prognosis after transplant [ 6 , 12 , 28 ] . these findings were , however , not supported by other investigations [ 12 , 29 ] . regarding biomarkers analyses and their correlation with transplant outcome , the authors found that patients with a low ki-67 and normal expression of e - cadherin had a more favorable prognosis . on the other hand , the analysis of the data from the united network for organ sharing between november 1988 and march 2011 showed that only 185 liver transplants were due to nlms . the 5-year overall survival was 57.8% , lower than patients transplanted for other reasons / with other pathologies [ 12 , 14 ] . in patients with diffuse and unresectable liver disease , with uncontrolled symptoms ( resistant to medical therapy support ) , the main criteria for selecting patients for liver transplantation are in these cases being not candidate for resection , diagnosis , and complete resection of the primary tumor at least one year before , no extrahepatic disease , stable disease at least for one year , and failure of nonoperative treatments . patients with net tumors can be selected to treatment with different types of ablative techniques such as cryoablation , alcohol ablation ( these two are less used ) , and radiofrequency ablation ( rfa ) . decreasing temperature lowers cell viability , and this depends both on the rate of colling and the spatial relationship between the ice formation and the cryoablation probe . the lower the temperature ( which should be 50c ) the highest the chance of achieving tissue necrosis . alcohol ablation also known as percutaneous alcohol injection ( pai ) has already been studied in different series [ 33 , 34 ] . this technique could be of advantage in patients whose liver metastases are located next to large vessels or the bile duct , that could be damaged by the heat released during rfa . pai should be used not as monotherapy but with other ablative techniques in order to achieve better results . rfa consists in intense heat production from radiofrequency waves through alternating electric current and can be performed by percutaneous or laparoscopic approach [ 12 , 13 ] . it is mainly used in patients with a small number of liver lesions that can not be resected or as an adjuvant to other therapies . the largest clinical study until involving patients with hepatic lesions treated with rfa was reported by the cleveland clinic . eighty - nine patients were followed and submitted to a total of 119 laparoscopic rfas . results showed that about 90% of the patients experienced immediate relief of symptoms after the procedure , with a mean progression - free survival of 1.3 years . from these 89 patients , 22% had recurrence , and from these 22% recurrences , 63% corresponded to new liver lesions and 59% to extrahepatic disease . an important limitation of the effectiveness of this technique is the size of the tumor . it seems to be difficult to totally eradicate lesions greater than 3 cm , and lesions greater than 5 cm are unsuitable for rfa , as demonstrated by mazzagalia et al . rfa is a procedure with low morbidity ( 5%12% ) and low 30-day mortality ( 0%-1% ) [ 12 , 17 ] . the most frequent complications include hepatic abscesses , carcinoid crisis , biliopleural fistula , bile leakage , and pleural effusions . hepatic artery embolization ( hae ) and hepatic artery chemoembolization ( hace ) are two different technics that can also be used in the treatment of liver metastasis . hepatic arterial embolization is frequently applied as a palliative technique in patients with hepatic - predominant metastatic net who are not candidates for surgical resection . it is based on the principle that tumors in the liver derive most of their blood supply from the hepatic artery , whereas healthy hepatocytes derive most of their blood supply from the portal vein . using this technic , we can block blood supply with nutrients and oxygen to the tumor with consequent ischemia / necrosis . this technique has also proven to achieve the reduction of lesions size as well as symptoms relive [ 1 , 12 ] . retrospective evaluation showed that a high rate and a prolonged disease regression were achieved with chemotherapy after hae . therefore , cytostatic agents were added to this technique resulting in hepatic artery chemoembolization ( hace ) . hace provides not only the embolic blocking but also the supply of direct chemotherapy to these cells , increasing the action / concentration of these agents ( more than 20 times of the systemic chemotherapy ) . its benefits have been documented by different authors , but there were no comparative studies between hae and hace demonstrating superiority of one of these techniques . both hae and hace are palliative techniques used in patients with unresectable lesions , with diffuse and progressive liver disease , with symptoms not controlled with medical treatment and without impaired liver function [ 13 , 24 ] . the main benefit is symptomatic relief ( 70%90% of the patients ) and tumor growth control ( 50% of the patients ) [ 1 , 24 ] . the mean duration of response may be short ( up to allow collateral circulation to develop ) ranging from 642 months in uncontrolled series of patients [ 5 , 6 ] . these therapies have proved to be a valuable option , particularly in patients with more than 75% of hepatic involvement , carefully selected , without additional risk factors . sepsis , liver and renal failure , carcinoid crisis , necrotizing cholecystitis , and peptic ulcer bleeding have been reported in about 7.5 to 23.8% of patients . the most frequent is the postembolization syndrome ( i.e. , fever , abdominal pain , leukocytosis , increased transaminases , and bilirubin ) , which occurs in 90% of patients , in most cases self - limited ( 2472 hours of duration ) [ 6 , 17 , 24 ] . in order to minimize the complications , it is recommended to split the area to be treated in small portions in each session ( one lobe per session ) [ 5 , 17 ] . multiple sessions are usually required and must be carried out with 46 week interval [ 6 , 13 ] . concomitant administration of antibiotics and somatostatin analogues , as well as aggressive hydration , to prevent liver abscesses , carcinoid crisis , and tumor lysis syndrome is further recommended [ 1 , 24 ] . the use of somatostatin analogues during the procedure is of extreme importance in order to prevent carcinoid crisis . the most relevant contraindications are bleeding disorders , renal failure , and/or hepatic vein occlusion . external radiation therapy has limited value in nets , being only indicated in the presence of symptomatic brain and bone metastases . sirt is a novel technique based on small microspheres radioisotopes , including yttrium 90 ( 90y ) , which are introduced directly into the hepatic artery , leading to embolic blocking of tumor cells and also exposing them to radioactive agents . saxena et al . investigated the safety and efficacy of this treatment in patients with nlms . in this study , 34 patients had long - term responses with a mean overall survival of 29.4 months with radiological improvement in 50% of the patients . cromogranina a levels decreased to 50% , with a maintained response for approximately 30 months [ 12 , 38 ] . kennedy et al . also showed that the major benefit seems to stabilize advanced disease allowing an increase in overall survival although still under investigation results have been promising . complications such as postembolization syndrome are common , with a small risk of radiation gastritis and ulceration . since nets are a rare pathology , large randomized studies in patients with nlms are difficult to perform . this limitation leads to an overall assessment of restricted systemic therapies that can not be translated in to a consensual treatment algorithm . however , the data obtained from a single center study showed that the aggressive medical treatment of nlms instead of surgical therapy increases the 3-year survival rate to 76.4% and the 5-year survival rate to 63.9% . therefore , conservative treatment , that is , the majority of nets is expressed on the surface of their cells somatostatin receptors ( sstr 15 ) . their activation inhibits the secretion of peptides and amines by tumor cells and also the effect of tumor growth factors inducing apoptosis [ 12 , 24 ] . since natural somatostatin has a very short half life ( 2 - 3 min ) analogues with longer half lives have been developed for clinical use . in 1980 , bauer et al . synthesized the first analogue - octreotide . the main use of ssa is in symptomatic control . in an initial study , the subcutaneous administration of octreotide at the dose of 150 mg tid improved symptoms , especially carcinoid syndrome , in 88% of patients . however , the short action formulations continue to play an important role in stabilizing the disease , especially in the situations of carcinoid crisis and its prevention . in more recent years , there has been evidence that ssa can have antitumor activity and also be able to reduce tumor growth by direct action on somatostatin receptors . the first randomized clinical study that demonstrates the possible anti - tumor effects of long - acting octreotide in comparison to placebo was the promid study . this study showed a decreased risk of disease progression of 66% and arrested tumor growth in 69% for a median of 14.3 months . treatment with lar octreotide seems to be more effective in patients with low hepatic tumor burden and resected primary tumor [ 4 , 41 ] . scintigraphy with somatostatin allows , beyond the level of self - uptake , to predict therapy 's patient response . there are other ssas in use such as lanreotide and others in the study as pasireotide ( this one can interact with four receptors ( ssts1 , ssts2 , ssts3 , and ssts5 ) while octreotide and lanreotide only interacted with two type of receptors ) . preliminary data suggest that pasireotide could be useful in patients that do not respond to octreotide and could be able to control symptoms in 27% patients . however , nausea , vomiting , diarrhea , steatorrhea , cardiac abnormalities , arrhythmias , hypothyroidism , and hypoglycemia were reported in different percentages [ 42 , 44 , 45 ] . cholecystitis can occur in more than 50% of cases due to its action as an inhibitor of the contractility of the gallbladder . in patients with risk factors , interferon is an immunomodulator that inhibits the production of several growth factors and also has antiangiogenic properties . when used in net 's treatment these agents seem to have an additional activity which is the hyperregulation of somatostatin receptors . interferon may be used in functioning and nonfunctioning nets , alone or in combination with ssa in view of the lack of response under maximum dose of these agents . in some clinical studies , treatment with inf- showed symptomatic control in 30%70% of cases , with stable disease in more than 70% of patients [ 12 , 47 ] . however , other studies have not demonstrated their effectiveness even in association with the ssa . a 37% increase in 5-year survival with inf- alone to 57% in combination with octreotide was observed in a single study . however , the results were not statistically significant due to the small number of patients . two other small randomized studies showed that the combined therapy increased toxicity without additional gain in survival [ 49 , 50 ] . prrt is a therapeutic option with particular advantages in patients with a symptomatic disease and tumors with positive somatostatin receptors , not candidates for surgery . this technique uses a radioactive peptide bound to ssa , which after the interaction with the receptor is internalized , releasing specific and localized radioactivity , allowing a precise destruction of tumor cells [ 42 , 51 , 52 ] . it has little interference with the nontumor tissue , except with the kidney , bladder , and bone marrow . the most used are lutetium and y , which differ from each other in particles emission , particles energy , and tissue penetration [ 6 , 12 ] . low uptake indicates 20% of the possibility of the effect on liver metastasis , and high uptake indicates the possibility of 60% effectiveness . nets sensitivity to cytotoxic therapy appears to have correlation with the primary location of the tumor and tumor grade . clinical studies show their use in nets in general , without specifications for metastatic liver disease . the great effectiveness of their use is in combination with other agents such as 5-fluorouracil and doxorubicin , but with results of mean response of 9.3 months [ 12 , 13 ] . dacarbazine is another cytostatic agent with proof of effectiveness in pancreatic nets , with a response rate of 34% in phase ii study . clinical use of dacarbazine with stz has had limitations due to its high toxicity . recently , the use of alkylating agents such as temozolomide appears to be promising in this tumor type . in a phase ii study thalidomide and temozolamide demonstrated a response rate of 45% . in a retrospective study with temozolamide and capecitabine , the response rate was 70% , with a mean progression - free survival of 18 months and 2 years , overall survival of 92% . platinum based chemotherapy may be useful in patients with high grade undifferentiated tumors and with liver metastases . in these patients response rate was 42%80% using cisplatin and etoposide and 78% with combination of oxaliplatin [ 5760 ] . although chemotherapy could be used as rescue treatment , it is not considered the first line of the nonsurgical treatment . furthermore , the presence of liver metastases is associated with a poor response compared to nets without liver metastasis . conventional chemotherapeutic agents have limited efficacy in metastasized nets . despite positive results in some way to the little differentiated carcinomas and primary for the pancreas in relation to other locations , overall response rates are low and the impact on survival is small . the growing knowledge about the biology of these tumors , along with the ability to synthesize new drugs that interfere with therapeutic targets , has given the possibility to develop new target therapies that can change the outcome of these patients . nets are highly vascular tumors that express on their cell surface vascular endothelial growth factor ( vegf ) receptors . tumor progression seems to be associated with high levels of circulating vegf , making this possible therapeutic target , such as in other neoplasms . bevacizumab ( a monoclonal antibody ) , sunitinib , sorafenib , and pazopanib ( tyrosine kinase inhibitors ) have been the most studied drugs in pancreatic nets . bevacizumab was introduced in a randomized clinical study with patients being treated with ssa and bevacizumab compared to the combined therapy with peg inf-. superiority was seen in bevacizumab treatment arm with 95% of patients with a progression - free survival of 18 months . based on positive results from phase ii studies , sunitinib was tested in a double - blind phase iii study in patients with well - differentiated and progressive pancreatic net compared with placebo . the primary endpoint ( progression - free survival ) was statistically higher among patients with sunitinib ( 11.4 months compared with 5.5 months in the placebo group ) . also positive was the objective response rate of 9.3% , with stabilization of the disease in 63% of patients . in this clinical trial , there were 95% of patients with metastases including hepatic metastases in the treatment arm and 94% in the placebo arm ( figure 1 ) . however , this study was stopped early because of the high number of adverse events and deaths in the placebo arm [ 1 , 12 ] . the mtor pathway is of great importance since it interferes with the control of cell growth , apoptosis , and protein synthesis . the presence of mtor pathway abnormalities is acknowledged in nets , and these abnormalities are also believed to be in part responsible for the development of these tumors . two mtor inhibitors have been studied in pancreatic nets , temsirolimus , and everolimus . in a phase ii clinical study with everolimus has demonstrated a response rate of 9.6% and stabilization of the disease in 67.8% of patients . the subsequent phase iii study ( radiant study ) confirmed its activity in patients with well and moderately differentiated progressive pancreatic net with a progression - free survival of 11.1 months and 4.6 months in the placebo arm . in the everolimus arm , 94.8% of the patients had hepatic metastases versus 93.3% of patients in the everolimus plus lar arm . most of the adverse effects were grade 1 - 2 ( stomatitis , diarrhea , rash , fatigue , and infections ) . the nets frequently metastasize to the liver , and the presence of liver metastases worsens the prognosis of patients , increasing morbidity and mortality . surgery resection remains the gold standard , especially in well - differentiated resectable lesions and also improves symptomatic control in selected cases . other liver - directed methods are also advantageous , especially in the presence of predominantly liver disease . in addition , they can be combined with each other and with surgery and systemic therapeutic , allowing a greater range of treatment and increased overall survival and progression - free survival . those modalities include the rfa , in situations of localized liver damage up to 3 cm of diameter , and hace , in the case of diffuse liver disease systemic therapy is important in controlling symptoms , particularly in carcinoid tumors , with the ssa . for those patients who are not candidates to surgery , ssa can also offer control of disease progression in low grade net . these agents remain the core of medical therapy for metastatic carcinoid tumors . in pnets and particularly in high grade nets with liver metastases , chemotherapy has a substantial role . more recently , the inhibitors of the mtor pathway and vegf pathway has been shown very promising in controlling disease progression in pnets . it is not known their role in high grade tumors and whenever necessary they can be used together with ssa . for instance , the prevention of tumor progression is one of the main investigating areas . in this area , the role of lar octreotide ( in advanced tumors other than small intestine ) , lanreotide ( investigation is already ongoing in nonfunctioning net ) , and pasireotide ( som230 in nets from all sites of origin ) is being evaluated . the use of everolimus and sunitinib in early stage tumors is another interesting issue , and its role in tumor progression prevention is still to be defined . physicians and net patients are also waiting for guidelines that acknowledge the best treatment approach for patients with grade 3 tumors from any origin and site . the combination of molecular target therapy and chemotherapy , namely , ssa plus chemotherapy , temozolamide and everolimus or temozolamide , and sunitinib in pnet patients should also be addressed in clinical trials .

neuroendocrine tumors ( nets ) comprise a heterogeneous group of tumors that form a distinct entity . approximately 7580% of patients present with liver metastases at the time of their diagnosis , and 20%25% will develop these lesions in the course of their disease . the presence of secondary deposits in the liver significantly increases the morbidity and mortality in these patients . the only potentially curative treatment is the surgical resection of the primary tumor and hepatic lesions . however , only 10% of patients presents under ideal conditions for that approach . several techniques aimed at localized liver lesions have been applied also with interesting results in terms of survival and symptom control . the same has been demonstrated with new systemic therapies ( target therapies ) . however , these are still under study , in order to define their true role in the management of these patients . this paper intends to address , in a general way , the various treatment options in patients with liver metastases from neuroendocrine tumors .

1. Introduction 2. The Clinical Significance and Prognosis of Liver Metastases 3. Liver-Targeted Therapies 4. Systemic Therapy 5. Conclusion

the neuroendocrine tumors ( nets ) are a heterogeneous group of neoplasms that share some biological characteristics , that have been addressed as a common entity . in 62%82% of the cases , the nets are located in the digestive system , being designated as gastroenteropancreatic neuroendocrine tumors ( gep - nets ) . the latest figures from the uk , sweden , and switzerland suggested that their incidence is 2 - 3/100000 with a higher prevalence in females . also data from the surveillance , epidemiology , and end results ( seer ) program database report an increased incidence between 1973 and 2004 of 1.09 to 5,25/100000 [ 3 , 4 ] . nets are classically characterized by their ability to secrete hormones and/or vasoactive peptides , which results in many of their clinical manifestations , ganging up on specific hormonal syndromes . the most frequent syndrome is the carcinoid syndrome , which results from the secretion of serotonin and includes symptoms such as skin flushing , severe diarrhea , abdominal cramping , and electrolyte abnormalities [ 4 , 5 ] . the liver is the most important place of metastatic disease . more than 75% of patients with nets of the small intestine and 30%85% of patients with pancreatic nets have liver metastases at the time of diagnosis or during the course of their disease . additionally 5%10% of patients with nets of unknown primary location have liver metastases at the time of the diagnosis [ 6 , 8 ] . patients with liver metastases have a worsening of their morbidity and mortality , as compared to patients without these lesions . survival rate to 5 years of patients with untreated liver metastasis is about 13%54% , against 75%99% in patients without liver metastases [ 6 , 9 ] . this revision aims to address the theme of gep - nets with metastatic liver , their clinical significance , prognosis , and its current therapeutic approach with a special focus on medical treatment . patients with nets often develop liver metastases ( nlms ) affecting significantly their morbidity and mortality . about 75%80% of patients present with hepatic metastasis at the time of diagnosis ( synchronous ) , while 20%25% the presence of liver metastases is closely related to the appearance / aggravation of symptoms . the presence of hepatic metastases leads to less metabolization of these peptides leading to an increase of their circulating levels . sometimes , only then its distribution , in the way it modifies therapeutic management , reflects the aggressiveness of the tumor . type ii metastases include the presence of dominant injury with small satellites contralateral ( 10%15% of cases ) . because of the possibility of surgical resection , type i is associated with a favorable outcome . in this group of patients however , the complete excision of secondary lesions is only an option for a small number of patients ( 10%20% ) . for that reason , several techniques have been developed by exploiting duality in the liver vasculature in order to control the disease process . these modalities are include the ablative techniques , the hepatic artery embolization , chemoembolization , and radioembolization ( selective internal radiation therapy ) . curative intention depends on the stage and presentation of the disease [ 13 , 14 ] . even with hepatic involvement , surgical approach , when feasible , is the best treatment option , with proven benefits in terms of overall survival and quality of life improvement [ 1 , 6 , 12 ] . patients treated with surgical resection of the primary tumor and liver metastases had a survival at 5 years of more than 60% , reaching 80% in some studies , with minimum mortality ( less than 5% ) and admissible morbidity ( less than 30% ) [ 6 , 12 ] . patients with liver metastasis that are not candidates to surgical treatment ( 80%90% of cases ) , still benefit from primary tumor resection . in these cases , where primary tumor was resected therefore , the palliation of the symptoms is also a criterion for surgery [ 5 , 6 ] . regarding the criteria for selecting patients with nlms , there is no consensus , since the several studies developed included a small number of patients and different surgical procedures . however , when the complete excision of a significant number of liver metastasis is possible , surgery must always be considered . in a recent retrospective study , 74 cases were analyzed and the 5-year survival rate was more than 60% in all patients who underwent surgical resection of liver metastases . in 65% of these patients , excision was incomplete , and there was no worsening of the prognosis [ 6 , 12 ] . however , the identification of other predictive variables in patients ' selection for surgery was needed . therefore , clinical studies were conducted , and patients were selected based on tumor grade . the analyses of the results from these studies showed that tumors with high grade had a worse postsurgical outcome when compared to low grade tumors . translating these results into clinical practice , in 2008 , the european neuroendocrine tumor society ( enets ) issued general guidelines for surgical resection based on the three types of liver involvement . for patients with hepatic involvement of type i surgical resection is the first therapeutic option , while in patients with the involvement of type iii it is totally excluded . in order to give the best possible treatment to patients with net tumors this evaluation should take into account the following aspects : performance status , localization of the primary tumor , tumor grade , possibility of primary tumor resection , presence and localization of metastases , possibility of complete resection of metastases , presence of extrahepatic disease ( extrahepatic disease should be excluded based on adequate image methods ) , and the presence or absence of carcinoid heart disease [ 6 , 17 ] . in patients with carcinoid heart disease , symptoms control and clinical stabilization should be achieved before aggressive surgery is considered . also , in patients with carcinoid syndrome , even in those under treatment with somatostatin analogues , intra- and postoperative carcinoid crisis must be prevented , using these same somatostatin analogues . preoperative administration of octreotide ( 300 micrograms subcutaneously ) can reduce the incidence of carcinoid crisis and is recommended for patients with a history of carcinoid syndrome who require surgical procedures . calcium and catecholamines may provoke the release of mediators from the tumor and worsen , rather than ameliorate , the syndrome . in patients with high risk of carcinoid crisis , an iv octreotide drip may be initiated preoperatively [ 1 , 1823 ] . the presence of abdominal lymph node involvement , including local recurrence , is not an absolute contraindication for surgery , if the excision of lymph nodes and liver metastases and/or recurrent lesions could be done simultaneously or in planned staggered phases . the use of intraoperative echography seems to be of additional value as it allows to define the extent of known lesions and to detect small additional lesions . it is usually recommended in cases where more than 90% of the tumor can be excised or in young patients [ 6 , 12 , 20 , 25 , 26 ] . liver transplantation is only indicated in cases of metastatic disease if the primary tumor is an net . in 1998 , lehnert analyzed a total of 103 net patients with liver transplant . however , in a more recent clinical trial , primary location in the duodenum or pancreas was associated with worse prognosis after transplant [ 6 , 12 , 28 ] . these findings were , however , not supported by other investigations [ 12 , 29 ] . regarding biomarkers analyses and their correlation with transplant outcome , the authors found that patients with a low ki-67 and normal expression of e - cadherin had a more favorable prognosis . on the other hand , the analysis of the data from the united network for organ sharing between november 1988 and march 2011 showed that only 185 liver transplants were due to nlms . in patients with diffuse and unresectable liver disease , with uncontrolled symptoms ( resistant to medical therapy support ) , the main criteria for selecting patients for liver transplantation are in these cases being not candidate for resection , diagnosis , and complete resection of the primary tumor at least one year before , no extrahepatic disease , stable disease at least for one year , and failure of nonoperative treatments . patients with net tumors can be selected to treatment with different types of ablative techniques such as cryoablation , alcohol ablation ( these two are less used ) , and radiofrequency ablation ( rfa ) . this technique could be of advantage in patients whose liver metastases are located next to large vessels or the bile duct , that could be damaged by the heat released during rfa . pai should be used not as monotherapy but with other ablative techniques in order to achieve better results . it is mainly used in patients with a small number of liver lesions that can not be resected or as an adjuvant to other therapies . the largest clinical study until involving patients with hepatic lesions treated with rfa was reported by the cleveland clinic . from these 89 patients , 22% had recurrence , and from these 22% recurrences , 63% corresponded to new liver lesions and 59% to extrahepatic disease . an important limitation of the effectiveness of this technique is the size of the tumor . hepatic artery embolization ( hae ) and hepatic artery chemoembolization ( hace ) are two different technics that can also be used in the treatment of liver metastasis . hepatic arterial embolization is frequently applied as a palliative technique in patients with hepatic - predominant metastatic net who are not candidates for surgical resection . it is based on the principle that tumors in the liver derive most of their blood supply from the hepatic artery , whereas healthy hepatocytes derive most of their blood supply from the portal vein . hace provides not only the embolic blocking but also the supply of direct chemotherapy to these cells , increasing the action / concentration of these agents ( more than 20 times of the systemic chemotherapy ) . its benefits have been documented by different authors , but there were no comparative studies between hae and hace demonstrating superiority of one of these techniques . both hae and hace are palliative techniques used in patients with unresectable lesions , with diffuse and progressive liver disease , with symptoms not controlled with medical treatment and without impaired liver function [ 13 , 24 ] . the main benefit is symptomatic relief ( 70%90% of the patients ) and tumor growth control ( 50% of the patients ) [ 1 , 24 ] . these therapies have proved to be a valuable option , particularly in patients with more than 75% of hepatic involvement , carefully selected , without additional risk factors . sepsis , liver and renal failure , carcinoid crisis , necrotizing cholecystitis , and peptic ulcer bleeding have been reported in about 7.5 to 23.8% of patients . , fever , abdominal pain , leukocytosis , increased transaminases , and bilirubin ) , which occurs in 90% of patients , in most cases self - limited ( 2472 hours of duration ) [ 6 , 17 , 24 ] . in order to minimize the complications , it is recommended to split the area to be treated in small portions in each session ( one lobe per session ) [ 5 , 17 ] . concomitant administration of antibiotics and somatostatin analogues , as well as aggressive hydration , to prevent liver abscesses , carcinoid crisis , and tumor lysis syndrome is further recommended [ 1 , 24 ] . the use of somatostatin analogues during the procedure is of extreme importance in order to prevent carcinoid crisis . external radiation therapy has limited value in nets , being only indicated in the presence of symptomatic brain and bone metastases . investigated the safety and efficacy of this treatment in patients with nlms . in this study , 34 patients had long - term responses with a mean overall survival of 29.4 months with radiological improvement in 50% of the patients . also showed that the major benefit seems to stabilize advanced disease allowing an increase in overall survival although still under investigation results have been promising . since nets are a rare pathology , large randomized studies in patients with nlms are difficult to perform . this limitation leads to an overall assessment of restricted systemic therapies that can not be translated in to a consensual treatment algorithm . however , the data obtained from a single center study showed that the aggressive medical treatment of nlms instead of surgical therapy increases the 3-year survival rate to 76.4% and the 5-year survival rate to 63.9% . therefore , conservative treatment , that is , the majority of nets is expressed on the surface of their cells somatostatin receptors ( sstr 15 ) . in an initial study , the subcutaneous administration of octreotide at the dose of 150 mg tid improved symptoms , especially carcinoid syndrome , in 88% of patients . however , the short action formulations continue to play an important role in stabilizing the disease , especially in the situations of carcinoid crisis and its prevention . in more recent years , there has been evidence that ssa can have antitumor activity and also be able to reduce tumor growth by direct action on somatostatin receptors . treatment with lar octreotide seems to be more effective in patients with low hepatic tumor burden and resected primary tumor [ 4 , 41 ] . there are other ssas in use such as lanreotide and others in the study as pasireotide ( this one can interact with four receptors ( ssts1 , ssts2 , ssts3 , and ssts5 ) while octreotide and lanreotide only interacted with two type of receptors ) . however , nausea , vomiting , diarrhea , steatorrhea , cardiac abnormalities , arrhythmias , hypothyroidism , and hypoglycemia were reported in different percentages [ 42 , 44 , 45 ] . cholecystitis can occur in more than 50% of cases due to its action as an inhibitor of the contractility of the gallbladder . in patients with risk factors , interferon is an immunomodulator that inhibits the production of several growth factors and also has antiangiogenic properties . interferon may be used in functioning and nonfunctioning nets , alone or in combination with ssa in view of the lack of response under maximum dose of these agents . a 37% increase in 5-year survival with inf- alone to 57% in combination with octreotide was observed in a single study . however , the results were not statistically significant due to the small number of patients . prrt is a therapeutic option with particular advantages in patients with a symptomatic disease and tumors with positive somatostatin receptors , not candidates for surgery . the most used are lutetium and y , which differ from each other in particles emission , particles energy , and tissue penetration [ 6 , 12 ] . low uptake indicates 20% of the possibility of the effect on liver metastasis , and high uptake indicates the possibility of 60% effectiveness . nets sensitivity to cytotoxic therapy appears to have correlation with the primary location of the tumor and tumor grade . in a phase ii study thalidomide and temozolamide demonstrated a response rate of 45% . in a retrospective study with temozolamide and capecitabine , the response rate was 70% , with a mean progression - free survival of 18 months and 2 years , overall survival of 92% . platinum based chemotherapy may be useful in patients with high grade undifferentiated tumors and with liver metastases . in these patients response rate was 42%80% using cisplatin and etoposide and 78% with combination of oxaliplatin [ 5760 ] . furthermore , the presence of liver metastases is associated with a poor response compared to nets without liver metastasis . the growing knowledge about the biology of these tumors , along with the ability to synthesize new drugs that interfere with therapeutic targets , has given the possibility to develop new target therapies that can change the outcome of these patients . bevacizumab ( a monoclonal antibody ) , sunitinib , sorafenib , and pazopanib ( tyrosine kinase inhibitors ) have been the most studied drugs in pancreatic nets . superiority was seen in bevacizumab treatment arm with 95% of patients with a progression - free survival of 18 months . based on positive results from phase ii studies , sunitinib was tested in a double - blind phase iii study in patients with well - differentiated and progressive pancreatic net compared with placebo . the primary endpoint ( progression - free survival ) was statistically higher among patients with sunitinib ( 11.4 months compared with 5.5 months in the placebo group ) . also positive was the objective response rate of 9.3% , with stabilization of the disease in 63% of patients . in this clinical trial , there were 95% of patients with metastases including hepatic metastases in the treatment arm and 94% in the placebo arm ( figure 1 ) . however , this study was stopped early because of the high number of adverse events and deaths in the placebo arm [ 1 , 12 ] . the presence of mtor pathway abnormalities is acknowledged in nets , and these abnormalities are also believed to be in part responsible for the development of these tumors . two mtor inhibitors have been studied in pancreatic nets , temsirolimus , and everolimus . in a phase ii clinical study with everolimus has demonstrated a response rate of 9.6% and stabilization of the disease in 67.8% of patients . the subsequent phase iii study ( radiant study ) confirmed its activity in patients with well and moderately differentiated progressive pancreatic net with a progression - free survival of 11.1 months and 4.6 months in the placebo arm . in the everolimus arm , 94.8% of the patients had hepatic metastases versus 93.3% of patients in the everolimus plus lar arm . most of the adverse effects were grade 1 - 2 ( stomatitis , diarrhea , rash , fatigue , and infections ) . the nets frequently metastasize to the liver , and the presence of liver metastases worsens the prognosis of patients , increasing morbidity and mortality . other liver - directed methods are also advantageous , especially in the presence of predominantly liver disease . those modalities include the rfa , in situations of localized liver damage up to 3 cm of diameter , and hace , in the case of diffuse liver disease systemic therapy is important in controlling symptoms , particularly in carcinoid tumors , with the ssa . in pnets and particularly in high grade nets with liver metastases , chemotherapy has a substantial role . more recently , the inhibitors of the mtor pathway and vegf pathway has been shown very promising in controlling disease progression in pnets . for instance , the prevention of tumor progression is one of the main investigating areas . in this area , the role of lar octreotide ( in advanced tumors other than small intestine ) , lanreotide ( investigation is already ongoing in nonfunctioning net ) , and pasireotide ( som230 in nets from all sites of origin ) is being evaluated . the use of everolimus and sunitinib in early stage tumors is another interesting issue , and its role in tumor progression prevention is still to be defined . physicians and net patients are also waiting for guidelines that acknowledge the best treatment approach for patients with grade 3 tumors from any origin and site . the combination of molecular target therapy and chemotherapy , namely , ssa plus chemotherapy , temozolamide and everolimus or temozolamide , and sunitinib in pnet patients should also be addressed in clinical trials .

enrollment of individuals in organizations such as health maintenance organizations ( hmos ) that supply medical care for a fixed periodic premium or a capitated rate continues to grow ; by 1991 enrollment in such organizations was about 13 percent of the population ( national center for health statistics , 1992 ) . moreover , the percentage of children enrolled is probably even higher because disproportionately few medicare enrollees are enrolled in hmos ( 2.8 percent ) , and because hmos have typically covered maternity and well - child care with less cost sharing than insurance plans in the fee - for - service ( ffs ) system ( mcmillan , lubitz , and russell , 1987 ) . with growing hmo enrollment , more attention is being paid to the method for setting the rate at which the government and the private sector pay hmos . in public - sector programs , most researchers have focused on medicare 's formula , the adjusted average per capita cost ( aapcc ) , and have consequently analyzed the behavior of those 65 years of age or over , or at least the behavior of adults ( anderson et al . , 1986 ; anderson and knickman , 1984a , 1984b ; ash et al . , 1989 ; beebe , lubitz , and eggers , 1985 ; gruenberg , wallack , and tompkins , 1986 ; howland et al . , 1987 ; lubitz , beebe , and riley , 1985 ; mccall and wai , 1983 ; mcclure , 1984 ; newhouse , 1986 ; newhouse et al . , 1989 ; thomas and lichtenstein , 1986a and 1986b ; thomas et al . , 1983 ) . however , 44 percent of the recipients in the other major public program , medicaid , were less than 18 years of age in 1989 , a figure that is likely to grow in light of the planned eligibility expansion for poor children ( reilly , clauser , and baugh , 1990 ) . moreover , many states are attempting to expand their use of capitated systems for the medicaid population . if each capitated group enrolled a representative mix of health risks among the medicaid population , the rate could simply be a fraction , or perhaps as much as 100 percent , of per capita ffs costs . however , it is unreasonable to expect that each group will do so . on the one hand , chronically ill patients who are under the care of an ffs physician will have incentives to continue with their physicians rather than to join an hmo ; on the other hand , hmos have incentives to avoid costly patients if they only receive payment that is based on the cost of the average patients . even if health risks were distributed randomly , chance alone would cause some hmos to have a mix of enrollees whose health characteristics differed from the population average . if all hmos received the same payment per patient , there would be windfall profits and losses . such profits and losses would , however , be more important for small hmos because of the law of large numbers . the natural approach to the issue of heterogeneous enrollees is to vary the amount paid an hmo according to an enrollee 's expected use of medical services , i.e. , to adjust the average rate . indeed , medicare makes such adjustments , thus the term adjusted average per capita cost ( aapcc ) . specifically , it adjusts for age , gender , welfare status , institutional status , county of residence , and basis for medicare eligibility ( old age , disability , or end stage renal disease ) . it is estimated that they may account for only 5 to 10 percent of the variation in expected cost and much less of actual cost across individuals ( lubitz , beebe , and riley , 1985 ; newhouse , 1986 ; newhouse et al . , 1989 ) . in our 1989 article , we examined how much additional adjusters , specifically measures of health and of use in the prior year , would improve the performance of an aapcc - type formula for adults under 65 years of age . using similar methods , we present herein results for children , although our results are limited to outpatient expenditures for children 5 - 13 years of age because our data set has few children that were hospitalized . outpatient expenditures , however , account for 55 percent of total expenditures by children , compared with 38 percent for adults ( manning et al . , 1987 ) , and we see little reason to believe that the relative performance of various adjusters would change much if we had sufficient inpatient data to analyze ( though the absolute amounts of explained variance may decrease ) . we begin by estimating how much of the actual variation in expenditure one could potentially explain , and how much instead is because of random or unforeseeable events . because adjusters can not predict variation due to future random events , we wish to ignore the influence of such events as we assess the performance of adjusters , and want only to explain variation in expected , not actual , expenditure . put another way , we estimate what the r would be if we regressed actual expenditures on a set of almost ideal adjusters . as the explanatory power of a set of adjusters approaches the maximum explainable variance , the incentives for risk selection fall to negligible levels . we begin with the analog for children of the demographic types of variables currently used in the aapcc formula . we then estimate the gain from also using several measures of health status and prior use to adjust the capitation rate . we analyze outpatient expenditures per child ; that is , we analyze individual , not family , expenditure . one might argue that because families typically enroll as a group , we should have analyzed family behavior . the explanatory variables whose importance we assess , however , are at the individual level . their relative importance would not have changed had we chosen to analyze data at the family level , but we would have had to impose additional assumptions to aggregate individual - level explanatory variables to the family level . indeed , the appropriate assumptions are not at all obvious . however , because family - member expenditures are not independent , any incentive to skim or dump will be increased if families enroll as a unit . expenditure can not be fully or even largely predicted , so any set of risk adjusters will not explain all variance . fortunately this does not cause a problem , as long as the hmo ( indeed , simply paying at the average for all is appropriate if all expenditures are unpredictable . ) problems potentially arise , however , if the hmo can determine that one person 's expected expenditure ( before the fact ) exceeds another 's . there is a financial incentive to enroll the low - cost person ( skimming ) if there is no adjustment in payment . thus , one criterion for judging a set of adjusters is how well they explain expected expenditure or predictable variance . if all predictable variance is accounted for , there should be no skimming . to judge against this criterion , therefore , we need to know the variance in expected expenditure , which will be less than the actual or observed variance by the variance in unpredictable expenditure . if children 's medical expenditures correspond to the following simple model , it would be straightforward to determine the variance in expected expenditure , which is the amount of variance one could possibly explain in a regression of annual expenditure , using cross - sectional data : xit is a vector of risk adjusters , is a vector of weights , i indexes the child , t indexes year , i is an unobserved child - specific , time - invariant ( stable ) component of variance , and it is a child - specific , time varying component of variance . if the last term it is random and can not be predicted by the hmo or by the family , the maximum variance that could be explained is that accounted for by the first two terms , and we shall make this assumption . in fact , this is a lower boundary on the maximum explainable variance because some elements of it might also be predictable . that is , there may be some time - varying variables , omitted from the x vector , that explain a non - trivial amount of variance . an example of a variable usually contained in it rather than in xit is an illness that has a partially predictable time pattern , such as leukemia . nonetheless , it seems plausible to assume that most of the variation in it is random . to the extent this is true , to the degree that it is predictable , however , our estimates understate the amount of variation one can explain . we have thus estimated the maximum explainable variance by estimating the proportion of variance in the i term of the right - hand side of equation 1 , assuming no adjusters ( i.e. , no x 's ) ; this is analogous to the r from using a dummy variable for each person or to the proportion of variance that is between - person variance . to estimate the between - person variance , we subtracted an estimate of within - person variance from total variance , correcting for the bias from estimating within - person variance from a finite time series ( searle , 1971 ) . an alternative method for computing the maximum r is to specify an x vector and estimate the amount of stable variation in the residuals . in principle , this method should lead to a higher maximum r because the method described in the previous paragraph omits any variation for the x term from covariates that change over time . for adults , however , we found that the estimated maximum r by the alternative method was less than the estimate using the method described in the previous paragraph ( newhouse et al . we have used r as a criterion variable , but some question the appropriateness of doing so . they argue that r shows the goodness of prediction at the individual level , but that the formula only needs to predict well for groups ( lubitz , 1987 ) . in other words , as long as the hmo receives adequate payment for its entire group of enrollees , the formula does not need to predict well at the individual level . this argument , however , ignores the behavioral incentives of the hmo , which can make more money by discouraging enrollment ( or encouraging disenrollment ) of any individual or family whose expected cost exceeds revenue . to blunt this incentive requires a premium that matches the expected cost of the hmo for each patient or family that it enrolls . a different criticism is that r may be on average high , but still perform badly for certain subgroups . that is , the payment formula may not fit well in some regions of the response surface , the functional form of the x vector may be specified incorrectly . this criticism has merit if one 's purpose is to develop a specific payment formula , but it is less relevant for our purposes . our aim is not to develop a specific formula , but only to compare in a gross way the performance of demographic , health status , and prior use variables . welch ( 1985 ) has proposed a model in which the errors follow a first order auto - regressive process : uit is an independently and identically distributed random term and 1 < < 1 . in equation 2 , the potential explainable variance is that explained by the adjusters plus that explained by the first term on the right - hand side of the equation ( because when one is predicting year t 's expenditures , one has an estimate of it1 ) . thus , in this model the maximum r is approximately the proportion of variance explained by the adjusters plus approximately [ 1/(1 )]var ( u ) . the consistency of equations 1 and 2 with the data can be tested straightforwardly by examining the pattern of correlation of the residuals over time . in the first model , the correlation between the residuals for time periods t and t + s for varying s should be constant ( up to sampling error ) and equal to variance [ ]/ ( variance [ ] + variance [ ] ) . in the second model , the correlation should decline geometrically ( specifically , it should equal ) . we later present results on the time pattern of the correlations in our data for children . for adults , the effect of regression to the mean , which equation 2 implies , appears modest ( newhouse et al . , 1989 ) . because our principal interest was to ascertain how useful various adjusters would be in further developing capitation rates rather than a particular payment formula , we have not performed a variety of specification tests for the accuracy of the functional form for the x vector . thus , variables are simply entered in a linear form , and no tests for interactions have been performed . a more complex specification would probably improve performance , but it seems unlikely to change our qualitative conclusions . moreover , going beyond a simple linear form risks overfitting our sample data , thereby distorting our results . using our results on explainable variance we consider a child whom the hmo predicts to be one standard deviation below or above the mean for expenditure based on the information available to it . we show how the profit or loss to the hmo diminishes as the payment formula changes to incorporate information from additional risk adjusters . in the limit , the additional risk adjusters would encompass all the information available to the hmo , and the hmo would not gain from selection . the data we use come from the rand health insurance experiment , the design of which has been described in many places ( brook et al . , 1983 ; manning et al . , 1987 ; newhouse et al . , 1981 ; newhouse et al . , 1993 ) . seattle , washington ; dayton , ohio ; charleston , south carolina ; fitchburg - leominster , massachusetts ; and two rural areas , franklin county , massachusetts , and georgetown county , south carolina to insurance plans that varied the cost sharing they faced . we have removed the effect in the sample of cost sharing from all observations because some of the variation in spending due to cost sharing was induced by the experiment ; i.e. , we have removed the between - plan variance . in effect , we ask how well various explanatory variables or adjusters account for within - plan variance . thus , our results apply to an insured group with no variation in cost sharing , which is a good approximation to groups covered by capitation arrangements . an aspect of the experiment that approximates capitation less well is that the experimental plans employed no utilization management techniques , such as pre - admission certification . this clearly raised the absolute level of spending relative to an hmo ( manning et al . , 1984 ) , and the question therefore arises as to whether results from this sample apply to children in a capitated group . although we can not be sure they do , it is not clear whether utilization management would much affect the proportion of variance explained by various personal characteristics . unless utilization management techniques differentially increase the predictable portions of expenditure , the conclusions of this article , with respect to how well one can predict , are unaffected . even if they were to increase the proportion of predictable variation , they would have to increase it differentially by type of covariate for our conclusions with respect to specific covariates to be changed . ( the number of children 5 - 13 years of age enrolled in the hmo portion of the experiment , a little more than 200 , were too few to use in this analysis . ) the families who participated in this experiment were randomly assigned to a 3-year or 5-year participation period , during which time the experiment acted as their insurance company . ( they formally assigned to the experiment the benefits of any insurance for which they were eligible . ) independent verification of physician office claims indicates that the families filed claims with the experiment for more than 90 percent of their utilization ; thus , we have a nearly complete record of utilization for the period of participation ( rogers and newhouse , 1985 ) . the families invited to participate in the experiment were randomly selected , subject to some qualifications that are not important for this article . specifically , the following groups were excluded : ( 1 ) those eligible for medicare ; ( 2 ) military personnel in active duty and retired ; ( 3 ) veterans with service - connected disabilities ; and ( 4 ) those institutionalized indefinitely ( those in prison and in long - term psychiatric hospitals are the principal groups excluded by this criterion ) . additionally , in five of the six sites ( all but seattle ) , low - income individuals were over - sampled to a limited extent . all those living at a given dwelling unit who met the eligibility requirements were offered enrollment . as a result , the utilization of the 1,844 observations are not all independent because the amount of utilization by children in the same family is positively correlated . our calculations of the explained proportion of variance do not account for intrafamily correlation , but that should have little effect on our estimates of the proportion of variance that various types of individual characteristics can explain . in addition , as we will show , there is dependence over time within child . indeed , the essence of the risk - adjustment problem is to account for the dependence in the residuals over time . the sample used for the regression equation included only those participants who completed the study and the physical examination at exit . although 93 percent of those children who began the study completed it , our analysis sample is considerably smaller . children who turned 14 years of age during the experiment were excluded from our analysis because they took an adult screening exam at exit , which differed from the children 's exam . moreover , children under 5 years of age at exit were not given physiologic tests and were also excluded . this excluded another 10 percent of the children who began the study . in the regression analysis , we did not use children in their first year of participation because we did not have comparable prior - use data for them . we did use first - year data in examining the stability of year - to - year correlations . we excluded those with any missing data for physiologic variables ; these were mainly children who moved out of area during the experiment , and so did not have a hearing test as part of their out - of - area screening examination . in all , our sample consisted of 2,185 person - years . only 84 of the person - years ( 3.8 percent ) had inpatient use . because inpatient use was so rare , we chose not to include it . our major interest was to predict annual expenditures per child on medical care services in constant dollars . for purposes of calculating the maximum r , we examined expenditure in both raw and trimmed form . the trim point was at the 98th percentile of total medical expenditure . for trimmed data , if an observation was in the upper 2 percent of the relevant distribution , it was set equal to the mean of the upper 2 percent of the observations . this preserved the overall mean . because we wished to ignore within - plan variation , we began by regressing expenditures on plan . plan was defined as the log of the nominal coinsurance rate plus a dummy variable for one particular plan ( a plan with outpatient - only cost sharing ) . by design , we then calculated : a indexes the specification with only the plan variables included , and b indexes any of the more complete specifications . in fact , the plan variables explained only 1 percent of the total variance , so this correction is in practice unimportant . first , we included the demographic kinds of variables used by medicare : age ( entered linearly ) ; gender ; aid to families with dependent children ( afdc ) status ( supplemental security income recipients are not in the sample population ) ; and site . then we added four different sets of variables to this basic set : dichotomous physiologic health . a set of dummy variables that indicate the presence or absence of the physiologic conditions shown in table 1 . variables defined in table 1 as ( 0,1 ) were included in the regression unchanged . variables defined in table 1 as the maximum of zero and the test value minus some cutting point were dichotomized according to whether the test value was above or below the cutting point . for example , a dummy variable for anemia assumes the value one if a child 5 - 8 years of age has a hemoglobin below 11.0 g / ml . these physiological measures are derived from data collected at exit from the study rather than at entrance because only a random 60 percent of the children were given an exam at entrance . we felt we would obtain more accurate estimates by using data measured at exit for the entire sample than the data measured at enrollment for a partial sample despite the possibility that use would affect the observed value . the experimental results showed that plan did not affect these values ( valdez , brook , and rogers , 1985 ) , and of course pre - experiment use could have affected the enrolled values . a set of variables that indicate the presence or absence of the physiologic conditions shown in table 1 , and for some conditions , a measure of the severity . variables , again measured at exit , were included in the regression as defined in table 1 . for example , two variables related to anemia were included in the regression : ( 1 ) low hemoglobin , coded as the maximum of 0.0 or ( for children 5 - 8 years of age ) 11.0hemoglobin value ; and ( 2 ) the dummy variable for anemia described in the preceding paragraph . in principle , the coefficient of the dummy variable measures the fixed costs of treating the condition , and the coefficient of the continuous variable measures the variable cost of increased severity . the cutting points reflect a judgment about values above or below which most physicians would not treat . for example , most physicians would probably not prescribe treatment for anemia with hemoglobin values above 11.0 . for values differing from the cutting point in an unhealthful direction it is quite possible , indeed probable , that the true functional form is non - linear , but theory does not specify a functional form , and we felt we would likely overfit the data if we experimented with non - linear functional forms . for example , we treated the effect of being anemic and having asthma as additive . any effort to create an actual payment formula using these variables would need to consider more complicated functional forms , though any such effort should employ a larger data set than the one used in this study . at this point the observed value of the physiologic health variables was used ; thus , an individual who had a hemoglobin value of 12.0 g/100 ml achieved through medications was not distinguished from one who had a natural hemoglobin value of 12.0 g/100 ml and who was not under treatment . in effect , our specification implies that the expected treatment cost of a child will increase with less healthy values , but that will not always be true . specifically , it will not be true if treatment alters the physiologic measure and less healthy patients use more resources ( or if not all individuals are under treatment ) . consider the above example of two persons with a hemoglobin value of 12 . although a physiologic condition that we measured was in fact responsible for treatment costs of one of the persons ( i.e. , the medication to raise the hemoglobin level ) , the physiologic variable we measured would not explain any variation in expenditure because it would be at 12.0 for both persons . ideally one would measure what the value of the physiologic health measure ( e.g. , hemoglobin ) would be if each child were untreated , but this can not be observed . an extension that partially allows for this difficulty is to enter a dichotomous variable for being in treatment . incorporating such an adjuster has the additional advantage that the relevant information can be collected solely from claims forms . nonetheless , such an approach is only a partial solution because it does not allow for bias within the treated group . for example , one child may have a hemoglobin value of 10 g/100ml without treatment , whereas another may have a value of 10.5 . if medication raises them both to 12.0 but the costs of treating the first person are greater , the cost difference would appear to the analyst as unexplained . this may be one reason that the measures of prior use described later achieve considerably more explanatory power than the measures of health status . a set of measures of functional status or physical health , general health perceptions , and mental health as rated by a parent , usually the mother . although the use of such variables as adjusters in a payment formula seems problematic because of the possibilities for fraud , we wished to ascertain the possible gains from using them in our data where there were no incentives for fraud . the same difficulty just described for the physiologic variables is present in these variables as well because medical care for a chronic problem can affect these measures , and medical care may be greater the more severe the problem . four variables measuring use of medical services in the previous year : whether there was any outpatient expenditure ; whether there was any inpatient expenditure ; and the logarithms of outpatients and inpatient expenditures . to determine the gain from using various adjusters , we use a variant of a two - equation model we have used in other work ( manning et al . , 1987 ; duan et al . , 1983 ) . this variant models the probability of outpatient expenditures and the logarithm of outpatient spending , but then retransforms the logarithm to raw dollars using a non - parametric method ( the smearing estimate ) described in the articles cited . armed with our estimated equations ( and estimated retransformation factor ) , we then compute the amount of explained variation as follows : we first predict the total outpatient expenditure of each person using the two - equation model . we then calculate a measure of r due to efron ( 1978 ) who uses the following formula : y - hati is the predicted yi using the two - equation model with alternative sets of explanatory variables , and y - bar is the sample mean of y. thus , the numerator of the expression in parentheses is the unexplained sum of squares , and the denominator is the total sum of squares . note that this measure of r can be negative when predicting from a non - linear model such as ours , but in this application it never was . we have used the two - part model and efron 's r rather than the more ordinary analysis of covariance because the two - part model has less tendency to overt it , and yields estimates with significantly lower mean square forecast error ( duan et al . , 1983 ) . thus , use of analysis of covariance , which is common in the literature , overstates how well one can do . our major interest was to predict annual expenditures per child on medical care services in constant dollars . for purposes of calculating the maximum r , we examined expenditure in both raw and trimmed form . the trim point was at the 98th percentile of total medical expenditure . for trimmed data , if an observation was in the upper 2 percent of the relevant distribution , it was set equal to the mean of the upper 2 percent of the observations . because we wished to ignore within - plan variation , we began by regressing expenditures on plan . plan was defined as the log of the nominal coinsurance rate plus a dummy variable for one particular plan ( a plan with outpatient - only cost sharing ) . by design , we then calculated : a indexes the specification with only the plan variables included , and b indexes any of the more complete specifications . in fact , the plan variables explained only 1 percent of the total variance , so this correction is in practice unimportant . first , we included the demographic kinds of variables used by medicare : age ( entered linearly ) ; gender ; aid to families with dependent children ( afdc ) status ( supplemental security income recipients are not in the sample population ) ; and site . then we added four different sets of variables to this basic set : dichotomous physiologic health . a set of dummy variables that indicate the presence or absence of the physiologic conditions shown in table 1 . variables defined in table 1 as ( 0,1 ) were included in the regression unchanged . variables defined in table 1 as the maximum of zero and the test value minus some cutting point were dichotomized according to whether the test value was above or below the cutting point . for example , a dummy variable for anemia assumes the value one if a child 5 - 8 years of age has a hemoglobin below 11.0 g / ml . these physiological measures are derived from data collected at exit from the study rather than at entrance because only a random 60 percent of the children were given an exam at entrance . we felt we would obtain more accurate estimates by using data measured at exit for the entire sample than the data measured at enrollment for a partial sample despite the possibility that use would affect the observed value . the experimental results showed that plan did not affect these values ( valdez , brook , and rogers , 1985 ) , and of course pre - experiment use could have affected the enrolled values . a set of variables that indicate the presence or absence of the physiologic conditions shown in table 1 , and for some conditions , a measure of the severity . variables , again measured at exit , were included in the regression as defined in table 1 . for example , two variables related to anemia were included in the regression : ( 1 ) low hemoglobin , coded as the maximum of 0.0 or ( for children 5 - 8 years of age ) 11.0hemoglobin value ; and ( 2 ) the dummy variable for anemia described in the preceding paragraph . in principle , the coefficient of the dummy variable measures the fixed costs of treating the condition , and the coefficient of the continuous variable measures the variable cost of increased severity . all variation on one side of a cutting point the cutting points reflect a judgment about values above or below which most physicians would not treat . for example , most physicians would probably not prescribe treatment for anemia with hemoglobin values above 11.0 . for values differing from the cutting point in an unhealthful direction , we simply entered the physiologic measure linearly . it is quite possible , indeed probable , that the true functional form is non - linear , but theory does not specify a functional form , and we felt we would likely overfit the data if we experimented with non - linear functional forms . for example , we treated the effect of being anemic and having asthma as additive . any effort to create an actual payment formula using these variables would need to consider more complicated functional forms , though any such effort should employ a larger data set than the one used in this study . at this point the observed value of the physiologic health variables was used ; thus , an individual who had a hemoglobin value of 12.0 g/100 ml achieved through medications was not distinguished from one who had a natural hemoglobin value of 12.0 g/100 ml and who was not under treatment . in effect , our specification implies that the expected treatment cost of a child will increase with less healthy values , but that will not always be true . specifically , it will not be true if treatment alters the physiologic measure and less healthy patients use more resources ( or if not all individuals are under treatment ) . consider the above example of two persons with a hemoglobin value of 12 . although a physiologic condition that we measured was in fact responsible for treatment costs of one of the persons ( i.e. , the medication to raise the hemoglobin level ) , the physiologic variable we measured would not explain any variation in expenditure because it would be at 12.0 for both persons . ideally one would measure what the value of the physiologic health measure ( e.g. , hemoglobin ) would be if each child were untreated , but this can not be observed . an extension that partially allows for this difficulty is to enter a dichotomous variable for being in treatment . incorporating such an adjuster has the additional advantage that the relevant information can be collected solely from claims forms . nonetheless , such an approach is only a partial solution because it does not allow for bias within the treated group . for example , one child may have a hemoglobin value of 10 g/100ml without treatment , whereas another may have a value of 10.5 . if medication raises them both to 12.0 but the costs of treating the first person are greater , the cost difference would appear to the analyst as unexplained . this may be one reason that the measures of prior use described later achieve considerably more explanatory power than the measures of health status . a set of measures of functional status or physical health , general health perceptions , and mental health as rated by a parent , usually the mother . although the use of such variables as adjusters in a payment formula seems problematic because of the possibilities for fraud , we wished to ascertain the possible gains from using them in our data where there were no incentives for fraud . the same difficulty just described for the physiologic variables is present in these variables as well because medical care for a chronic problem can affect these measures , and medical care may be greater the more severe the problem . four variables measuring use of medical services in the previous year : whether there was any outpatient expenditure ; whether there was any inpatient expenditure ; and the logarithms of outpatients and inpatient expenditures . to determine the gain from using various adjusters , we use a variant of a two - equation model we have used in other work ( manning et al . , 1987 ; duan et al . , 1983 ) , with the variables in table 1 as explanatory variables . this variant models the probability of outpatient expenditures and the logarithm of outpatient spending , but then retransforms the logarithm to raw dollars using a non - parametric method ( the smearing estimate ) described in the articles cited . armed with our estimated equations ( and estimated retransformation factor ) , we then compute the amount of explained variation as follows : we first predict the total outpatient expenditure of each person using the two - equation model . we then calculate a measure of r due to efron ( 1978 ) who uses the following formula : y - hati is the predicted yi using the two - equation model with alternative sets of explanatory variables , and y - bar is the sample mean of y. thus , the numerator of the expression in parentheses is the unexplained sum of squares , and the denominator is the total sum of squares . note that this measure of r can be negative when predicting from a non - linear model such as ours , but in this application it never was . we have used the two - part model and efron 's r rather than the more ordinary analysis of covariance because the two - part model has less tendency to overt it , and yields estimates with significantly lower mean square forecast error ( duan et al . , 1983 ) . thus , use of analysis of covariance , which is common in the literature , overstates how well one can do . the maximum r for children is 37 percent for untrimmed data and 35 percent for trimmed data ( table 2 ) . because the results are not sensitive to use of trimmed or untrimmed data , we present only the untrimmed results . age , gender , site , and afdc status explain only about 6 percent of the explainable variance in expected outpatient costs , i.e. , 6 percent of what one could hope to explain . measures of subjective health status do not much improve on the demographic variables of age , gender , site , and afdc status . only 14 percent of the explainable variance in expected costs is explained if those measures are also included . the variance in expected costs that is explained rises into the 25 to 30 percent range . results using the continuous specification of the health variable differ little from those using the dichotomous version . when paired with measures of health , this fraction rises into the two - thirds region . the year - to - year correlations decline as the time period extends ( table 3 ) , but the decline appears to bottom out rather than continue geometrically , especially for ambulatory expenses . if there were simple regression to the mean , the values of the average of diagonal column should fall geometrically when reading from bottom to top ( one is averaging increasingly longer intervals ) , but they clearly do not . the expected gain from including measures of prior use is substantial in reducing incentives to select favorable risks . as table 4 shows , for a child one standard deviation from the mean , the gain or loss is $ 672 per child if hmos know only the variance from prior use and our health measures , and only demographic variables are used to adjust premiums . this is about one - half the comparable figure for adults , but is enhanced by the usual practice of enrolling all children in a family because of the positive correlation among children . for a three - child family with an inter - child correlation of 0.25the correlation between expenditures of children in the same family in the health insurance experiment data the profit or loss would be 22 percent again as large as for a 3-child family with no correlation , or $ 1,426 at one standard deviation from the mean of 3-child families ( 1,426=6724.5 ) . simple demographic variables such as age , gender , site , and welfare status account for only about 6 percent of the explainable variance in the expected annual ambulatory care expenditure of children . indeed , they performed even less well than they did for adults , where they accounted for about 15 percent of the explainable variance of outpatient expenditure . it is perhaps not surprising that age would explain more variation in expenditure among those 14 - 65 years of age than among those 5 - 13 years of age , simply because of the greater variation in age among the adult group . despite their poor performance , this set of demographic variables is an administratively straightforward one to include in a payment formula . put another way , although there is little reason not to vary capitation rates based on these demographic characteristics , simply adjusting for them will not solve the problem of heterogeneity among enrollees . a frequently advocated step is to add measures of health status as adjusters ( howland et al . , 1987 ; mcclure , 1984 ; thomas and lichtenstein , 1986a , 1986b ; thomas , lichtenstein , and wyszewianski , 1983 ) . those measures of health status that we included did not much help . in the case of physiologic measures , this could be because our list of variables was quite short , limited to six types of medical problems . nonetheless , most remaining chronic diseases affecting children are not very prevalent . because the percentage of variance explained is proportional to the percentage of children with the problem furthermore , a much longer list of physiologic variables used for adults did not perform markedly better . although few children in a general population are physically limited or have serious mental illness , the general health index has been shown to be a reliable and valid measure of child health , and its distribution is not as non - normal as the other measures ( eisen et al . , 1980 ) . it is striking that even a quite limited set of six physiologic conditions measured at exit can explain notably more variance in outpatient expenditure than the three subjective measures taken at enrollment . this finding also held for adults ; subjective measures performed less well than physiologic measures . from a practical point of view , it may not be so serious that subjective measures of health status are of little help . implementing them as part of a payment formula would pose possibly insuperable problems because of the possibility of fraud . for that reason , and because we believe the scope for improving our physiologic measures is considerably greater than the scope for improving our subjective measures of health , we suggest that in the case of children the preponderance of any further effort spent to develop health - status adjusters be devoted to physiologic measures . although none of the measures of health status performed very well , measures of prior use did . more than one - half the variation in expected ambulatory care costs ( i.e. , the stable variance ) could be explained by our four measures of prior use . indeed , prior use did even better for children than it did for adults , where the comparable figure was somewhat less than one - half . incorporating measures of prior use into the payment formula poses both a mundane and a philosophical issue . many capitated organizations do not have readily available data on outpatient use ; thus , there would be a serious implementation problem . the philosophical issue relates to the appropriateness of paying on the basis of prior use . one commonly heard argument for capitation is that the ffs fee structure is distorted ( i.e. , it is profitable for physicians to treat more intensively , and they can act on these incentives because of consumer ignorance ) and that these distortions can be internalized to the organization by means of capitation . although the existing fee structure may well be distorted ( i.e. , many fees depart markedly from marginal cost ) , capitation means that the marginal revenue of an additional service is zero . necessary services that were contracted for , unless these services attract or retain healthy ( low cost ) patients who pay an average rate or unless they reduce future expenditure . ( preventive care is an example of a service that may both attract healthy patients and reduce future expenditure . ) against the point - in - time incentive to underserve are the possible consequences of current enrollees ' withdrawing and potential damage to the organization 's reputation , reducing the number of new enrollees . these threats , however , rely upon information , and information may not be very good . if the consumer is not knowledgeable enough to detect overservice from an ffs physician , will he or she be knowledgeable enough to detect underservice ? indeed , the potential of capitated organizations to underserve has been widely noted ( pauly , 1980 ) . the second problem with the philosophical argument is that it simply ignores the mismatch between an enrollee 's cost to the hmo and the revenue received for that enrollee , if the revenue is not tailored to the individual 's characteristics . thus , it does not address the incentive of the capitated organization to seek good risks and the resulting market failure for the poor risk ( table 4 ) . including prior use in the payment formula mitigates both problems the incentive to underserve an enrollee , and the incentive to select good risks . it mitigates the problem of incentives at the time of use by paying some positive amount , provided the child remains an enrollee in the next year . it also addresses the mismatch between marginal revenue and cost at the individual level , as the results in table 2 demonstrate ; i.e. , prior use picks up unmeasured variation across individuals in the amount of current use . thus , its inclusion reduces the incentives to select patients whose expected costs are less than average . for the same reason , it does not equally pay hmos whose mixes of health risks differ , whether through deliberate action on the hmo 's part or simply through random events . including prior use and our measures of health status raises the proportion of explained variance to the 65 to 70 percent range , but , as table 4 shows , there are nonetheless substantial profits to be made from selective enrollment and disenrollment . if one wants to do better , one will have to include a measure of current use in the payment formula ( newhouse , 1986 ) . for example , payment could be a weighted average of an adjusted capitation rate and a current use payment . basing the formula on current use rather than prior use also means the hmo does not lose revenue if a high - spending child disenrolls . the objections many have to including any measure of use in the payment formula are twofold . this is true , but points up that we are dealing with a second - best solution . second , many feel ffs is inherently a flawed system that provides incentives for overservice . as pauly ( 1980 ) pointed out , the incentive is not inherent . ignoring the moral hazard on the patient 's side , a fee at the marginal cost of delivering the service would provide the physician ( or capitated organization ) no incentive for overservice . paying a partial capitation and a partial fee reduces the likelihood that the fee will induce overservice . the moral hazard can be addressed through the incentives of capitation to the provider . in short , systems that vary payment with the amount of use although one can conceptualize fees at marginal cost , in practice a regulator will not know the true marginal cost . thus , one can not claim a priori that a mixed payment system , part ffs and part capitation , will definitely improve on either pure system , but our results suggest it might . we conclude that some experimentation with a mixed payment system is indicated ( selden , 1990 ; newhouse , 1991 ) .

few capitation arrangements vary premiums by a child 's health characteristics , yielding an incentive to discriminate against children with predictably high expenditures from chronic diseases . in this article , we explore risk adjusters for the 35 percent of the variance in annual outpatient expenditure we find to be potentially predictable . demographic factors such as age and gender only explain 5 percent of such variance ; health status measures explain 25 percent , prior use and health status measures together explain 65 to 70 percent . the profit from risk selection falls less than proportionately with improved ability to adjust for risk . partial capitation rates may be necessary to mitigate skimming and dumping .

Introduction Maximum Explainable Variance Data Dependent Variables Potential Adjusters Estimation Methods Results Discussion

enrollment of individuals in organizations such as health maintenance organizations ( hmos ) that supply medical care for a fixed periodic premium or a capitated rate continues to grow ; by 1991 enrollment in such organizations was about 13 percent of the population ( national center for health statistics , 1992 ) . however , 44 percent of the recipients in the other major public program , medicaid , were less than 18 years of age in 1989 , a figure that is likely to grow in light of the planned eligibility expansion for poor children ( reilly , clauser , and baugh , 1990 ) . if each capitated group enrolled a representative mix of health risks among the medicaid population , the rate could simply be a fraction , or perhaps as much as 100 percent , of per capita ffs costs . on the one hand , chronically ill patients who are under the care of an ffs physician will have incentives to continue with their physicians rather than to join an hmo ; on the other hand , hmos have incentives to avoid costly patients if they only receive payment that is based on the cost of the average patients . , to adjust the average rate . it is estimated that they may account for only 5 to 10 percent of the variation in expected cost and much less of actual cost across individuals ( lubitz , beebe , and riley , 1985 ; newhouse , 1986 ; newhouse et al . in our 1989 article , we examined how much additional adjusters , specifically measures of health and of use in the prior year , would improve the performance of an aapcc - type formula for adults under 65 years of age . using similar methods , we present herein results for children , although our results are limited to outpatient expenditures for children 5 - 13 years of age because our data set has few children that were hospitalized . we begin by estimating how much of the actual variation in expenditure one could potentially explain , and how much instead is because of random or unforeseeable events . because adjusters can not predict variation due to future random events , we wish to ignore the influence of such events as we assess the performance of adjusters , and want only to explain variation in expected , not actual , expenditure . as the explanatory power of a set of adjusters approaches the maximum explainable variance , the incentives for risk selection fall to negligible levels . we begin with the analog for children of the demographic types of variables currently used in the aapcc formula . we then estimate the gain from also using several measures of health status and prior use to adjust the capitation rate . one might argue that because families typically enroll as a group , we should have analyzed family behavior . however , because family - member expenditures are not independent , any incentive to skim or dump will be increased if families enroll as a unit . to judge against this criterion , therefore , we need to know the variance in expected expenditure , which will be less than the actual or observed variance by the variance in unpredictable expenditure . if children 's medical expenditures correspond to the following simple model , it would be straightforward to determine the variance in expected expenditure , which is the amount of variance one could possibly explain in a regression of annual expenditure , using cross - sectional data : xit is a vector of risk adjusters , is a vector of weights , i indexes the child , t indexes year , i is an unobserved child - specific , time - invariant ( stable ) component of variance , and it is a child - specific , time varying component of variance . that is , there may be some time - varying variables , omitted from the x vector , that explain a non - trivial amount of variance . an example of a variable usually contained in it rather than in xit is an illness that has a partially predictable time pattern , such as leukemia . nonetheless , it seems plausible to assume that most of the variation in it is random . we have thus estimated the maximum explainable variance by estimating the proportion of variance in the i term of the right - hand side of equation 1 , assuming no adjusters ( i.e. to estimate the between - person variance , we subtracted an estimate of within - person variance from total variance , correcting for the bias from estimating within - person variance from a finite time series ( searle , 1971 ) . for adults , however , we found that the estimated maximum r by the alternative method was less than the estimate using the method described in the previous paragraph ( newhouse et al . to blunt this incentive requires a premium that matches the expected cost of the hmo for each patient or family that it enrolls . that is , the payment formula may not fit well in some regions of the response surface , the functional form of the x vector may be specified incorrectly . our aim is not to develop a specific formula , but only to compare in a gross way the performance of demographic , health status , and prior use variables . in equation 2 , the potential explainable variance is that explained by the adjusters plus that explained by the first term on the right - hand side of the equation ( because when one is predicting year t 's expenditures , one has an estimate of it1 ) . thus , in this model the maximum r is approximately the proportion of variance explained by the adjusters plus approximately [ 1/(1 )]var ( u ) . the consistency of equations 1 and 2 with the data can be tested straightforwardly by examining the pattern of correlation of the residuals over time . because our principal interest was to ascertain how useful various adjusters would be in further developing capitation rates rather than a particular payment formula , we have not performed a variety of specification tests for the accuracy of the functional form for the x vector . using our results on explainable variance we consider a child whom the hmo predicts to be one standard deviation below or above the mean for expenditure based on the information available to it . we show how the profit or loss to the hmo diminishes as the payment formula changes to incorporate information from additional risk adjusters . in the limit , the additional risk adjusters would encompass all the information available to the hmo , and the hmo would not gain from selection . , we have removed the between - plan variance . in effect , we ask how well various explanatory variables or adjusters account for within - plan variance . thus , our results apply to an insured group with no variation in cost sharing , which is a good approximation to groups covered by capitation arrangements . an aspect of the experiment that approximates capitation less well is that the experimental plans employed no utilization management techniques , such as pre - admission certification . unless utilization management techniques differentially increase the predictable portions of expenditure , the conclusions of this article , with respect to how well one can predict , are unaffected . even if they were to increase the proportion of predictable variation , they would have to increase it differentially by type of covariate for our conclusions with respect to specific covariates to be changed . ( the number of children 5 - 13 years of age enrolled in the hmo portion of the experiment , a little more than 200 , were too few to use in this analysis . ) independent verification of physician office claims indicates that the families filed claims with the experiment for more than 90 percent of their utilization ; thus , we have a nearly complete record of utilization for the period of participation ( rogers and newhouse , 1985 ) . the families invited to participate in the experiment were randomly selected , subject to some qualifications that are not important for this article . additionally , in five of the six sites ( all but seattle ) , low - income individuals were over - sampled to a limited extent . as a result , the utilization of the 1,844 observations are not all independent because the amount of utilization by children in the same family is positively correlated . our calculations of the explained proportion of variance do not account for intrafamily correlation , but that should have little effect on our estimates of the proportion of variance that various types of individual characteristics can explain . indeed , the essence of the risk - adjustment problem is to account for the dependence in the residuals over time . the sample used for the regression equation included only those participants who completed the study and the physical examination at exit . although 93 percent of those children who began the study completed it , our analysis sample is considerably smaller . this excluded another 10 percent of the children who began the study . in the regression analysis , we did not use children in their first year of participation because we did not have comparable prior - use data for them . only 84 of the person - years ( 3.8 percent ) had inpatient use . because inpatient use was so rare , we chose not to include it . for trimmed data , if an observation was in the upper 2 percent of the relevant distribution , it was set equal to the mean of the upper 2 percent of the observations . because we wished to ignore within - plan variation , we began by regressing expenditures on plan . plan was defined as the log of the nominal coinsurance rate plus a dummy variable for one particular plan ( a plan with outpatient - only cost sharing ) . by design , we then calculated : a indexes the specification with only the plan variables included , and b indexes any of the more complete specifications . in fact , the plan variables explained only 1 percent of the total variance , so this correction is in practice unimportant . first , we included the demographic kinds of variables used by medicare : age ( entered linearly ) ; gender ; aid to families with dependent children ( afdc ) status ( supplemental security income recipients are not in the sample population ) ; and site . a set of dummy variables that indicate the presence or absence of the physiologic conditions shown in table 1 . for example , a dummy variable for anemia assumes the value one if a child 5 - 8 years of age has a hemoglobin below 11.0 g / ml . these physiological measures are derived from data collected at exit from the study rather than at entrance because only a random 60 percent of the children were given an exam at entrance . a set of variables that indicate the presence or absence of the physiologic conditions shown in table 1 , and for some conditions , a measure of the severity . for example , we treated the effect of being anemic and having asthma as additive . any effort to create an actual payment formula using these variables would need to consider more complicated functional forms , though any such effort should employ a larger data set than the one used in this study . at this point the observed value of the physiologic health variables was used ; thus , an individual who had a hemoglobin value of 12.0 g/100 ml achieved through medications was not distinguished from one who had a natural hemoglobin value of 12.0 g/100 ml and who was not under treatment . in effect , our specification implies that the expected treatment cost of a child will increase with less healthy values , but that will not always be true . ideally one would measure what the value of the physiologic health measure ( e.g. this may be one reason that the measures of prior use described later achieve considerably more explanatory power than the measures of health status . a set of measures of functional status or physical health , general health perceptions , and mental health as rated by a parent , usually the mother . although the use of such variables as adjusters in a payment formula seems problematic because of the possibilities for fraud , we wished to ascertain the possible gains from using them in our data where there were no incentives for fraud . the same difficulty just described for the physiologic variables is present in these variables as well because medical care for a chronic problem can affect these measures , and medical care may be greater the more severe the problem . to determine the gain from using various adjusters , we use a variant of a two - equation model we have used in other work ( manning et al . armed with our estimated equations ( and estimated retransformation factor ) , we then compute the amount of explained variation as follows : we first predict the total outpatient expenditure of each person using the two - equation model . note that this measure of r can be negative when predicting from a non - linear model such as ours , but in this application it never was . for purposes of calculating the maximum r , we examined expenditure in both raw and trimmed form . for trimmed data , if an observation was in the upper 2 percent of the relevant distribution , it was set equal to the mean of the upper 2 percent of the observations . because we wished to ignore within - plan variation , we began by regressing expenditures on plan . by design , we then calculated : a indexes the specification with only the plan variables included , and b indexes any of the more complete specifications . in fact , the plan variables explained only 1 percent of the total variance , so this correction is in practice unimportant . first , we included the demographic kinds of variables used by medicare : age ( entered linearly ) ; gender ; aid to families with dependent children ( afdc ) status ( supplemental security income recipients are not in the sample population ) ; and site . a set of dummy variables that indicate the presence or absence of the physiologic conditions shown in table 1 . for example , a dummy variable for anemia assumes the value one if a child 5 - 8 years of age has a hemoglobin below 11.0 g / ml . these physiological measures are derived from data collected at exit from the study rather than at entrance because only a random 60 percent of the children were given an exam at entrance . we felt we would obtain more accurate estimates by using data measured at exit for the entire sample than the data measured at enrollment for a partial sample despite the possibility that use would affect the observed value . in principle , the coefficient of the dummy variable measures the fixed costs of treating the condition , and the coefficient of the continuous variable measures the variable cost of increased severity . for example , we treated the effect of being anemic and having asthma as additive . any effort to create an actual payment formula using these variables would need to consider more complicated functional forms , though any such effort should employ a larger data set than the one used in this study . in effect , our specification implies that the expected treatment cost of a child will increase with less healthy values , but that will not always be true . although a physiologic condition that we measured was in fact responsible for treatment costs of one of the persons ( i.e. ideally one would measure what the value of the physiologic health measure ( e.g. this may be one reason that the measures of prior use described later achieve considerably more explanatory power than the measures of health status . although the use of such variables as adjusters in a payment formula seems problematic because of the possibilities for fraud , we wished to ascertain the possible gains from using them in our data where there were no incentives for fraud . the same difficulty just described for the physiologic variables is present in these variables as well because medical care for a chronic problem can affect these measures , and medical care may be greater the more severe the problem . to determine the gain from using various adjusters , we use a variant of a two - equation model we have used in other work ( manning et al . armed with our estimated equations ( and estimated retransformation factor ) , we then compute the amount of explained variation as follows : we first predict the total outpatient expenditure of each person using the two - equation model . we then calculate a measure of r due to efron ( 1978 ) who uses the following formula : y - hati is the predicted yi using the two - equation model with alternative sets of explanatory variables , and y - bar is the sample mean of y. thus , the numerator of the expression in parentheses is the unexplained sum of squares , and the denominator is the total sum of squares . note that this measure of r can be negative when predicting from a non - linear model such as ours , but in this application it never was . the maximum r for children is 37 percent for untrimmed data and 35 percent for trimmed data ( table 2 ) . because the results are not sensitive to use of trimmed or untrimmed data , we present only the untrimmed results . age , gender , site , and afdc status explain only about 6 percent of the explainable variance in expected outpatient costs , i.e. , 6 percent of what one could hope to explain . only 14 percent of the explainable variance in expected costs is explained if those measures are also included . the variance in expected costs that is explained rises into the 25 to 30 percent range . the expected gain from including measures of prior use is substantial in reducing incentives to select favorable risks . as table 4 shows , for a child one standard deviation from the mean , the gain or loss is $ 672 per child if hmos know only the variance from prior use and our health measures , and only demographic variables are used to adjust premiums . this is about one - half the comparable figure for adults , but is enhanced by the usual practice of enrolling all children in a family because of the positive correlation among children . simple demographic variables such as age , gender , site , and welfare status account for only about 6 percent of the explainable variance in the expected annual ambulatory care expenditure of children . indeed , they performed even less well than they did for adults , where they accounted for about 15 percent of the explainable variance of outpatient expenditure . it is perhaps not surprising that age would explain more variation in expenditure among those 14 - 65 years of age than among those 5 - 13 years of age , simply because of the greater variation in age among the adult group . put another way , although there is little reason not to vary capitation rates based on these demographic characteristics , simply adjusting for them will not solve the problem of heterogeneity among enrollees . a frequently advocated step is to add measures of health status as adjusters ( howland et al . those measures of health status that we included did not much help . because the percentage of variance explained is proportional to the percentage of children with the problem furthermore , a much longer list of physiologic variables used for adults did not perform markedly better . it is striking that even a quite limited set of six physiologic conditions measured at exit can explain notably more variance in outpatient expenditure than the three subjective measures taken at enrollment . implementing them as part of a payment formula would pose possibly insuperable problems because of the possibility of fraud . for that reason , and because we believe the scope for improving our physiologic measures is considerably greater than the scope for improving our subjective measures of health , we suggest that in the case of children the preponderance of any further effort spent to develop health - status adjusters be devoted to physiologic measures . although none of the measures of health status performed very well , measures of prior use did . , the stable variance ) could be explained by our four measures of prior use . indeed , prior use did even better for children than it did for adults , where the comparable figure was somewhat less than one - half . the philosophical issue relates to the appropriateness of paying on the basis of prior use . against the point - in - time incentive to underserve are the possible consequences of current enrollees ' withdrawing and potential damage to the organization 's reputation , reducing the number of new enrollees . thus , it does not address the incentive of the capitated organization to seek good risks and the resulting market failure for the poor risk ( table 4 ) . including prior use in the payment formula mitigates both problems the incentive to underserve an enrollee , and the incentive to select good risks . , prior use picks up unmeasured variation across individuals in the amount of current use . thus , its inclusion reduces the incentives to select patients whose expected costs are less than average . for the same reason , it does not equally pay hmos whose mixes of health risks differ , whether through deliberate action on the hmo 's part or simply through random events . including prior use and our measures of health status raises the proportion of explained variance to the 65 to 70 percent range , but , as table 4 shows , there are nonetheless substantial profits to be made from selective enrollment and disenrollment . basing the formula on current use rather than prior use also means the hmo does not lose revenue if a high - spending child disenrolls .

the cytoplasmic and nuclear compartments of eukaryotic cells are separated by the nuclear envelope ( ne ) . the double membrane of the ne is perforated by nuclear pore complexes ( npcs ) , which are large multiprotein complexes that support passive diffusion of small molecules , and facilitate receptor - mediated translocation of proteins and ribonucleoprotein complexes . overall , the vertebrate npc is a 120 mda protein complex made of 30 different proteins called nucleoporins ( or nups ) that are repetitively arranged as distinct subcomplexes to form the npc ( cronshaw et al . 2002 ; lim and fahrenkrog 2006 ; rout et al . 2000 ; schwartz 2005 ; tran and wente 2006 ) . in the plane of the ne , the eightfold symmetric central framework of the npc , also known as the spoke complex , encloses a central pore that is 50 nm long and is narrowest ( 40 nm ) at the ne midplane ( beck et al . 2004 , 2007 ; stoffler et al . 2003 ) . attached to the central framework are cytoplasmic filaments and a nuclear basket ( figs . 1 , 2 ) . fig . a cytoplasmic face of negatively stained and b a stretch along a nuclear envelope of embedded and thin - sectioned nuclei from triturus alpestrus . c cytoplasmic face of negatively stained and d a stretch along a nuclear envelope of embedded and thin - sectioned nuclei from xenopus laevis . view of the nuclear basket of isolated nuclei from xenopus oocytes prepared by e critical point drying and field emission scanning electron microscopy , f quick - freeze / freeze - drying / rotary metal shadowing and g thin - sectioning and transmission electron microscopy . a reproduced with permission from gall ( 1967 ) . three - dimensional reconstruction of the central framework of negatively stained npcs after detergent treatment a from xenopus oocytes , and b its adaptation for a consensus model of the npc architecture and c from yeast nuclei and d its adaptation into a model . e consensus model of the npc based on a reconstruction of native npcs embedded in thick amorphous ice . f reconstruction of npcs from intact nuclei of dictyostelium discoideum examined by cryo - electron tomography . ( 2007 ) electron micrographs of npcs over time . a cytoplasmic face of negatively stained and b a stretch along a nuclear envelope of embedded and thin - sectioned nuclei from triturus alpestrus . c cytoplasmic face of negatively stained and d a stretch along a nuclear envelope of embedded and thin - sectioned nuclei from xenopus laevis . view of the nuclear basket of isolated nuclei from xenopus oocytes prepared by e critical point drying and field emission scanning electron microscopy , f quick - freeze / freeze - drying / rotary metal shadowing and g thin - sectioning and transmission electron microscopy . three - dimensional reconstruction of the central framework of negatively stained npcs after detergent treatment a from xenopus oocytes , and b its adaptation for a consensus model of the npc architecture and c from yeast nuclei and d its adaptation into a model . e consensus model of the npc based on a reconstruction of native npcs embedded in thick amorphous ice . f reconstruction of npcs from intact nuclei of dictyostelium discoideum examined by cryo - electron tomography . a reprinted with permission from hinshaw et al . ( 2007 ) we begin , here , with a retrospective that summarizes the analysis and elucidation of npc architecture by electron microscopy ( em ) techniques . we will discuss how the dissection of npc structure by electron tomography and x - ray crystallography has in a methodical and progressive manner reached the ultrastructural , molecular and atomic scale . in parallel , we will overview the progress that has contributed to the contemporary understanding of how the npc functions as a selective gate - barrier . setting our sights on the future , we will close by highlighting the importance of reconciling both npc structure and function , as we strive towards converging on a single conceptual , mechanistic understanding of the npc . npc structure was initially dissected using transmission em ( tem ) , but later expanded to scanning transmission as well as to scanning em and , most recently , to cryo - electron tomography ( cet ) . the first em study on the ne in 1950 revealed that it is perforated by pores ( callan and tomlin 1950 ) . gall ( 1967 ) showed for the first time that npcs exhibit an octagonal structure ( fig . this was later confirmed by a number of studies in the following years ( aaronson and blobel 1974 ; franke and scheer 1970 ; maul 1971 ) , whereby its overall architecture , particularly its eightfold symmetry , appeared to be evolutionarily conserved ( franke and scheer 1970 ) . although its functional significance was ambiguous at that time , it was already evident from these early studies that the npc is composed of a membranous structure , specifically with a part residing within the ne , and nonmembranous , filamentous structures being attached to the cytoplasmic and the nuclear face of the npc ( fig . 1b ) ( franke and scheer 1970 ; kessel 1969 ) . according to today s consensus , the npc consists of an approximately cylindrical central framework , eight cytoplasmic filaments and a nuclear basket ( fig . 1c g ) that is composed of eight filaments that join into a distal ring ( fig . refinement in em techniques using negatively stained and frozen - hydrated npcs from xenopus laevis oocyte nes ( akey and radermacher 1993 ; hinshaw et al . 2c ) ( yang et al . 1998 ) provided early 3d reconstructions of the central framework . the central framework of the npc ( also known as the spoke complex ) resides within the ne , and is anchored to the region where the inner and outer nuclear membranes fuse . early structural studies showed that the cytoplasmic and nuclear ring moieties are integral to the central framework . 2003 ) and intact , transport - competent nuclei isolated from dictyostelium discoideum ( fig . 2004 , 2007 ) have improved the resolution of the central framework to 6 nm and revealed the first reconstructions of peripheral , flexible components of the npc , i.e. the cytoplasmic filaments and the nuclear basket . in dictyostelium , the cytoplasmic filaments are about 35 nm in length and the nuclear basket is about 60 nm long . together with the 50 nm central framework , the npc has an overall length of about 150 nm and an outer diameter of 125 nm ( beck et al . although the overall linear dimensions of the npc vary between species , the overall 3d architecture appears to be evolutionarily conserved ( fahrenkrog et al . 1998 ; kiseleva et al . 2004 ; yang et al . 1998 ) . enclosed by the central framework is the hourglass - shaped central pore of the npc , which has a diameter of 6070 nm at its cytoplasmic and nuclear periphery and is 45 nm in the midplane of the npc / ne ( beck et al . 2004 , 2007 ; pante and kann 2002 ; stoffler et al . 2003 ) . this central pore mediates the traffic between the cytoplasm and the nucleus , i.e. diffusion of small molecules and ions , as well as the selective transport of signal - carrying macromolecular cargo with diameters up to 39 nm ( pante and kann 2002 ) ( see later section ) . the peripheral channels of the npc have a diameter of about 8 nm and have been implicated in the diffusion of small molecules and ions ( feldherr and akin 1997 ; hinshaw et al . 1992 ) and/or in trafficking of integral membrane proteins to the inner nuclear membrane ( soullam and worman 1995 ) . structurally , the cytoplasmic and the nuclear openings of the peripheral channels are not topologically continuous ( stoffler et al . however , since biochemical studies suggest that passive and facilitated transport across the npc proceed via routes that are sterically nonoverlapping ( naim et al . 2007 ) , it remains debated if ( 1 ) there are two routes existing in the central pore [ i.e. , facilitated transport along the walls of the central pore and passive diffusion through a narrow diffusion tube located at the pore center ( peters 2005 ) ] , or ( 2 ) whether passive diffusion might somehow also utilize the peripheral channels ( akey and radermacher 1993 ; beck et al . other potential roles for the peripheral channels have been proposed , such as in maintaining the ne electrical conductance ( danker et al . 1999 ; enss et al . 2003 ; mazzanti et al . 2001 ; shahin et al . 2001 ) or as mechanical buffer zones that accommodate deformations of the central framework upon translocation of large cargoes ( stoffler et al . yeast cells have 200 npcs / nucleus , proliferating human cells have 1020 npcs/m ( i.e. 2,0005,000 npcs / nucleus ) and a mature xenopus oocyte has about 60 npcs/m yielding 5 10 npcs / nucleus ( gerace and burke 1988 ; gorlich and kutay 1999 ) . a comprehensive ultrastructural study using freeze - fracture em of yeast cells in combination with 3d reconstruction has shown that the distribution of yeast npcs in the ne is not equidistant , but rather cluster into regions of higher density ( winey et al . this observation is not limited to yeast and is present in other cell types as well ( franke 1974 ) . in yeast , the number of npcs was found to increase steadily , beginning in the g1-phase and peaking in the s - phase of the cell cycle , suggesting that npc assembly occurs continuously throughout the cell cycle ( winey et al . similarly , the density of npcs increases throughout the cell cycle in hela s3 cells ( maeshima et al . 2006 ) . the molecular building blocks of the npc comprise 30 different proteins known as nucleoporins or nups , which are present in at least eight copies per npc ( cronshaw et al . the transmembrane group , which contains transmembrane -helices and a cadherin fold , comprises the outermost features of the npc central framework and is thought to assist in anchoring the npc to the ne . the second group of nucleoporins contain -propeller and -solenoid folds , which localizes towards the inside of the npc , whereas the third class harbors the conserved sequence motif of phenylalanine glycine ( fg)-repeats in combination with a coiled - coil fold and likely contributes to the formation of the npc s inner central framework and the peripheral structures ( devos et al . 2006 ; schwartz 2005 ; tran and wente 2006 ) . other less frequent structural motifs found in nucleoporins are zinc - finger domains as in nup153 and ranbp2/nup358 ( higa et al . 2007 ) or rna - recognition motifs as in nup35 ( handa et al . -propellers are predicted in the third class of the nucleoporins , and in fact seven - bladed -propellers have been resolved from the n - terminal domains ( ntd ) of the human nucleoporins nup133 and nup214 as well as from the ntd of nup159p in yeast by x - ray crystallography ( berke et al . proteins with -propeller folds participate in diverse cellular functions and serve as platforms for multiple dynamic protein protein interactions . along this line , yeast nup133p and nup159p both play roles in mrna export from the nucleus , given that deletion or mutations in their ntds impair their functions in mrna export , probably by preventing the association of multiple mrna export factors with the npc ( berke et al . the ntd of human nup133 furthermore contains an amphipathic -helical motif capable of sensing membrane curvature ( drin et al . this motif corresponds to an exposed loop , which connects two blades of the -propeller and folds to an -helix upon interacting with small liposomes . whether the -helical motif in nup133 serves to recognize the curved topology of the nuclear pore membrane to anchor the npc during interphase or to recognize small vesicles containing ne fragments critical for ne reassembly after mitosis , or both , remains to be clarified ( drin et al . the ntd of human nup214 , in comparison to its yeast homolog nup159p , consists of two distinct structural elements : the -propeller and a 30-residue c - terminal extended peptide segment ( napetschnig et al . this extension binds to the bottom of the -propeller with low affinity and has been suggested to play an auto - inhibitory role in npc assembly . the first crystal structure obtained for a nucleoporin was the npc targeting domain of human nup98 ( hodel et al . this domain , similar to the nuclear pore - targeting domain of its yeast homolog nup116p , consists of a six - stranded -sheet sandwiched against a two - stranded -sheet and is flanked by two -helical regions ( hodel et al . this domain exhibits multiple conformations and is stabilized only when bound to a ligand , i.e. nup96 and nup145p - c in the case of nup98 and nup116p , respectively ( robinson et al . 2005 ) . conformational diversity may allow nup98 and nup116p to bind to multiple targets within the npc or to associate and dissociate quickly from the npc to increase the mobility of the nucleoporins , as described for nup98 , which shuttles in a transcription - dependent manner ( griffis et al . an attempt to crystallize the first subcomplex of the npc , the nup62 complex , yielded the structure of the -helical coiled - coil domain of rat nup58/45 ( melcak et al . the intradimer interface is hydrophobic , whereas dimer - dimer interactions occur through large hydrophilic residues . the tetramer can adopt various conformations leading to a lateral displacement between tetramers suggesting an intermolecular sliding mechanism ( melcak et al . the nup62 complex has recently been mapped to the cytoplasmic periphery of the npc s central pore ( schwarz - herion et al . 2007 ) , so that sliding of nup58/45 , and possibly of nup62 and nup54 as well , could contribute in modulating the diameter of the central pore in response to transport activity ( melcak et al . fg - repeat domains ( also known as fg - domains ) are found in about one - third of the nucleoporins and mediate the interaction between soluble transport receptors loaded with signal - bearing cargo and the npc . these fg - domains constitute the key components of the selective gate - barrier that limits the diffusion of cargoes through the npc ( see following section ) . atomic structures of short fg - repeat peptides in complex with , for example , the import receptor importin ( bayliss et al . 2002a ) or the putative mrna export factor tap / nxf1 ( grant et al . 2002 , 2003 ) , have consistently shown that the interaction between fg - repeats and the different transport receptors involves primarily the phenylalanine ring of the fg - repeat core and hydrophobic residues on the surface of the receptor ( isgro and schulten 2005 , 2007a , b ) . hydrophilic linker regions between individual fg - motifs , which constitute the majority of amino acid mass in the overall fg - domain , appear to influence the strength of the binding and allow simultaneous binding of several fg - cores to the receptor ( liu and stewart 2005 ) . based on biophysical measurements , the fg - domains of yeast nucleoporins have been found to be natively unfolded ( denning et al . similarly , fg - domains of human , fly , worm and other yeast species are most likely disordered based on their amino acid composition ( denning and rexach 2007 ) . this notion is further supported by immuno - em studies of vertebrate fg - repeat nucleoporins , which suggest that the fg - domains are flexible and mobile within the npc ( fahrenkrog et al . single molecule studies using atomic force microscopy ( afm ) on the recombinantly expressed fg - domain of human nup153 further revealed that this 700 residue domain is in fact an 180 nm long unfolded molecule when adsorbed on the surface of mica ( lim et al . , it is now estimated that each npc is populated by at least 128 fg - domains together displaying 3,500 fg - repeats ( strawn et al . 2004 ) . nup153 and nup214 are both known to play roles in distinct nucleocytoplasmic transport ( nct ) pathways and interact with a number of nuclear transport receptors via their fg - repeats ( ball and ullman 2005 ; bernad et al . 2006 ; hutten and kehlenbach 2006 ; sabri et al . 2007 ; van deursen et al . 1996 ) . the location of the fg - domains of nup153 and nup214 shifts in a transport - dependent manner in the npc , further supporting their role in nct ( paulillo et al . 2005 ) . systematic deletion of fg - repeat regions in yeast nucleoporins revealed , however , that yeast npcs suffer from only slight changes in distinct nuclear transport pathways , but otherwise remain functional despite having removed up to 50% of their fg - repeats ( strawn et al . this suggests that the fg - repeats exhibit a functional redundancy in bulk nct , except for specific nuclear transport pathways , which may require individual fg - nucleoporins , and/or that other interaction sites for transport receptors exist within the npc ( terry and wente 2007 ) . besides playing important roles in nct , the crystal structure of the rrm domain of mouse nup35 revealed that all three fg - sequences of this nucleoporin are in ordered secondary structure elements and that these fg - sequences do not interact with transport receptors , such as importin , but rather with , for example , the integral membrane protein ndc1 . thus , the fg - sequences of nup35 may contribute to the formation of the npc s central framework ( handa et al . npcs are porous to small molecules ( e.g. water and ions ) , which freely diffuse through the npc , while more massive cargoes ( i.e. > 40 kda in size ) [ a recent study has showed that this limit may extend to 100 kda ( wang and brattain 2007 ) ] require the assistance of soluble transport receptor proteins to be effectively chaperoned through the npc . a large number of these receptor proteins , known collectively as karyopherins ( kaps ) or more specifically as importins ( imp ) and exportins ( exp ) , were discovered around the 1990s ( rexach and blobel 1995 ; wozniak et al . today , the biochemical role of the receptors in unlocking the npc barrier as part of the nuclear trafficking machinery is relatively well understood . as illustrated in fig . 3 , appropriate cargoes are identified through a short sequence of residues known as nuclear localization / export signals ( i.e. nls / nes ) for import and export , respectively , which exhibit binding interactions with the karyopherins [ sometimes using an adaptor such as , for example , importin- ( gorlich et al . 1994 ) ] . otherwise , passage through the npc is obstructed for large , non - nls / nes harboring molecules that do not bind to the karyopherins ( i.e. passive ) ( paine et al . the directionality of nct is driven by an asymmetric distribution of the two nucleotide states of the small gtpase ran ( melchior et al . being predominant in the nucleus , rangtp functions to release the cargo from its import receptor by binding to the receptor itself ( gorlich et al . exportins bind to their cargo in the presence of rangtp , which ferries the complex back into the cytoplasm . once in the cytoplasm , gtp hydrolysis causes the disassembly of the export complex , thereby recycling the export receptor and fueling the cytoplasmic rangdp pool . passage through the npc is restricted to transport receptors , which bind and chaperone nls - cargo through the npc , while access is prohibited for passive ( non - nls ) molecules of similar size . binding of rangtp to the transport receptor releases the nls - cargo into the nucleus . the gray shaded area emphasizes the location of the fg - domains and the question mark highlights the uncertainty with regard to the biophysical aspects of the selective gating mechanism within the npc , which operates to restrict or promote cargo translocation schematic representation identifying the main biochemical constituents of the nuclear import machinery . passage through the npc is restricted to transport receptors , which bind and chaperone nls - cargo through the npc , while access is prohibited for passive ( non - nls ) molecules of similar size . binding of rangtp to the transport receptor releases the nls - cargo into the nucleus . the gray shaded area emphasizes the location of the fg - domains and the question mark highlights the uncertainty with regard to the biophysical aspects of the selective gating mechanism within the npc , which operates to restrict or promote cargo translocation early models of the npc gating mechanism were derived from initial em studies , which linked the biophysical origin of the selective gate to the presence of a central plug or central transporter the central transporter was suggested to consist of an iris - like mechanism that is hinged to the central channel ( akey 1990 ) ( fig . although recent evidence indicates that this feature represents to a large extent cargo complexes arrested during translocation ( beck et al . 2004 , 2007 ; stoffler et al . 2003 ) , this model suggested a translocation mechanism that has largely defined the criteria for subsequent npc models in the field ( akey and goldfarb 1989 ) . these steps include a peripheral binding to the npc , followed by a docking step , and then a translocation step ( e.g. by a dilation of the transporter ) . fig . a the central transporter consisting of an iris - like mechanism that is hinged to the central channel . b being natively unfolded and located at the nuclear / cytoplasmic peripheries of the npc , the virtual gating model suggests that the entropic movements of the fg - domains can act as a barrier to inert cargo . c the affinity gradient model proposes that receptor - cargo complexes traverse a pathway that is lined by fg - domains of increasing affinity . d the oily - spaghetti model predicts that the fg - domains can only be pushed aside by receptor - cargo complexes . e the undersaturated and subsequently the saturated hydrogel model purport that the fg - domains crosslink via a dense number of inter - fg interactions to form a highly organized three - dimensional network within the npc . f the nanomechanical reversible collapse model asserts that the selective gating mechanism consists of a stochastic flux of collapsing and distending fg - domains that is regulated by binding and unbinding interactions between the fg - domain and the transport receptors . a the central transporter consisting of an iris - like mechanism that is hinged to the central channel . b being natively unfolded and located at the nuclear / cytoplasmic peripheries of the npc , the virtual gating model suggests that the entropic movements of the fg - domains can act as a barrier to inert cargo . c the affinity gradient model proposes that receptor - cargo complexes traverse a pathway that is lined by fg - domains of increasing affinity . d the oily - spaghetti model predicts that the fg - domains can only be pushed aside by receptor - cargo complexes . e the undersaturated and subsequently the saturated hydrogel model purport that the fg - domains crosslink via a dense number of inter - fg interactions to form a highly organized three - dimensional network within the npc . model asserts that the selective gating mechanism consists of a stochastic flux of collapsing and distending fg - domains that is regulated by binding and unbinding interactions between the fg - domain and the transport receptors . ( 1995b ) recognized that the fg - domains represent the key constituents of the npc selective gate by determining that the fg - repeats act as docking sites for kaps . by identifying and binding to the fg - domains , the kaps ( 2000 ) showed that each npc consists of up to 12 different fg - domains ( i.e. nucleoporins ) , which are located near the nuclear and cytoplasmic peripheries of the npc . to further emphasize the functional redundancy between the various fg - domains in the npc ( 1 ) the asymmetric fg - nups have been shown to be dispensable for nct ( zeitler and weis 2004 ) ; ( 2 ) the direction of transport through the npc can be inverted ( nachury and weis 1999 ) ; ( 3 ) active transport is able to proceed in npcs lacking cytoplasmic filaments ( i.e. fg - rich nup358 ) ( walther et al . 2002 ) ; ( 4 ) the selective gating mechanism has been found to remain functional even after 50% of the fg - repeats had been depleted ( strawn et al . , it is generally agreed that the fg - domains act as the physical constituents of the npc barrier ( ben - efraim and gerace 2001 ; macara 2001 ; ribbeck and gorlich 2002 ; rout et al . 2000 ) ( fig . 3 ) . however , the mechanistic manner in which the fg - domains contribute to the selective gating of the npc is widely speculated and has been the subject of several reviews ( fahrenkrog and aebi 2003 ; lim et al . 2006a ; stewart 2007 ; suntharalingam and wente 2003 ; weis 2003 ) . akin to the entropic fluctuations of unstructured microtubule - associated proteins ( maps ) ( mukhopadhyay and hoh 2001 ) and neurofilament sidearms ( brown and hoh 1997 ) , the brownian affinity gating model ( rout et al . 2000 ) , later called virtual gating ( rout et al . 2003 ) , proposes that the entropic behavior of peripheral fg - domains acts as a substantial barrier to inert cargo ( fig . translocation is anticipated for receptor - mediated cargoes due to interactions between the fg - repeats and the transport receptors , which increases the residence time and probability of entry into the npc . in a similar manner , the oily - spaghetti model ( macara 2001 ) postulates that noninteracting fg - domains are pushed aside by cargo complexes but otherwise obstruct the passage of passive cargo ( fig . , the dualistic ability of each npc to restrict or promote cargo translocation ( i.e. to simultaneously act as a barrier and a vectorial transport facilitator ) is only figuratively understood . cargo complex movement through the npc has been compared to stepping from one fg - repeat to the next ( rexach and blobel 1995 ) , or sliding over a surface comprised of fg - repeats ( peters 2005 ) . alternatively , the affinity gradient model ( ben - efraim and gerace 2001 ) suggests that transport complexes step through npcs lined with fg - nucleoporins exhibiting increasing binding affinities with the cargo complexes ( fig . finally , the selective phase model ( ribbeck and gorlich 2002 ) predicts that fg - domains attract each other via hydrophobic inter - fg - repeat interactions to form a hydrophobic gel or meshwork ( fig . this interpretation is based on experiments , which show that the addition of hydrophobic solvents disrupts the meshwork and triggers a nonselective opening of the pore ( ribbeck and gorlich 2002 ; shulga and goldfarb 2003 ) . hence , it is predicted that passive , more hydrophilic material is obstructed while hydrophobic cargo complexes are able to dissolve through the sieve - like meshwork . frey et al . ( 2006 ) showed that the yeast fg - nup , nsp1 , can be cast in the form of a macroscopic hydrogel to lend support to the selective phase model ( ribbeck and gorlich 2002 ) . however , the same authors reported that the nsp1 hydrogel lacked any discriminatory effects between inert , non - kap binding proteins and kap - complexed proteins unless the fg concentration within the gel was raised ( 50 mm ) to the point where it formed a saturated hydrogel ( frey and gorlich 2007 ) . in this manner , they explained that an efficient permeability barrier was recovered due to the formation of a highly ordered meshwork consisting of cohesive inter - fg repeats ( fig . in addition to preventing the translocation of inert molecules , it was found that such a saturated hydrogel could reproduce the diffusion rates of receptor - driven transport in the npc ( kubitscheck et al . ( 2007 ) reported evidence that refutes the gel - forming quality of nsp1 . to investigate the cohesiveness of different fg - domains , patel et al . devised a low affinity assay , which could detect the binding of cfp - nups to gst - nups immobilized on sepharose beads . by avoiding the non - physiological conditions used to form the hydrogels ( frey et al . 2006 ; frey and gorlich 2007 ) , they found that only glfg - domains show weak cohesive interactions , whereas fxfg - domains ( such as nsp1 ) do not bind together . based on their findings , the authors suggested that the fxfg domains on both cytoplasmic and nuclear peripheries act as an entropic repulsive barrier , while the glfg - domains form a cohesive meshwork in the central npc channel ( patel et al . therefore , they proposed a two - gate model that combines elements of both brownian gating and the selective phase ( patel et al . 2007 ) . such a two - gate model , however , is likely to be more appropriate to yeast npcs , because vertebrate npcs are known to be composed almost entirely of fxfg - domains ( except for the glfg - domain of nup98 ) ( suntharalingam and wente 2003 ) . furthermore , by a systematic depletion of fg - domains in yeast , the authors showed that the npcs displayed similar qualitative leakiness in all the cases studied , which indicates that all the fg - domains exhibit a functional redundancy in that both peripherally and centrally anchored fg - nups play equal roles in maintaining the selective gating mechanism . ( 2006b , 2007a ) developed an experimental platform that allowed for the collective behavior of surface - tethered fg - domains to be probed at the nanoscopic length scale . by reproducing the physical dimensions of the npc , they found that the fxfg - domains of the vertebrate nup153 resembles a polymer brush ( halperin et al . this is caused by packing constraints between the entropy - dominated fg - domains , which force the surface - tethered fg - domains to extend in a net directionality away from the surface ( i.e. entropic barrier ) . this creates a significant repulsive barrier that can repel large macromolecules while allowing small molecules such as ions and water to diffuse more easily through . this may explain why a reduced number of fg - domains is enough to maintain an effective barrier in the npc . by showing that the extended brush - like fg - domains collapsed in hexanediol , they were further able to explain why the npcs could reversibly open and close when similar reagents were added / removed in nuclear transport assays ( patel et al . 2007 ; ribbeck and gorlich 2002 ; shulga and goldfarb 2003 ) . this was further substantiated with atomic force microscope ( afm ) single molecule force spectroscopy ( smfs ) analysis , which showed that individual nup153 fg - domain molecules ( 1 ) lack intra - fg interactions ; ( 2 ) are natively unfolded ; ( 3 ) can be reversibly stretched and relaxed without any change to its intrinsic entropic elasticity , i.e. resembling a worm - like chain ( wlc ) ( bustamante et al . smfs analysis revealed complex plateau - like ( un)binding topologies between kap1 ( importin ) and nup153 when kap1-modified afm tips were used ( lim et al . 2001 ; tompa 2002 ) in fg - receptor interactions and is in agreement with the fact that kap1 consists of five experimentally verified hydrophobic fg - binding sites ( bayliss et al . ; liu and stewart 2005 ) , with additional five binding sites predicted by molecular dynamics ( md ) simulations that can be simultaneously occupied ( isgro and schulten 2005 ) . to further correlate the governing biochemical interactions to the biophysical behavior of the fg - domains , lim et al . ( 2007a ) studied the nanomechanical response of the brush - like fg - domains under the influence of transport factors such as kap1 , rangtp and rangdp . consequently , they found that the entropic barrier collapsed in vitro due to kap1-fg binding interactions ( i.e. causing a reduction in conformational entropy of the fg - domains ) and was reversed by the sequestration of kap1 by rangtp ( but not rangdp ) . by validating similar effects in situ using an immunogold - em labeling assay in xenopus oocytes nuclei , they hypothesized that the selective gating mechanism consists of a rapid , stochastic flux of collapsing and distending fg - domains that regulates passage through the npc ( fig . such seamlessness may explain the reversibility of nct and apparent open communication between the cytoplasm and nucleus ( kopito and elbaum 2007 ) . besides resolving the overall structure of the npc , an accurate picture of how selective gating is achieved by the ( structureless ) fg - domains remains unclear due to a general lack of understanding with regard to their behavior and function within the npc . so far , only the reversible collapse of the fg - domains has been directly observed to occur in the npc ( lim et al . the source of this ambiguity stems in part from the difficulty in trying to visualize the fg - domains in vivo , which is evident given the lack of resolution even when using state - of - the - art structural techniques such as cryo - tomography ( cryo - et ) to detect the fg - domains ( beck et al . although immunogold - em techniques can provide positional information of the fg - domains within the npc , this is limited to static views of fg - domain behavior ( fahrenkrog et al . 2002 ; paulillo et al . 2005 , 2006 ; schwarz - herion et al . new microscopic techniques such as the afm is also usually limited in resolution and chemical sensitivity due to the complexity of the npc and its cellular environment ( jaggi et al . 2002 , 2003 ) , the fg - domains themselves have so far only been directly visualized as individual bioploymers by afm ( lim et al . in combination with biochemical efforts to identify whether different transport receptors use preferential fg - domains during nct ( terry and wente 2007 ) , it may be beneficial to use bottom - up strategies to investigate how the fg - domains behave on a biophysical level to give rise to the selective gating of the npc . this is because biochemical approaches can only provide a marginal mechanistic description of the selective gating process . moreover , because nct occurs over tens of nanometers in and around the npc , i.e. a near - field effect , this in fact necessitates a consideration of such physical details , including ( 1 ) the geometry and nanoscopic dimensions of the npc , ( 2 ) the fg - domain anchoring sites within the npc and ( 3 ) the limited number of fg - domains located within the npc . thus , it will be essential to establish fg - domain conformations at the npc - relevant length scale . to critically underscore this point , can macroscopic assays that address the characteristics of enormously large numbers of fg - domains be interpreted to represent and describe the biophysical behavior of 100150 natively unfolded fg - domains anchored within the npc ? how does one reconcile the notion of having extended fg - domains in the npc when the diameter of the central pore is about ten times larger than the size of a fg - domain at dynamic equilibrium [ stokes radius typically a few nanometers ( denning et al . what physical basis can allow for the fg - domains on opposing sides of the central pore to extend so far past their stokes radius so as to touch each other , much less to form an inter - fg crosslinked meshwork spanning the diameter of the central pore ( frey and gorlich 2007 ) ( fig . fg binding effects prevent the formation of such a meshwork by causing individual fg - domains to collapse or conversely , if a brush - like conformation is a valid prerequisite for the extension of the fg - domains to form a barrier in the npc ( lim et al . 2006b ) , does this necessarily preclude the possibility of inter - fg interactions occurring between neighboring fg - domains ? bearing in mind that the fg - domains are anchored to the npc instead of free floating in solution , whether or not fxfg- and glfg - domains cohere in the npc ( patel et al . 2007 ) will not only depend on how strongly ( and dynamically , i.e. frequently ) they interact with each other , but also on their physical proximity within the npc ( i.e. anchoring site ) . therefore , other contextual effects that require consideration include the following : ( 1 ) how confinement effects based on the hourglass - like geometry affect the entropic behavior of the fg - domains ( e.g. preferring to remain in the central pore or outside ) ? ( 2 ) the strength of fg fg interactions in the midst of competing effects such as the steric hindrance between the hydrophilic regions of neighboring fg - domains ; ( 3 ) how nonspecific interactions can bias their behavior ( paradise et al . 2007 ; timney et al . 2006 ) , e.g. macromolecular crowding ( zimmerman and minton 1993 ) ? ( 4 ) more generally , how does the complex cellular environment ( exhibiting a multitude of specific / nonspecific interactions ) in and around the npc near - field affect fg - domain conformations ? ( 5 ) finally , how do these effects influence and affect the interactions between the fg - domains and transport receptors ? given these arguments , the modus operandi of the npc selective gating mechanism requires careful substantiation at the near - field , mesoscopic scale ( i.e. the length scale relevant to the properties of a material or phenomenon ) . to emphasize its relevance , the outermost surface of a hydrogel at the gel liquid interface has been shown to consist of noncrosslinked polymer chains ( kim et al . moreover , fg - domain behavior will have to be assessed at the relevant time scales , since selective gating appears to be a rapid kinetic process over a time scale of the order of 5 ms ( kubitscheck et al . this emphasizes the need to move beyond conventional biological methods and to adopt more interdisciplinary experimental approaches ( dutta and belfort 2007 ) . in addition , the use of molecular dynamics ( md ) simulations ( isgro and schulten 2005 , 2007a , b ) and other theoretical frameworks ( zilman et al . 2007 ) may be able to illuminate aspects of fg - domain behavior and the nuclear trafficking machinery that experimentalists can then look out for and validate . perhaps most importantly , the desire to conceptually reconcile and reach a single objective understanding of npc function and structure will require that such aspects of nuclear transport processes be scrutinized in individual npcs in vivo . however , beyond the technical challenges involved , a difficulty remains as to how a ground - state can be defined in the npc so as to monitor subsequent changes in the nebulous haze of fg - domains without any quantitative information regarding the endogenous receptors and cargo already bound to the fg - domains , which will influence fg - domain behavior . in any case , such attention to detail will be key to defining a unified picture of the npc . as noted by paine et al . ( 1975 ) in nature more than 30 years ago , as solute size approaches the dimensions of the pore , solute pore wall interactions become increasingly important .

the spatial separation between the cytoplasm and the cell nucleus necessitates the continuous exchange of macromolecular cargo across the double - membraned nuclear envelope . being the only passageway in and out of the nucleus , the nuclear pore complex ( npc ) has the principal function of regulating the high throughput of nucleocytoplasmic transport in a highly selective manner so as to maintain cellular order and function . here , we present a retrospective review of the evidence that has led to the current understanding of both npc structure and function . looking towards the future , we contemplate on how various outstanding effects and nanoscopic characteristics ought to be addressed , with the goal of reconciling structure and function into a single unified picture of the npc .

Introduction Nuclear pore complex architecture Biochemical composition of the nuclear pore complex: the nucleoporins Nucleocytoplasmic transport and nuclear pore complex function Modeling NPC function Unraveling FG-domain behavior Outlook

the cytoplasmic and nuclear compartments of eukaryotic cells are separated by the nuclear envelope ( ne ) . the double membrane of the ne is perforated by nuclear pore complexes ( npcs ) , which are large multiprotein complexes that support passive diffusion of small molecules , and facilitate receptor - mediated translocation of proteins and ribonucleoprotein complexes . overall , the vertebrate npc is a 120 mda protein complex made of 30 different proteins called nucleoporins ( or nups ) that are repetitively arranged as distinct subcomplexes to form the npc ( cronshaw et al . in the plane of the ne , the eightfold symmetric central framework of the npc , also known as the spoke complex , encloses a central pore that is 50 nm long and is narrowest ( 40 nm ) at the ne midplane ( beck et al . view of the nuclear basket of isolated nuclei from xenopus oocytes prepared by e critical point drying and field emission scanning electron microscopy , f quick - freeze / freeze - drying / rotary metal shadowing and g thin - sectioning and transmission electron microscopy . three - dimensional reconstruction of the central framework of negatively stained npcs after detergent treatment a from xenopus oocytes , and b its adaptation for a consensus model of the npc architecture and c from yeast nuclei and d its adaptation into a model . view of the nuclear basket of isolated nuclei from xenopus oocytes prepared by e critical point drying and field emission scanning electron microscopy , f quick - freeze / freeze - drying / rotary metal shadowing and g thin - sectioning and transmission electron microscopy . three - dimensional reconstruction of the central framework of negatively stained npcs after detergent treatment a from xenopus oocytes , and b its adaptation for a consensus model of the npc architecture and c from yeast nuclei and d its adaptation into a model . e consensus model of the npc based on a reconstruction of native npcs embedded in thick amorphous ice . ( 2007 ) we begin , here , with a retrospective that summarizes the analysis and elucidation of npc architecture by electron microscopy ( em ) techniques . in parallel , we will overview the progress that has contributed to the contemporary understanding of how the npc functions as a selective gate - barrier . setting our sights on the future , we will close by highlighting the importance of reconciling both npc structure and function , as we strive towards converging on a single conceptual , mechanistic understanding of the npc . npc structure was initially dissected using transmission em ( tem ) , but later expanded to scanning transmission as well as to scanning em and , most recently , to cryo - electron tomography ( cet ) . although its functional significance was ambiguous at that time , it was already evident from these early studies that the npc is composed of a membranous structure , specifically with a part residing within the ne , and nonmembranous , filamentous structures being attached to the cytoplasmic and the nuclear face of the npc ( fig . according to today s consensus , the npc consists of an approximately cylindrical central framework , eight cytoplasmic filaments and a nuclear basket ( fig . the central framework of the npc ( also known as the spoke complex ) resides within the ne , and is anchored to the region where the inner and outer nuclear membranes fuse . 2004 , 2007 ) have improved the resolution of the central framework to 6 nm and revealed the first reconstructions of peripheral , flexible components of the npc , i.e. in dictyostelium , the cytoplasmic filaments are about 35 nm in length and the nuclear basket is about 60 nm long . together with the 50 nm central framework , the npc has an overall length of about 150 nm and an outer diameter of 125 nm ( beck et al . although the overall linear dimensions of the npc vary between species , the overall 3d architecture appears to be evolutionarily conserved ( fahrenkrog et al . enclosed by the central framework is the hourglass - shaped central pore of the npc , which has a diameter of 6070 nm at its cytoplasmic and nuclear periphery and is 45 nm in the midplane of the npc / ne ( beck et al . this central pore mediates the traffic between the cytoplasm and the nucleus , i.e. the peripheral channels of the npc have a diameter of about 8 nm and have been implicated in the diffusion of small molecules and ions ( feldherr and akin 1997 ; hinshaw et al . structurally , the cytoplasmic and the nuclear openings of the peripheral channels are not topologically continuous ( stoffler et al . in yeast , the number of npcs was found to increase steadily , beginning in the g1-phase and peaking in the s - phase of the cell cycle , suggesting that npc assembly occurs continuously throughout the cell cycle ( winey et al . similarly , the density of npcs increases throughout the cell cycle in hela s3 cells ( maeshima et al . the molecular building blocks of the npc comprise 30 different proteins known as nucleoporins or nups , which are present in at least eight copies per npc ( cronshaw et al . the transmembrane group , which contains transmembrane -helices and a cadherin fold , comprises the outermost features of the npc central framework and is thought to assist in anchoring the npc to the ne . the second group of nucleoporins contain -propeller and -solenoid folds , which localizes towards the inside of the npc , whereas the third class harbors the conserved sequence motif of phenylalanine glycine ( fg)-repeats in combination with a coiled - coil fold and likely contributes to the formation of the npc s inner central framework and the peripheral structures ( devos et al . along this line , yeast nup133p and nup159p both play roles in mrna export from the nucleus , given that deletion or mutations in their ntds impair their functions in mrna export , probably by preventing the association of multiple mrna export factors with the npc ( berke et al . whether the -helical motif in nup133 serves to recognize the curved topology of the nuclear pore membrane to anchor the npc during interphase or to recognize small vesicles containing ne fragments critical for ne reassembly after mitosis , or both , remains to be clarified ( drin et al . this domain , similar to the nuclear pore - targeting domain of its yeast homolog nup116p , consists of a six - stranded -sheet sandwiched against a two - stranded -sheet and is flanked by two -helical regions ( hodel et al . conformational diversity may allow nup98 and nup116p to bind to multiple targets within the npc or to associate and dissociate quickly from the npc to increase the mobility of the nucleoporins , as described for nup98 , which shuttles in a transcription - dependent manner ( griffis et al . an attempt to crystallize the first subcomplex of the npc , the nup62 complex , yielded the structure of the -helical coiled - coil domain of rat nup58/45 ( melcak et al . the nup62 complex has recently been mapped to the cytoplasmic periphery of the npc s central pore ( schwarz - herion et al . fg - repeat domains ( also known as fg - domains ) are found in about one - third of the nucleoporins and mediate the interaction between soluble transport receptors loaded with signal - bearing cargo and the npc . these fg - domains constitute the key components of the selective gate - barrier that limits the diffusion of cargoes through the npc ( see following section ) . 2002 , 2003 ) , have consistently shown that the interaction between fg - repeats and the different transport receptors involves primarily the phenylalanine ring of the fg - repeat core and hydrophobic residues on the surface of the receptor ( isgro and schulten 2005 , 2007a , b ) . hydrophilic linker regions between individual fg - motifs , which constitute the majority of amino acid mass in the overall fg - domain , appear to influence the strength of the binding and allow simultaneous binding of several fg - cores to the receptor ( liu and stewart 2005 ) . based on biophysical measurements , the fg - domains of yeast nucleoporins have been found to be natively unfolded ( denning et al . the location of the fg - domains of nup153 and nup214 shifts in a transport - dependent manner in the npc , further supporting their role in nct ( paulillo et al . besides playing important roles in nct , the crystal structure of the rrm domain of mouse nup35 revealed that all three fg - sequences of this nucleoporin are in ordered secondary structure elements and that these fg - sequences do not interact with transport receptors , such as importin , but rather with , for example , the integral membrane protein ndc1 . thus , the fg - sequences of nup35 may contribute to the formation of the npc s central framework ( handa et al . today , the biochemical role of the receptors in unlocking the npc barrier as part of the nuclear trafficking machinery is relatively well understood . otherwise , passage through the npc is obstructed for large , non - nls / nes harboring molecules that do not bind to the karyopherins ( i.e. being predominant in the nucleus , rangtp functions to release the cargo from its import receptor by binding to the receptor itself ( gorlich et al . binding of rangtp to the transport receptor releases the nls - cargo into the nucleus . the gray shaded area emphasizes the location of the fg - domains and the question mark highlights the uncertainty with regard to the biophysical aspects of the selective gating mechanism within the npc , which operates to restrict or promote cargo translocation schematic representation identifying the main biochemical constituents of the nuclear import machinery . the gray shaded area emphasizes the location of the fg - domains and the question mark highlights the uncertainty with regard to the biophysical aspects of the selective gating mechanism within the npc , which operates to restrict or promote cargo translocation early models of the npc gating mechanism were derived from initial em studies , which linked the biophysical origin of the selective gate to the presence of a central plug or central transporter the central transporter was suggested to consist of an iris - like mechanism that is hinged to the central channel ( akey 1990 ) ( fig . b being natively unfolded and located at the nuclear / cytoplasmic peripheries of the npc , the virtual gating model suggests that the entropic movements of the fg - domains can act as a barrier to inert cargo . e the undersaturated and subsequently the saturated hydrogel model purport that the fg - domains crosslink via a dense number of inter - fg interactions to form a highly organized three - dimensional network within the npc . f the nanomechanical reversible collapse model asserts that the selective gating mechanism consists of a stochastic flux of collapsing and distending fg - domains that is regulated by binding and unbinding interactions between the fg - domain and the transport receptors . b being natively unfolded and located at the nuclear / cytoplasmic peripheries of the npc , the virtual gating model suggests that the entropic movements of the fg - domains can act as a barrier to inert cargo . e the undersaturated and subsequently the saturated hydrogel model purport that the fg - domains crosslink via a dense number of inter - fg interactions to form a highly organized three - dimensional network within the npc . model asserts that the selective gating mechanism consists of a stochastic flux of collapsing and distending fg - domains that is regulated by binding and unbinding interactions between the fg - domain and the transport receptors . ( 1995b ) recognized that the fg - domains represent the key constituents of the npc selective gate by determining that the fg - repeats act as docking sites for kaps . by identifying and binding to the fg - domains , the kaps ( 2000 ) showed that each npc consists of up to 12 different fg - domains ( i.e. nucleoporins ) , which are located near the nuclear and cytoplasmic peripheries of the npc . to further emphasize the functional redundancy between the various fg - domains in the npc ( 1 ) the asymmetric fg - nups have been shown to be dispensable for nct ( zeitler and weis 2004 ) ; ( 2 ) the direction of transport through the npc can be inverted ( nachury and weis 1999 ) ; ( 3 ) active transport is able to proceed in npcs lacking cytoplasmic filaments ( i.e. , it is generally agreed that the fg - domains act as the physical constituents of the npc barrier ( ben - efraim and gerace 2001 ; macara 2001 ; ribbeck and gorlich 2002 ; rout et al . however , the mechanistic manner in which the fg - domains contribute to the selective gating of the npc is widely speculated and has been the subject of several reviews ( fahrenkrog and aebi 2003 ; lim et al . akin to the entropic fluctuations of unstructured microtubule - associated proteins ( maps ) ( mukhopadhyay and hoh 2001 ) and neurofilament sidearms ( brown and hoh 1997 ) , the brownian affinity gating model ( rout et al . translocation is anticipated for receptor - mediated cargoes due to interactions between the fg - repeats and the transport receptors , which increases the residence time and probability of entry into the npc . in a similar manner , the oily - spaghetti model ( macara 2001 ) postulates that noninteracting fg - domains are pushed aside by cargo complexes but otherwise obstruct the passage of passive cargo ( fig . cargo complex movement through the npc has been compared to stepping from one fg - repeat to the next ( rexach and blobel 1995 ) , or sliding over a surface comprised of fg - repeats ( peters 2005 ) . alternatively , the affinity gradient model ( ben - efraim and gerace 2001 ) suggests that transport complexes step through npcs lined with fg - nucleoporins exhibiting increasing binding affinities with the cargo complexes ( fig . in this manner , they explained that an efficient permeability barrier was recovered due to the formation of a highly ordered meshwork consisting of cohesive inter - fg repeats ( fig . in addition to preventing the translocation of inert molecules , it was found that such a saturated hydrogel could reproduce the diffusion rates of receptor - driven transport in the npc ( kubitscheck et al . by reproducing the physical dimensions of the npc , they found that the fxfg - domains of the vertebrate nup153 resembles a polymer brush ( halperin et al . this may explain why a reduced number of fg - domains is enough to maintain an effective barrier in the npc . to further correlate the governing biochemical interactions to the biophysical behavior of the fg - domains , lim et al . such seamlessness may explain the reversibility of nct and apparent open communication between the cytoplasm and nucleus ( kopito and elbaum 2007 ) . besides resolving the overall structure of the npc , an accurate picture of how selective gating is achieved by the ( structureless ) fg - domains remains unclear due to a general lack of understanding with regard to their behavior and function within the npc . although immunogold - em techniques can provide positional information of the fg - domains within the npc , this is limited to static views of fg - domain behavior ( fahrenkrog et al . new microscopic techniques such as the afm is also usually limited in resolution and chemical sensitivity due to the complexity of the npc and its cellular environment ( jaggi et al . in combination with biochemical efforts to identify whether different transport receptors use preferential fg - domains during nct ( terry and wente 2007 ) , it may be beneficial to use bottom - up strategies to investigate how the fg - domains behave on a biophysical level to give rise to the selective gating of the npc . moreover , because nct occurs over tens of nanometers in and around the npc , i.e. a near - field effect , this in fact necessitates a consideration of such physical details , including ( 1 ) the geometry and nanoscopic dimensions of the npc , ( 2 ) the fg - domain anchoring sites within the npc and ( 3 ) the limited number of fg - domains located within the npc . how does one reconcile the notion of having extended fg - domains in the npc when the diameter of the central pore is about ten times larger than the size of a fg - domain at dynamic equilibrium [ stokes radius typically a few nanometers ( denning et al . what physical basis can allow for the fg - domains on opposing sides of the central pore to extend so far past their stokes radius so as to touch each other , much less to form an inter - fg crosslinked meshwork spanning the diameter of the central pore ( frey and gorlich 2007 ) ( fig . fg binding effects prevent the formation of such a meshwork by causing individual fg - domains to collapse or conversely , if a brush - like conformation is a valid prerequisite for the extension of the fg - domains to form a barrier in the npc ( lim et al . bearing in mind that the fg - domains are anchored to the npc instead of free floating in solution , whether or not fxfg- and glfg - domains cohere in the npc ( patel et al . ( 4 ) more generally , how does the complex cellular environment ( exhibiting a multitude of specific / nonspecific interactions ) in and around the npc near - field affect fg - domain conformations ? given these arguments , the modus operandi of the npc selective gating mechanism requires careful substantiation at the near - field , mesoscopic scale ( i.e. moreover , fg - domain behavior will have to be assessed at the relevant time scales , since selective gating appears to be a rapid kinetic process over a time scale of the order of 5 ms ( kubitscheck et al . 2007 ) may be able to illuminate aspects of fg - domain behavior and the nuclear trafficking machinery that experimentalists can then look out for and validate . perhaps most importantly , the desire to conceptually reconcile and reach a single objective understanding of npc function and structure will require that such aspects of nuclear transport processes be scrutinized in individual npcs in vivo . however , beyond the technical challenges involved , a difficulty remains as to how a ground - state can be defined in the npc so as to monitor subsequent changes in the nebulous haze of fg - domains without any quantitative information regarding the endogenous receptors and cargo already bound to the fg - domains , which will influence fg - domain behavior . in any case , such attention to detail will be key to defining a unified picture of the npc .

comparative studies of chloroplast genome sequences have investigated divergences spanning an enormous range of evolutionary times . these have included studies of intraspecific variation in domesticated plants ( yamane et al . 2003 ) , studies of early land plant evolution ( kugita et al . 2003 ) and also the earliest events of oxygenic photosynthesis ( martin et al . this range of comparisons has been possible because of the conservative nature of chloroplast ( cp ) genome evolution ( palmer 1985 ) , which involves relatively slow rates of sequence evolution in some parts of the cp genome ( sammut and huttley 2011 ) and elevated rates in other parts ( magee et al . 2010 ; sammut and huttley 2011 ) . molecular evolution of the cp genome sequences is typically modeled as a time reversible substitution process , in which changes at any one site are independent of changes at any other site ( li and goldman 1998 ; drouin et al . however , observations have suggested more complex processes of evolution in which both lineage - specific and nonrandom spatial patterns of substitution occur ( li and goldman 1998 ; lee et al . 2007 ; gruenheit et al . 2008 ; magee et al . 2010 ; wu et al . 2011 ; zhong et al . 2011 ) . such observations have practical significance for understanding the limitations of cp genomes in phylogenetic analyses of highly diverged lineages ( gruenheit et al . 2008 ) , and for understanding the mutational dynamics of hotspot regions studied in comparisons of closely related taxa ( shaw et al . 2007 ; it has been suggested that certain genome regions are predisposed to mutational events such as substitutions and insertion / deletionsthe regional difference hypothesis ( silva and kondrashov 2002 ; hardison et al . 2003 ) . a second hypothesis explaining the association between indels and substitutions is that certain ( large ) indels act to induce substitutions through a dna repair process that recruits error - prone dna polymerasesthe indel - induced mutation hypothesis ( tian et al . a third and related hypothesis is that it is the presence of repeat sequences rather than indels per se , that actually promotes replication fork arrest , causing the recruitment of the error - prone dna polymerases , and in doing so generates nucleotide substitutions ( mcdonald et al . these hypotheses have not been explicitly investigated in cp genomes yet these genomes are known to contain very high densities of direct and inverted oligonucleotide repeats . associations between repeats , indels , and substitutions have also been reported in cp genomes ( mclenachan et al . cp genome repeats include simple sequence repeats ( ssrs , also known as microsatellites ) and other moderate to long ( 848 bp ) repeats . contraction and expansion of the ssr units , caused by slipped strand mispairing during dna replication ( levinson and gutman 1987 ) , frequently produces short indels at these ssr loci ( masood et al . the moderate - to - long repeats have also been suggested to cause indels ( kawata et al . 1997 ) and inversions ( kim and lee 2005 ; whitlock et al . 2010 ) . most angiosperms also contain two large inverted repeat ( ir ) regions , commonly known as ira and irb ( 576 kb ; palmer 1991 ) . here , we report the cp genome sequences of two morphotypes of taro ( colocasia esculenta ; var . gp ; matthews 1985 ) and examine the genome wide association of repeats ( excluding ira and irb ) , indels and substitutions in the cp genomes of these taro morphotypes and four other distantly related aroids in the duckweed ( lemnoideae ) subfamily . colocasia esculenta ( l. ) schott , commonly known as taro , is an ancient root crop in subfamily aroideae of the monocot family araceae . this species is distributed in the tropical to subtropical and some temperate regions of the world ( bown 1988 ) . gene arrangement and other features of the c. esculenta cp genome are shown in figure 1 . size of the cp genome was 162,546 bp ( gc content : 36.1% ) in var . the gc content varied from 42.4% in irs to 34.4% in the large single copy ( lsc ) and only 28.4% in the small single copy ( ssc ) regions of the taro cp genomes . higher gc content in the ir regions corresponded to the presence of the ribosomal dna locus . pair - wise sequence alignment between the taro cp genomes revealed 99.5% identical sequence , 241 substitutions , and 92 indels . the lsc region contained 141 ( 58.6% ) substitutions and 65 ( 71% ) indels , the ssc region contained 83 ( 34.4% ) substitutions and 25 ( 27% ) indels , whereas the ira and irb regions collectively contained only 17 ( 7% ) substitutions and 2 ( 2% ) indels , indicating that the ir was the most evolutionarily stable region . prominent differences between the two taro cp genomes were found at the irb ssc boundary ( numerous indels making up a 91 bp difference in size ) , and at the ssc ira boundary ( a shift of 64 bp in the repeat boundary without causing indels ) . thus , the ir boundaries at both ends of the ssc region were polymorphic at intraspecific level in taro . polymorphism between the two taro cp genomes included 59 substitutions in 29 protein coding genes . among these , the most polymorphic gene was ycf1 even when normalized for its size , showing 16 substitutions between the two genomes . some protein coding genes ( including atph , psbm , and psbz ) and trna genes ( including trnh , trng , and trnw ) in particular showed a relatively high density of substitutions and indels within 20 bp upstream of their respective coding regions . whether this observation has functional significance a set of 30 functional trna genes covering all 20 amino acids required for protein synthesis was present in the taro cp genome . brown lines in the outer circle represent the lsc and ssc regions , cyan lines represent the irs , whereas inner green lines show at and blue lines show gc percentage throughout the cp genome . brown lines in the outer circle represent the lsc and ssc regions , cyan lines represent the irs , whereas inner green lines show at and blue lines show gc percentage throughout the cp genome . the overall gene arrangement was similar between taro ( c. esculenta ) and the duckweed ( lemna minor ; mardanov et al . however , notable differences were as follows : trnh gene is reported in the lsc region in duckweed , whereas the 5-end of this gene extended into the ira region in taro.infa gene is completely missing in duckweed , but a pseudo - copy of this gene with internal stop codons was observed in taro.a single functional rpl2 gene spanning the irb lsc boundary is reported in duckweed , whereas two functional copies of this gene were found in taro , one in each of the ir regions.a pseudo - copy of ycf68 gene is reported in duckweed ; however , a functional copy of this gene was observed in each ir region in taro.duckweed has ycf1 and rps15 genes within its ir regions , whereas these genes were placed within the ssc region in taro . trnh gene is reported in the lsc region in duckweed , whereas the 5-end of this gene extended into the ira region in taro . infa gene is completely missing in duckweed , but a pseudo - copy of this gene with internal stop codons was observed in taro . a single functional rpl2 gene spanning the irb lsc boundary is reported in duckweed , whereas two functional copies of this gene were found in taro , one in each of the ir regions . a pseudo - copy of ycf68 gene is reported in duckweed ; however , a functional copy of this gene was observed in each ir region in taro . duckweed has ycf1 and rps15 genes within its ir regions , whereas these genes were placed within the ssc region in taro . multiple independent gene transfers from cp to nuclear genomes are thought to have occurred during angiosperm evolution ( millen et al . the ycf68 gene is present in a range of plant families as a functional or a pseudo - gene , and may have functional significance even in its noncoding form ( raubeson et al . other genes showing variation in comparison with l. minor include trnh , rpl2 , ycf1 , and rps15 . these boundaries are well known to exhibit expansion and contraction in angiosperms ( whitlock et al . 2010 ) as well as in gymnosperms ( lin et al . a comparison of the size and percentage proportions of lsc , ssc , and ir regions in taro and other aroid cp genomes is given in table 1 . characterization of these boundaries is likely to provide useful insights into the dynamics of single copy ir boundary shifts in colocasia and other aroid cp genomes . table 1comparison among total size ( bp ) and sizes of the lsc , ssc , and two ir regions in taro and other aroid chloroplast genomesspeciesgenbank idgenome sizelscsscircolocasia esculenta var . rrjn105690162,54689,817 ( 55.26)22,075 ( 13.58)25,327 ( 31.16)lemna minornc010109165,95589,906 ( 54.17)13,603 ( 8.20)31,223 ( 37.63)spirodela polyrhizajn160603168,78891,222 ( 54.04)14,056 ( 8.33)31,755 ( 37.63)wolffiella lingulatajn160604169,33792,015 ( 54.34)13,956 ( 8.24)31,683 ( 37.42)wolffia australianajn160605168,70491,454 ( 54.21)13,394 ( 7.94)31,930 ( 37.85)note . percentage proportions of the lsc , ssc , and irs are given in parenthesis . comparison among total size ( bp ) and sizes of the lsc , ssc , and two ir regions in taro and other aroid chloroplast genomes note.percentage proportions of the lsc , ssc , and irs are given in parenthesis . we have visualized the extent to which indel and substitution mutations are nonrandomly distributed between taro and other aroid cp genomes , using a circos ( krzywinski et al . this plot shows that substitutions are very closely correlated in their distribution with moderate ( 15 bp ) to long ( 48 bp ) repeat sequences mainly found in noncoding regions . correlation ( r ) and related values for these data are given in table 2 . correlations were highly significant in comparisons of three types of mutations , including 1 ) repeats and substitutions , 2 ) substitutions and indels , and 3 ) repeats and indels . in a pairwise comparison of the two closely related taro genomes , the strength of correlations was greatest for repeats and indels followed by substitutions and indels and then repeats and substitutions . in contrast , when pairwise comparison was made between a taro genome and a more distantly related aroid genome , the strength of correlations reversed . substitutions and indels and then repeats and indels ( table 2 ) . the strongest correlation value observed was for repeats and indels in comparison of the two taro genomes . 2011 ) and have led to a hypothesis that repeat sequences play a pivotal role in generation of indel and substitution mutations ( mcdonald et al . rr showing the relationship between short repeats within the chloroplast genome and distribution of indels and substitutions in pairwise comparisons of taro var . all data in the histogram tracks are shown in nonoverlapping 250 bp bins , with the taro var . rr chloroplast ideogram ( lsc in purple , ssc in red , and irs in cyan ) ; genome annotation on the positive and negative strand ( genes in green ; trnas in blue and rrnas in purple ) ; five circular plots showing comparisons between c. esculenta var . gp , 2 ) lemna minor , 3 ) spirodela polyrhiza , 4 ) wolffia australiana , and 5 ) wolffiella lingulata . for each genome comparison , the number of indels in each 250 bp bin is shown in orange ( scale of 010 ) , and the number of substitutions is shown in blue ( scale of 0160 ) . across these five plots , two ends of a red line mark the two locations of the forward ( direct ) repeats , whereas those of a green line mark the two locations of the reverse ( inverted ) repeats on the genome . in this part of the figure , the large irs are not plotted , as they would obscure a large part of the figure . number of repeats shown in the diagram is 667 , with a size range from 15 to 48 bp ( average repeat size : 16 bp ) . rr taken as a reference ) to calculate the correlations between 1 ) repeats and substitutions , 2 ) insertion - deletions ( indels ) and substitutions , and 3 ) repeats and indelscomparisonc . gpwolffiella lingulatawolffia australianalemna minorspirodela polyrhizarepeats and substitutions correlation between repeats and substitutions ( r)0.2450.3910.4160.4240.491 significance of correlation ( t)6.44***10.81***11.657***11.92***14.37 * * * coefficient of determination ( r)0.0600.1520.1730.1800.241insertion deletions ( indels ) and substitutions correlation between indels and substitutions ( r)0.3910.2200.2450.3230.387 significance of correlation ( t)10.82***5.75***6.43***8.71***10.69 * * * coefficient of determination ( r)0.1530.0480.0600.1050.150repeats and indels correlation between repeats and indels ( r)0.6400.1680.1780.2240.212 significance of correlation ( t)21.20***4.33***4.59***5.87***5.51 * * * coefficient of determination ( r)0.4090.0280.0320.0500.045note . the alignments compared closely related ( var . rr to w. lingulata , w. australiana , l. minor , and s. polyrhiza ) aroid chloroplast genomes . the alignments were partitioned into 651 nonoverlapping bins of 250 bp size each to calculate these correlations.***all correlations were highly significant at 0.001 and 649 degree of freedom . rr showing the relationship between short repeats within the chloroplast genome and distribution of indels and substitutions in pairwise comparisons of taro var . all data in the histogram tracks are shown in nonoverlapping 250 bp bins , with the taro var . rr chloroplast ideogram ( lsc in purple , ssc in red , and irs in cyan ) ; genome annotation on the positive and negative strand ( genes in green ; trnas in blue and rrnas in purple ) ; five circular plots showing comparisons between c. esculenta var . gp , 2 ) lemna minor , 3 ) spirodela polyrhiza , 4 ) wolffia australiana , and 5 ) wolffiella lingulata . for each genome comparison , the number of indels in each 250 bp bin is shown in orange ( scale of 010 ) , and the number of substitutions is shown in blue ( scale of 0160 ) . two ends of a red line mark the two locations of the forward ( direct ) repeats , whereas those of a green line mark the two locations of the reverse ( inverted ) repeats on the genome . in this part of the figure , the large irs are not plotted , as they would obscure a large part of the figure . number of repeats shown in the diagram is 667 , with a size range from 15 to 48 bp ( average repeat size : 16 bp ) . comparisons among the pairwise alignments ( colocasia esculenta var . rr taken as a reference ) to calculate the correlations between 1 ) repeats and substitutions , 2 ) insertion - deletions ( indels ) and substitutions , and 3 ) repeats and indels note.the alignments compared closely related ( var . rr to w. lingulata , w. australiana , l. minor , and s. polyrhiza ) aroid chloroplast genomes . the alignments were partitioned into 651 nonoverlapping bins of 250 bp size each to calculate these correlations . * * * all correlations were highly significant at 0.001 and 649 degree of freedom . since tian et al . sized indels induce substitutions in their surrounding sequences , we also investigated this relationship in a multiple sequence alignment ( parental alignment ) of all six aroid cp genomes . from the this parental alignment , we extracted data partitions containing distinct indel location points ( ilps ) to make mutually exclusive partitions with respect to locations of the ilps . partition b contained ilps associated with large ( oligonucleotide long , non - ssr ) indels in both coding and noncoding regions . partition c contained ilps in noncoding regions , associated with both ssr indels and large indels . partition d contained ilps in coding regions , associated with both ssr and large indels . the density of substitutions in all partitions was highly dependent upon inverse of distance from the ilps ( r ranged from 0.85 to 0.97 for all bin sizes ; supplementary fig . s1 , supplementary material online ) . higher substitution density in bins closer to the ilps was a general trend in all five comparisons above , including the partition in which coding regions were removed ( partition c ) ; however , in this case , distance from the ilps was relatively shorter than in the other four comparisons . the indel - induced mutation hypothesis was further explored in a comparison including the parental alignment and partitions a and b , as shown in figure 3 . from this comparison , it is evident that the partition b ( containing ilps associated with large indels ) displayed a higher density of substitutions closer to ilps , and the density of substitutions decreased with an increase in distance from the ilps . in contrast , the partition a ( containing ilps associated with ssrs ) exhibited a low density of substitutions close to ilps , and the density of substitutions showed a net increase with increase in distance from the ilps . these observations are consistent with the indel - induced mutation hypothesis suggested for diploid eukaryote ( tian et al . 3.results showing ( a ) the number of mean non - zero data points used to calculate the substitution density and ( b ) the values of substitution density in 125 bp sequence adjacent to the ilps for the parental alignment as well as two of its partitions , a and b ( partition a contains ilps associated with ssr indels in coding and noncoding regions , while partition b contains ilps associated with large indels in coding and noncoding regions ) . lower than 150 values for non - zero data points at > 25 bp distance in ( a ) represents that taking an average for 1,000 random iterations , lesser than 150 ilps are 125 bp apart from each other in all three types of comparisons . results showing ( a ) the number of mean non - zero data points used to calculate the substitution density and ( b ) the values of substitution density in 125 bp sequence adjacent to the ilps for the parental alignment as well as two of its partitions , a and b ( partition a contains ilps associated with ssr indels in coding and noncoding regions , while partition b contains ilps associated with large indels in coding and noncoding regions ) . lower than 150 values for non - zero data points at > 25 bp distance in ( a ) represents that taking an average for 1,000 random iterations , lesser than 150 ilps are 125 bp apart from each other in all three types of comparisons . it is well known that certain regions of the chloroplast genome show different rates of mutations ( lee et al . 2007 ; gruenheit et al . 2008 2011 ) . these are observations consistent with a regional difference hypothesis ( silva and kondrashov 2002 ; hardison et al . 2003 ) and the suggestion that purifying selection operates at both coding and noncoding regions ( petersen et al . . however , these explanations are alone insufficient to explain substitution and indel patterns of the chloroplast genome . the extent of genome wide correlations reported here for indels , repeats , and substitution provides further support for the hypothesis by mcdonald et al . ( 2011 ) , which emphasizes the evolutionary importance of the repeats in causing mutations . in addition , our observations on substitution densities also provide support for an indel - induced mutation hypothesis ( tian et al . 2009 ) and further our understanding for the sometimes poor fit between time reversible substitution models and chloroplast sequence data . perhaps , most interestingly , the relationship between repeats , substitutions , and indels implies that , if the distribution of repeat sequences in a chloroplast genome is determined , there is a possibility to predict the mutational hotspot regions and other sequences that are most appropriate for population genetic , phylogeographic , and phylogenetic analyses . taro plants ( c. esculenta var rr ; voucher number mpn:46548 , and var gp ; voucher number mpn:46549 in the dame ella campbell herbarium , massey university , new zealand ) were obtained from the university of auckland campus . dna was extracted using a dneasy plant mini kit ( qiagen , usa ) and quantified using a qubit fluorometer ( invitrogen ) and quant - it - ds dna hs assay kit ( invitrogen ) . illumina sequence reads were generated using the gaiix platform at the massey genome service , massey university , new zealand . iilumina sequencing produced 33 million reads of 75 base long ( 16.5 million paired - end reads ) for var . rr , and 26.4 million reads of 75 base long ( 13.2 million paired - end reads ) for var . the reads were mapped to the duckweed cp genome ( l. minor ; mardanov et al . rr were de novo assembled into contiguous sequences ( contigs ) of variable lengths using velvet ( v.0.7.60 ; zerbino and birney 2008 ) , as described elsewhere ( collins et al . these contigs were blast - searched ( altschul et al . 1997 ) to determine homology to the duckweed cp genome . the two irs were distinguished by visual inspection of the boundaries between the repeat and single copy regions . genome annotation was carried out using dual organellar genome annotator ( dogma ; wyman et al . rr cp genome was then used as our reference genome to help assemble the var . to verify integrity of the de novo assembly process , the original 75 base long reads from both taro samples were mapped back to their respective , assembled cp genomes . summary statistics for the bwa mapping of 75 base long reads to the l. minor cp genome , as well as to their respective assembled var . table 3summary statistics for bwa mapping of 75 base , paired - end reads obtained from the colocasia esculenta var . the acronyms rr1 , rr2 , and rpe represent mapping with the read 1 , read 2 , and paired - end ( reads 1 and 2 taken together ) reads obtained from the var . similarly , gr1 , gr2 , and gpe represent mapping with the read 1 , read 2 , and paired - end reads obtained from the var . summary statistics for bwa mapping of 75 base , paired - end reads obtained from the colocasia esculenta var . gp morphotypes to the lemna minor chloroplast genome and to their assembled chloroplast genomes note.the acronyms rr1 , rr2 , and rpe represent mapping with the read 1 , read 2 , and paired - end ( reads 1 and 2 taken together ) reads obtained from the var . similarly , gr1 , gr2 , and gpe represent mapping with the read 1 , read 2 , and paired - end reads obtained from the var . gp cp genome , as well as to four aroid cp genomes from the lemnoideae subfamily , using dialign alignment ( morgenstern 2004 ) . the four aroid cp genomes included l. minor ( genbank i d : nc010109 ; mardanov et al . 2008 ) , spirodela polyrhiza ( genbank i d : jn160603 ) , wolffiella lingulata ( genbank i d : jn160604 ) , and wolffia australiana ( genbank i d : jn160605 ; wang and messing 2011 ) . selecting c. esculenta var . rr cp genome as a reference for the coordinate positions , indels , and substitutions were counted in pairwise comparisons in nonoverlapping bins of 250 bp through the entire length of the aligned cp genomes ( partitioning each of the five alignments into 651 bins ) . for the substitution count , indels in the var . similar patterns of indel and substitution counts were obtained using a mafft alignment ( katoh et al . a total of 5,000 forward ( direct ) and reverse ( inverted ) repeats with a minimum size of 14 bp , a maximum size of 48 bp , and a maximum of three nucleotide mismatch between the two repeat copies in the taro var . 2001 ) . of these 5,000 repeats , 667 locations of the forward and reverse in var . rr ( minimum size : 15 bp ; zero mismatch between the two copies ) , as well as polymorphic sites ( indels and substitutions ) in all five pairwise comparisons with respect to the var . rr cp genome were plotted as a circular diagram using circos ( krzywinski et al . 2009 ) . correlations ( r ) were calculated between numbers of 1 ) repeats and substitutions , 2 ) substitutions and indels , and 3 ) repeats and indels . this was done for comparisons of closely related ( two taro genomes ) and distantly related ( taro with other lemnoideae ) cp genomes . the correlation values ( r ) were used to determine the significance of correlation ( t ) and the coefficient of determination ( r ) , according to lowry ( 2012 ) . to further investigate the relationships between substitutions and indels , a multiple sequence alignment of all six aroid cp genomes was generated using dialign alignment ( morgenstern 2004 ) . this parental alignment was used to generate mutually exclusive alignment combinations with respect to locations of the ilps , to include ilps associated with coding and noncoding regions and ssr indels ( 171 ilps ; partition a ) and coding and noncoding regions and large indels ( 286 ilps ; partition b ) . the parental alignment was also used to generate two further mutually exclusive alignment combinations to include ilps associated with ssr indels and large indels in noncoding regions ( 376 ilps ; partition c ) and ssr indels and large indels in coding regions ( 81 ilps ; partition d ) . using a perl script , we counted the number and positions of substitutions with respect to the ilps , and plotted the substitution density as a function of distance from the ilps in nonoverlapping bins of 50 , 100 , 150 , 200 , and 250 bp each for the parental alignment as well as partitions a , b , and d ; and 10 , 20 , 30 , 40 , and 50 bp for the partition c. the effect of large indels in causing substitutions was further explored by comparing first three alignment combinations ( parental alignment along with partitions a and b ) and plotting the substitution density as a function of distance from the ilps in 125 bp sequence adjacent to the ilps . for this purpose , a jacknifing approach was used to randomly select 150 ilps from each of these three partitions with 1,000 random iterations to count substitutions within the 125 bp distance , divided into five nonoverlapping bins of 25 bp in size . plots showing the relationship between substitutions and ilps were generated using ms excel 2010 worksheets .

a characteristic feature of eukaryote and prokaryote genomes is the co - occurrence of nucleotide substitution and insertion / deletion ( indel ) mutations . although similar observations have also been made for chloroplast dna , genome - wide associations have not been reported . we determined the chloroplast genome sequences for two morphotypes of taro ( colocasia esculenta ; family araceae ) and compared these with four publicly available aroid chloroplast genomes . here , we report the extent of genome - wide association between direct and inverted repeats , indels , and substitutions in these aroid chloroplast genomes . we suggest that alternative but not mutually exclusive hypotheses explain the mutational dynamics of chloroplast genome evolution .

Introduction The Correlations among Repeats, Indels, and Substitutions in Aroid cp Genomes Materials and Methods Supplementary Material

comparative studies of chloroplast genome sequences have investigated divergences spanning an enormous range of evolutionary times . 2003 ) and also the earliest events of oxygenic photosynthesis ( martin et al . this range of comparisons has been possible because of the conservative nature of chloroplast ( cp ) genome evolution ( palmer 1985 ) , which involves relatively slow rates of sequence evolution in some parts of the cp genome ( sammut and huttley 2011 ) and elevated rates in other parts ( magee et al . molecular evolution of the cp genome sequences is typically modeled as a time reversible substitution process , in which changes at any one site are independent of changes at any other site ( li and goldman 1998 ; drouin et al . however , observations have suggested more complex processes of evolution in which both lineage - specific and nonrandom spatial patterns of substitution occur ( li and goldman 1998 ; lee et al . 2007 ; gruenheit et al . 2010 ; wu et al . such observations have practical significance for understanding the limitations of cp genomes in phylogenetic analyses of highly diverged lineages ( gruenheit et al . 2008 ) , and for understanding the mutational dynamics of hotspot regions studied in comparisons of closely related taxa ( shaw et al . 2007 ; it has been suggested that certain genome regions are predisposed to mutational events such as substitutions and insertion / deletionsthe regional difference hypothesis ( silva and kondrashov 2002 ; hardison et al . a second hypothesis explaining the association between indels and substitutions is that certain ( large ) indels act to induce substitutions through a dna repair process that recruits error - prone dna polymerasesthe indel - induced mutation hypothesis ( tian et al . a third and related hypothesis is that it is the presence of repeat sequences rather than indels per se , that actually promotes replication fork arrest , causing the recruitment of the error - prone dna polymerases , and in doing so generates nucleotide substitutions ( mcdonald et al . these hypotheses have not been explicitly investigated in cp genomes yet these genomes are known to contain very high densities of direct and inverted oligonucleotide repeats . associations between repeats , indels , and substitutions have also been reported in cp genomes ( mclenachan et al . cp genome repeats include simple sequence repeats ( ssrs , also known as microsatellites ) and other moderate to long ( 848 bp ) repeats . the moderate - to - long repeats have also been suggested to cause indels ( kawata et al . 1997 ) and inversions ( kim and lee 2005 ; whitlock et al . here , we report the cp genome sequences of two morphotypes of taro ( colocasia esculenta ; var . gp ; matthews 1985 ) and examine the genome wide association of repeats ( excluding ira and irb ) , indels and substitutions in the cp genomes of these taro morphotypes and four other distantly related aroids in the duckweed ( lemnoideae ) subfamily . colocasia esculenta ( l. ) schott , commonly known as taro , is an ancient root crop in subfamily aroideae of the monocot family araceae . the gc content varied from 42.4% in irs to 34.4% in the large single copy ( lsc ) and only 28.4% in the small single copy ( ssc ) regions of the taro cp genomes . pair - wise sequence alignment between the taro cp genomes revealed 99.5% identical sequence , 241 substitutions , and 92 indels . the lsc region contained 141 ( 58.6% ) substitutions and 65 ( 71% ) indels , the ssc region contained 83 ( 34.4% ) substitutions and 25 ( 27% ) indels , whereas the ira and irb regions collectively contained only 17 ( 7% ) substitutions and 2 ( 2% ) indels , indicating that the ir was the most evolutionarily stable region . prominent differences between the two taro cp genomes were found at the irb ssc boundary ( numerous indels making up a 91 bp difference in size ) , and at the ssc ira boundary ( a shift of 64 bp in the repeat boundary without causing indels ) . polymorphism between the two taro cp genomes included 59 substitutions in 29 protein coding genes . some protein coding genes ( including atph , psbm , and psbz ) and trna genes ( including trnh , trng , and trnw ) in particular showed a relatively high density of substitutions and indels within 20 bp upstream of their respective coding regions . whether this observation has functional significance a set of 30 functional trna genes covering all 20 amino acids required for protein synthesis was present in the taro cp genome . the overall gene arrangement was similar between taro ( c. esculenta ) and the duckweed ( lemna minor ; mardanov et al . the ycf68 gene is present in a range of plant families as a functional or a pseudo - gene , and may have functional significance even in its noncoding form ( raubeson et al . other genes showing variation in comparison with l. minor include trnh , rpl2 , ycf1 , and rps15 . a comparison of the size and percentage proportions of lsc , ssc , and ir regions in taro and other aroid cp genomes is given in table 1 . characterization of these boundaries is likely to provide useful insights into the dynamics of single copy ir boundary shifts in colocasia and other aroid cp genomes . table 1comparison among total size ( bp ) and sizes of the lsc , ssc , and two ir regions in taro and other aroid chloroplast genomesspeciesgenbank idgenome sizelscsscircolocasia esculenta var . rrjn105690162,54689,817 ( 55.26)22,075 ( 13.58)25,327 ( 31.16)lemna minornc010109165,95589,906 ( 54.17)13,603 ( 8.20)31,223 ( 37.63)spirodela polyrhizajn160603168,78891,222 ( 54.04)14,056 ( 8.33)31,755 ( 37.63)wolffiella lingulatajn160604169,33792,015 ( 54.34)13,956 ( 8.24)31,683 ( 37.42)wolffia australianajn160605168,70491,454 ( 54.21)13,394 ( 7.94)31,930 ( 37.85)note . percentage proportions of the lsc , ssc , and irs are given in parenthesis . comparison among total size ( bp ) and sizes of the lsc , ssc , and two ir regions in taro and other aroid chloroplast genomes note.percentage proportions of the lsc , ssc , and irs are given in parenthesis . we have visualized the extent to which indel and substitution mutations are nonrandomly distributed between taro and other aroid cp genomes , using a circos ( krzywinski et al . correlation ( r ) and related values for these data are given in table 2 . correlations were highly significant in comparisons of three types of mutations , including 1 ) repeats and substitutions , 2 ) substitutions and indels , and 3 ) repeats and indels . in a pairwise comparison of the two closely related taro genomes , the strength of correlations was greatest for repeats and indels followed by substitutions and indels and then repeats and substitutions . the strongest correlation value observed was for repeats and indels in comparison of the two taro genomes . 2011 ) and have led to a hypothesis that repeat sequences play a pivotal role in generation of indel and substitution mutations ( mcdonald et al . rr showing the relationship between short repeats within the chloroplast genome and distribution of indels and substitutions in pairwise comparisons of taro var . rr chloroplast ideogram ( lsc in purple , ssc in red , and irs in cyan ) ; genome annotation on the positive and negative strand ( genes in green ; trnas in blue and rrnas in purple ) ; five circular plots showing comparisons between c. esculenta var . gp , 2 ) lemna minor , 3 ) spirodela polyrhiza , 4 ) wolffia australiana , and 5 ) wolffiella lingulata . for each genome comparison , the number of indels in each 250 bp bin is shown in orange ( scale of 010 ) , and the number of substitutions is shown in blue ( scale of 0160 ) . across these five plots , two ends of a red line mark the two locations of the forward ( direct ) repeats , whereas those of a green line mark the two locations of the reverse ( inverted ) repeats on the genome . rr taken as a reference ) to calculate the correlations between 1 ) repeats and substitutions , 2 ) insertion - deletions ( indels ) and substitutions , and 3 ) repeats and indelscomparisonc . gpwolffiella lingulatawolffia australianalemna minorspirodela polyrhizarepeats and substitutions correlation between repeats and substitutions ( r)0.2450.3910.4160.4240.491 significance of correlation ( t)6.44***10.81***11.657***11.92***14.37 * * * coefficient of determination ( r)0.0600.1520.1730.1800.241insertion deletions ( indels ) and substitutions correlation between indels and substitutions ( r)0.3910.2200.2450.3230.387 significance of correlation ( t)10.82***5.75***6.43***8.71***10.69 * * * coefficient of determination ( r)0.1530.0480.0600.1050.150repeats and indels correlation between repeats and indels ( r)0.6400.1680.1780.2240.212 significance of correlation ( t)21.20***4.33***4.59***5.87***5.51 * * * coefficient of determination ( r)0.4090.0280.0320.0500.045note . rr to w. lingulata , w. australiana , l. minor , and s. polyrhiza ) aroid chloroplast genomes . rr showing the relationship between short repeats within the chloroplast genome and distribution of indels and substitutions in pairwise comparisons of taro var . rr chloroplast ideogram ( lsc in purple , ssc in red , and irs in cyan ) ; genome annotation on the positive and negative strand ( genes in green ; trnas in blue and rrnas in purple ) ; five circular plots showing comparisons between c. esculenta var . gp , 2 ) lemna minor , 3 ) spirodela polyrhiza , 4 ) wolffia australiana , and 5 ) wolffiella lingulata . for each genome comparison , the number of indels in each 250 bp bin is shown in orange ( scale of 010 ) , and the number of substitutions is shown in blue ( scale of 0160 ) . two ends of a red line mark the two locations of the forward ( direct ) repeats , whereas those of a green line mark the two locations of the reverse ( inverted ) repeats on the genome . comparisons among the pairwise alignments ( colocasia esculenta var . rr taken as a reference ) to calculate the correlations between 1 ) repeats and substitutions , 2 ) insertion - deletions ( indels ) and substitutions , and 3 ) repeats and indels note.the alignments compared closely related ( var . rr to w. lingulata , w. australiana , l. minor , and s. polyrhiza ) aroid chloroplast genomes . * * * all correlations were highly significant at 0.001 and 649 degree of freedom . sized indels induce substitutions in their surrounding sequences , we also investigated this relationship in a multiple sequence alignment ( parental alignment ) of all six aroid cp genomes . from the this parental alignment , we extracted data partitions containing distinct indel location points ( ilps ) to make mutually exclusive partitions with respect to locations of the ilps . the density of substitutions in all partitions was highly dependent upon inverse of distance from the ilps ( r ranged from 0.85 to 0.97 for all bin sizes ; supplementary fig . from this comparison , it is evident that the partition b ( containing ilps associated with large indels ) displayed a higher density of substitutions closer to ilps , and the density of substitutions decreased with an increase in distance from the ilps . in contrast , the partition a ( containing ilps associated with ssrs ) exhibited a low density of substitutions close to ilps , and the density of substitutions showed a net increase with increase in distance from the ilps . 3.results showing ( a ) the number of mean non - zero data points used to calculate the substitution density and ( b ) the values of substitution density in 125 bp sequence adjacent to the ilps for the parental alignment as well as two of its partitions , a and b ( partition a contains ilps associated with ssr indels in coding and noncoding regions , while partition b contains ilps associated with large indels in coding and noncoding regions ) . it is well known that certain regions of the chloroplast genome show different rates of mutations ( lee et al . 2003 ) and the suggestion that purifying selection operates at both coding and noncoding regions ( petersen et al . however , these explanations are alone insufficient to explain substitution and indel patterns of the chloroplast genome . the extent of genome wide correlations reported here for indels , repeats , and substitution provides further support for the hypothesis by mcdonald et al . 2009 ) and further our understanding for the sometimes poor fit between time reversible substitution models and chloroplast sequence data . perhaps , most interestingly , the relationship between repeats , substitutions , and indels implies that , if the distribution of repeat sequences in a chloroplast genome is determined , there is a possibility to predict the mutational hotspot regions and other sequences that are most appropriate for population genetic , phylogeographic , and phylogenetic analyses . taro plants ( c. esculenta var rr ; voucher number mpn:46548 , and var gp ; voucher number mpn:46549 in the dame ella campbell herbarium , massey university , new zealand ) were obtained from the university of auckland campus . dna was extracted using a dneasy plant mini kit ( qiagen , usa ) and quantified using a qubit fluorometer ( invitrogen ) and quant - it - ds dna hs assay kit ( invitrogen ) . rr , and 26.4 million reads of 75 base long ( 13.2 million paired - end reads ) for var . table 3summary statistics for bwa mapping of 75 base , paired - end reads obtained from the colocasia esculenta var . the acronyms rr1 , rr2 , and rpe represent mapping with the read 1 , read 2 , and paired - end ( reads 1 and 2 taken together ) reads obtained from the var . similarly , gr1 , gr2 , and gpe represent mapping with the read 1 , read 2 , and paired - end reads obtained from the var . summary statistics for bwa mapping of 75 base , paired - end reads obtained from the colocasia esculenta var . gp morphotypes to the lemna minor chloroplast genome and to their assembled chloroplast genomes note.the acronyms rr1 , rr2 , and rpe represent mapping with the read 1 , read 2 , and paired - end ( reads 1 and 2 taken together ) reads obtained from the var . similarly , gr1 , gr2 , and gpe represent mapping with the read 1 , read 2 , and paired - end reads obtained from the var . 2008 ) , spirodela polyrhiza ( genbank i d : jn160603 ) , wolffiella lingulata ( genbank i d : jn160604 ) , and wolffia australiana ( genbank i d : jn160605 ; wang and messing 2011 ) . rr cp genome as a reference for the coordinate positions , indels , and substitutions were counted in pairwise comparisons in nonoverlapping bins of 250 bp through the entire length of the aligned cp genomes ( partitioning each of the five alignments into 651 bins ) . for the substitution count , indels in the var . a total of 5,000 forward ( direct ) and reverse ( inverted ) repeats with a minimum size of 14 bp , a maximum size of 48 bp , and a maximum of three nucleotide mismatch between the two repeat copies in the taro var . of these 5,000 repeats , 667 locations of the forward and reverse in var . rr ( minimum size : 15 bp ; zero mismatch between the two copies ) , as well as polymorphic sites ( indels and substitutions ) in all five pairwise comparisons with respect to the var . correlations ( r ) were calculated between numbers of 1 ) repeats and substitutions , 2 ) substitutions and indels , and 3 ) repeats and indels . this was done for comparisons of closely related ( two taro genomes ) and distantly related ( taro with other lemnoideae ) cp genomes . the correlation values ( r ) were used to determine the significance of correlation ( t ) and the coefficient of determination ( r ) , according to lowry ( 2012 ) . to further investigate the relationships between substitutions and indels , a multiple sequence alignment of all six aroid cp genomes was generated using dialign alignment ( morgenstern 2004 ) . this parental alignment was used to generate mutually exclusive alignment combinations with respect to locations of the ilps , to include ilps associated with coding and noncoding regions and ssr indels ( 171 ilps ; partition a ) and coding and noncoding regions and large indels ( 286 ilps ; partition b ) . the parental alignment was also used to generate two further mutually exclusive alignment combinations to include ilps associated with ssr indels and large indels in noncoding regions ( 376 ilps ; partition c ) and ssr indels and large indels in coding regions ( 81 ilps ; partition d ) . using a perl script , we counted the number and positions of substitutions with respect to the ilps , and plotted the substitution density as a function of distance from the ilps in nonoverlapping bins of 50 , 100 , 150 , 200 , and 250 bp each for the parental alignment as well as partitions a , b , and d ; and 10 , 20 , 30 , 40 , and 50 bp for the partition c. the effect of large indels in causing substitutions was further explored by comparing first three alignment combinations ( parental alignment along with partitions a and b ) and plotting the substitution density as a function of distance from the ilps in 125 bp sequence adjacent to the ilps .

although the orchestrated rearrangements occurring during cell division provide a vivid example , responses to altered metabolic states are equally impressive . some subcellular architectural elements , such as membranes and the cytoskeleton , are maintained as stable elements held together by sets of relatively strong and thus persistent interactions ( e.g. , the interaction energy of tubulin dimers is 1015 kilocalories / mole ; caplow and fee , 2002 ) , whereas others , such as signaling networks , rely on weak , transient interactions ( e.g. , fractions to a few kilocalories / mole , in the realm of van der waals interactions ) . because weak interactions are numerous and in many cases cooperative ( all surfaces are sticky ! ) , when taken together they have a profound effect on the temporal and spatial distribution of macromolecules in the cell . all these interactions are under evolutionary pressure because they are essential for the fitness of an organism . mcconkey ( 1982 ) coined the term quinary to describe the set of evolutionarily selected weak interactions that are essential to maintaining the functional organization of biological macromolecules in living cells and eloquently pointed out that even the gentlest separation methods may disrupt them . challenge # 1 : to study the roles of weak , quinary interactions ( biological glue ) , we need to interrogate them without significantly perturbing them , which generally requires that we keep the living system intact ( gierasch and gershenson , 2009 ; wirth and gruebele , 2013 ) . therefore we need not only knowledge of the network of weak interactions in which any biomacromolecule participates , but also the time evolution of these interactions , particularly in response to a trigger ( e.g. , stress or normal conditions ) . whereas the physics of weak interactions is a mature science for well - defined molecular systems , the heterogeneity and complexity of cellular assemblies present significant challenges , such as measuring these interactions in vivo without additional perturbations unavoidably generated by introducing fluorescent reporters ( landgraf et al . challenge # 2 : to follow the network of weak interactions over time in response to changing cellular states , we need experimental methods that provide dynamic information about weak interactions . this challenge is driving the development of new optical methods and new biochemical tools to determine interactomes , but these tools are still in their infancy . the experimental data will feed physical analyses that can in turn address the temporal and spatial complexity of simultaneous interactions with energies that are only fractions of a kilocalorie / mole . challenge # 3 : living systems function over a broad range of length , time , and energy scales . to elucidate the physical underpinnings of biology one must decipher the extent to which events at one length , time , or energy scale affect those at others , and , conversely , determine when it is justified to consider only a subset of the length , time , and energetic ranges in analyzing a given process . of importance , interactions at the short range ( angstrom / molecular level ) have long - range implications ( micrometer / cellular level ) when considered in the proper cellular context , as illustrated later by the cellular cell wall biosynthesis machinery . fortunately , we are witnessing impressive progress in several labs that are tackling cell biological questions linked to quinary interactions . these studies are yielding both new methods and new experimental data about weak interactions and their biological roles . in the following vignettes , we briefly describe three examples in which the biological consequences of weak interactions are being explored and revealed . the concentration of proteins in the bacterial cytosol reaches a staggering 250 mg / ml by some estimates ( li et al . , 2014 ) ; factoring in other biological molecules ( e.g. , nucleic acids ) , the concentration of macromolecules within the cellular environment begins to reach 400 mg / ml , resulting in substantial loss of free water . at first glance these concentrated conditions should result in catastrophic aggregation or extremely high viscosity , yet enzyme complexes orchestrate complicated chemistry requiring free diffusion and mobility . of interest , recent work from the jacobs - wagner lab has shown that under atp - limiting conditions , the bacterial cytoplasm indeed forms a glassy state , where motions of particles are severely restricted ( figure 1a ; parry et al . , 2014 ) . their work suggests that this fluidization is driven through the action of multiple energy - dependent enzymes and their dynamics , and thus the cell is investing energy to maintain cellular fluidity . this work is in excellent agreement with a prior report from the theriot lab showing that jiggling motions of chromosomal loci in eukaryotic nuclei and in bacterial nucleoids are fueled by atp - dependent processes ( weber et al . , 2012 ) . for both cases , increases in motion can not be explained by a simple increase in thermal fluctuations due to heat release by energy consumption . instead , these experiments point to a model in which energy - dependent increases in other dynamical processes drive intracellular fluidity . proteins are constantly undergoing conformational changes ( squares / circles ) and breathing ( wavy lines ) . in many cases these dynamics are fueled directly by energy consumption ( e.g. , conformational changes in aaa+ proteins ) or indirectly ( e.g. , client protein remodeling by atp - dependent chaperones ) . ( 2014 ) , in the absence of atp , these interactions may be dampened , freezing proteins into static conformations . at the high solute concentrations in the cell , weak quinary interactions between these proteins can force a phase transition into a more glass - like state . on restoration of atp , motions resume , breaking these weak interactions and fluidizing the entire pool of cellular biomolecules . ( b ) formation of large - scale biomolecule assemblies can occur with a collection of weakly interacting proteins . at low concentrations , weak interactions cooperate to generate a higher - order assembly ( li et al . , 2012 ) . in the case of rna granules , low - complexity regions within multiple proteins produce a dynamic hydrogel that cages rna ( han et al . , 2012 ) . hydrogels can be disassembled rapidly upon phosphorylation of the proteins ( as shown by yellow circles ) or by changes in temperature and concentration ( kato et al . , 2012 ) . ( c ) transient short - range interactions can tune long - range effects across the cell . proper incorporation of peptidoglycan monomers into bacterial cell envelopes requires cross - linking enzymes ( red ) recruited by a moving assembly platform ( gray ) . at high concentrations ( left ) , there are sufficient cross - linking enzymes to saturate the platforms . if the interaction of cross - linking enzymes with the platform is strong / stable , then a decrease in enzyme levels would result in regions that lack the cross - linking enzyme and thus are unable to add monomers . over a long enough time interval ( t ) by contrast , dynamic weak interactions enable transient association of cross - linking enzymes with platforms and thus buffer changes in enzyme concentration so as to sustain cell - wide synthesis by rapid redistribution of enzymes across many platforms ( lee et al . , 2014 ) . we suggest that some aspect of this increased motion may arise from changes in quinary interactions upon changes in metabolism . proteins and protein complexes constantly undergo changes in structure that can stem directly or indirectly from atp consumption . for example , in the bacterium caulobacter crescentus , the atp - dependent aaa+ protease clpxp unfolds , remodels , and degrades hundreds of proteins during differentiation and the cell cycle ( bhat et al . , 2013 ) . this oligomeric enzyme undergoes dramatic conformational changes upon atp hydrolysis and also increases protein dynamics more globally due to its actions on its client proteins . on depletion of atp , these various direct and indirect dynamics are suppressed , paving the way for weak interactions among multiple unrelated proteins to gradually accrue and locally cage biomolecules , essentially freezing out movement during energy depletion . metabolic restart would increase the supply of atp , fueling protein conformational changes that lead to fluidization of the cytosol . in this light , quinary interactions can be viewed as angstrom - scale frictional forces that hold proteins within micrometer - scale subcellular neighborhoods . inherently weak interactions can produce long - lived associations especially when present in multivalent complexes and suitably high concentrations . similar to the phase transitions seen in any chemical solution close to saturation , the crowded environment of a cytoplasm can foster the abrupt condensation of biomolecules via quinary interactions into stable intracellular clusters . work from the rosen lab recapitulated these phase transitions and showed how they may be controlled by signals such as phosphorylation ( li et al . , 2012 ) . using tandem repeats of weakly interacting peptide ligands and their binding partners , this lab demonstrated how multivalent display can partition proteins into discrete assemblies converting low - affinity interactions into stable complexes ( figure 1b ) . the speed and degree of assembly can be tuned by valency and posttranslational modifications of the binding partners . evidence of these phase transitions has also been seen in heterogeneous biomolecular systems , as shown by the recent characterization of eukaryotic rna granules by the mcknight lab ( han et al . , 2012 ; kato et al . , these granules are critical for spatial and temporal control of translation , and thus their assembly can profoundly alter programming of the entire cell . collections of rna are caged by dynamic protein hydrogels formed in a concentration- , temperature- , and modification - dependent manner by coalescence of weakly interacting , intrinsically unstructured , low - complexity protein regions found within multiple proteins . both of these studies illustrate how short - range weak interactions presented at high enough valency can drive collections of biomolecules to generate large - scale assemblies . macromolecular complexes that perform biosynthetic operations are generally considered to be stable assemblies optimized not only for chemical reactivity , but also for overall stability ( e.g. , the ribosome ) . in contrast to this notion , quinary interactions can promote the formation of transient complexes assembled for specific functions . for decades it has been known that dynamic clustering of enzymes required for sequential steps of a metabolic process into a metabolon promotes substrate reactivity , presumably through a combination of substrate channeling and limiting diffusion of intermediates ( srere , 2000 ; clegg et al . , 2001 ) . however , these individual enzymes interact only weakly in dilute solution , requiring immobilization or enzyme fusions to capture this enhancement in vitro . recent work from k. c. huang 's lab illustrates how similar assemblies built from transient weak interactions orchestrate bacterial cell wall synthesis ( lee et al . , 2014 ) . formation of the peptidoglycan mesh of proteins and glycans that maintains bacterial cell shape requires multiple enzymes to break the existing mesh , insert new monomers , and cross - link those subunits into the growing matrix . a protein platform coordinates cell wall synthesizing enzymes by moving circumferentially around the inner membrane and guiding monomer insertion . the enzyme responsible for cross - linking newly inserted monomers is required for cell wall integrity , and inhibition of this enzyme blocks cell wall synthesis . of interest , single - particle tracking in living escherichia coli shows that the cross - linking enzyme exhibits rapid diffusive motions , in contrast to the slower , circumferential motions of the assembly platform ( lee et al . , 2014 ) . these highly dynamic interactions turn out to be beneficial for the cells , as there are only 100 copies of cross - linking enzyme per cell . if they were tightly associated with the assembly platform , then small decreases in enzyme concentration would result in empty assembly platforms that lack the ability to properly synthesize peptidoglycan due to the long - lived nature of the existing loaded platforms ( figure 1c ) . by contrast , weak binding to the platforms enables the cross - linking enzyme to cross - link the inserted monomers upon binding to a platform at one site and immediately hop to the next unoccupied platform to perform its function there . in this case , substantial decreases in enzyme concentration would be buffered by these dynamics , ensuring robust cell growth . thus the transient quality of the binding interactions of single proteins at the molecular level is responsible for long - range effects across the entire cell wall . the concentration of proteins in the bacterial cytosol reaches a staggering 250 mg / ml by some estimates ( li et al . , 2014 ) ; factoring in other biological molecules ( e.g. , nucleic acids ) , the concentration of macromolecules within the cellular environment begins to reach 400 mg / ml , resulting in substantial loss of free water . at first glance these concentrated conditions should result in catastrophic aggregation or extremely high viscosity , yet enzyme complexes orchestrate complicated chemistry requiring free diffusion and mobility . of interest , recent work from the jacobs - wagner lab has shown that under atp - limiting conditions , the bacterial cytoplasm indeed forms a glassy state , where motions of particles are severely restricted ( figure 1a ; parry et al . , 2014 ) . their work suggests that this fluidization is driven through the action of multiple energy - dependent enzymes and their dynamics , and thus the cell is investing energy to maintain cellular fluidity . this work is in excellent agreement with a prior report from the theriot lab showing that jiggling motions of chromosomal loci in eukaryotic nuclei and in bacterial nucleoids are fueled by atp - dependent processes ( weber et al . , 2012 ) . for both cases , increases in motion can not be explained by a simple increase in thermal fluctuations due to heat release by energy consumption . instead , these experiments point to a model in which energy - dependent increases in other dynamical processes drive intracellular fluidity . proteins are constantly undergoing conformational changes ( squares / circles ) and breathing ( wavy lines ) . in many cases these dynamics are fueled directly by energy consumption ( e.g. , conformational changes in aaa+ proteins ) or indirectly ( e.g. , client protein remodeling by atp - dependent chaperones ) . as illustrated in the work of parry et al . ( 2014 ) , in the absence of atp , these interactions may be dampened , freezing proteins into static conformations . at the high solute concentrations in the cell , weak quinary interactions between these proteins can force a phase transition into a more glass - like state . on restoration of atp , motions resume , breaking these weak interactions and fluidizing the entire pool of cellular biomolecules . ( b ) formation of large - scale biomolecule assemblies can occur with a collection of weakly interacting proteins . at low concentrations , weak interactions cooperate to generate a higher - order assembly ( li et al . , 2012 ) . in the case of rna granules , low - complexity regions within multiple proteins produce a dynamic hydrogel that cages rna ( han et al . , 2012 ) . hydrogels can be disassembled rapidly upon phosphorylation of the proteins ( as shown by yellow circles ) or by changes in temperature and concentration ( kato et al . , 2012 ) . ( c ) transient short - range interactions can tune long - range effects across the cell . proper incorporation of peptidoglycan monomers into bacterial cell envelopes requires cross - linking enzymes ( red ) recruited by a moving assembly platform ( gray ) . at high concentrations ( left ) , there are sufficient cross - linking enzymes to saturate the platforms . if the interaction of cross - linking enzymes with the platform is strong / stable , then a decrease in enzyme levels would result in regions that lack the cross - linking enzyme and thus are unable to add monomers . over a long enough time interval ( t ) by contrast , dynamic weak interactions enable transient association of cross - linking enzymes with platforms and thus buffer changes in enzyme concentration so as to sustain cell - wide synthesis by rapid redistribution of enzymes across many platforms ( lee et al . , 2014 ) . we suggest that some aspect of this increased motion may arise from changes in quinary interactions upon changes in metabolism . proteins and protein complexes constantly undergo changes in structure that can stem directly or indirectly from atp consumption . for example , in the bacterium caulobacter crescentus , the atp - dependent aaa+ protease clpxp unfolds , remodels , and degrades hundreds of proteins during differentiation and the cell cycle ( bhat et al . , 2013 ) . this oligomeric enzyme undergoes dramatic conformational changes upon atp hydrolysis and also increases protein dynamics more globally due to its actions on its client proteins . on depletion of atp , these various direct and indirect dynamics are suppressed , paving the way for weak interactions among multiple unrelated proteins to gradually accrue and locally cage biomolecules , essentially freezing out movement during energy depletion . metabolic restart would increase the supply of atp , fueling protein conformational changes that lead to fluidization of the cytosol . in this light , quinary interactions can be viewed as angstrom - scale frictional forces that hold proteins within micrometer - scale subcellular neighborhoods . inherently weak interactions can produce long - lived associations especially when present in multivalent complexes and suitably high concentrations . similar to the phase transitions seen in any chemical solution close to saturation , the crowded environment of a cytoplasm can foster the abrupt condensation of biomolecules via quinary interactions into stable intracellular clusters . work from the rosen lab recapitulated these phase transitions and showed how they may be controlled by signals such as phosphorylation ( li et al . , 2012 ) . using tandem repeats of weakly interacting peptide ligands and their binding partners , this lab demonstrated how multivalent display can partition proteins into discrete assemblies converting low - affinity interactions into stable complexes ( figure 1b ) . the speed and degree of assembly can be tuned by valency and posttranslational modifications of the binding partners . evidence of these phase transitions has also been seen in heterogeneous biomolecular systems , as shown by the recent characterization of eukaryotic rna granules by the mcknight lab ( han et al . , 2012 ; kato et al . , these granules are critical for spatial and temporal control of translation , and thus their assembly can profoundly alter programming of the entire cell . collections of rna are caged by dynamic protein hydrogels formed in a concentration- , temperature- , and modification - dependent manner by coalescence of weakly interacting , intrinsically unstructured , low - complexity protein regions found within multiple proteins . both of these studies illustrate how short - range weak interactions presented at high enough valency can drive collections of biomolecules to generate large - scale assemblies . macromolecular complexes that perform biosynthetic operations are generally considered to be stable assemblies optimized not only for chemical reactivity , but also for overall stability ( e.g. , the ribosome ) . in contrast to this notion , quinary interactions can promote the formation of transient complexes assembled for specific functions . for decades it has been known that dynamic clustering of enzymes required for sequential steps of a metabolic process into a metabolon promotes substrate reactivity , presumably through a combination of substrate channeling and limiting diffusion of intermediates ( srere , 2000 ; clegg et al . , 2001 ) . however , these individual enzymes interact only weakly in dilute solution , requiring immobilization or enzyme fusions to capture this enhancement in vitro . recent work from k. c. huang 's lab illustrates how similar assemblies built from transient weak interactions orchestrate bacterial cell wall synthesis ( lee et al . , 2014 ) . formation of the peptidoglycan mesh of proteins and glycans that maintains bacterial cell shape requires multiple enzymes to break the existing mesh , insert new monomers , and cross - link those subunits into the growing matrix . a protein platform coordinates cell wall synthesizing enzymes by moving circumferentially around the inner membrane and guiding monomer insertion . the enzyme responsible for cross - linking newly inserted monomers is required for cell wall integrity , and inhibition of this enzyme blocks cell wall synthesis . of interest , single - particle tracking in living escherichia coli shows that the cross - linking enzyme exhibits rapid diffusive motions , in contrast to the slower , circumferential motions of the assembly platform ( lee et al . , 2014 ) . these highly dynamic interactions turn out to be beneficial for the cells , as there are only 100 copies of cross - linking enzyme per cell . if they were tightly associated with the assembly platform , then small decreases in enzyme concentration would result in empty assembly platforms that lack the ability to properly synthesize peptidoglycan due to the long - lived nature of the existing loaded platforms ( figure 1c ) . by contrast , weak binding to the platforms enables the cross - linking enzyme to cross - link the inserted monomers upon binding to a platform at one site and immediately hop to the next unoccupied platform to perform its function there . in this case , substantial decreases in enzyme concentration would be buffered by these dynamics , ensuring robust cell growth . thus the transient quality of the binding interactions of single proteins at the molecular level is responsible for long - range effects across the entire cell wall . as the foregoing examples show , combinations of powerful new microscopy methods , genetic strategies , and proteomics have revealed the spatial and dynamic organization and reorganization of cellular components during different physiological events . these approaches have pointed out the complexity of the highly concentrated and organized cellular contents . furthermore , these examples have helped the field to realize and appreciate the complexity of biology that arises from collections of weak interactions . these scenarios illustrate the profound concepts that can be more deeply informed by understanding biological glue the panoply of weak quinary interactions . this leaves us with a yearning for the realization of dream experiments : we can imagine the insights that could be gained by knowing the nearest - neighbor distribution for each macromolecule in a cell as a function of time and physiological condition . who spends time with whom ? in what orientation , and with which partners ? are the interactions multivalent or one - to - one ? is the picture that emerges one of zones at higher interaction density than other zones ? is energy consumed to generate or disrupt these zones ? how do we achieve this goal ? methods to detect these interactions could include in - cell nuclear magnetic resonance to follow isotopically labeled molecules ( freedberg and selenko , 2014 ) ; particle tracking of fluorescently labeled molecules using light microscopy ( huang et al . , 2009 ) ; in situ measurements of pairwise interactions using fragment complementation at a systems level ( remy and michnick , 2004 ; tchekanda et al . , 2014 ) ; cross - linking studies that reveal neighboring proteins both in stable complexes and those that are only transiently interacting ; proximity - induced chemical modifications to capture accumulated snapshots of transient interactions ( slavoff et al . , 2011 ) ; and more that we expect are in development . the outcome of these approaches must be incorporated into robust computational models to frame the results in the proper complex cellular context ( chang et al . , 2013 ; all of these approaches and more will help shed light on the fabric of weak interactions controlling biology . new and more powerful physical understanding must accompany the development and application of these experimental methods so that we can elucidate how higher - order levels of cell biology can derive from these weak molecular interactions .

biological systems display stunning capacities to self - organize . moreover , their subcellular architectures are dynamic and responsive to changing needs and conditions . key to these properties are manifold weak quinary interactions that have evolved to create specific spatial networks of macromolecules . these specific arrangements of molecules enable signals to be propagated over distances much greater than molecular dimensions , create phase separations that define functional regions in cells , and amplify cellular responses to changes in their environments . a major challenge is to develop biochemical tools and physical models to describe the panoply of weak interactions operating in cells . we also need better approaches to measure the biases in the spatial distributions of cellular macromolecules that result from the integrated action of multiple weak interactions . partnerships between cell biologists , biochemists , and physicists are required to deploy these methods . together these approaches will help us realize the dream of understanding the biological glue that sustains life at a molecular and cellular level .

INTRODUCTION Churning the cytoplasm Subcellular phase transitions Brief but meaningful interactions WHERE SHOULD WE GO NEXT? DREAM EXPERIMENTS

although the orchestrated rearrangements occurring during cell division provide a vivid example , responses to altered metabolic states are equally impressive . some subcellular architectural elements , such as membranes and the cytoskeleton , are maintained as stable elements held together by sets of relatively strong and thus persistent interactions ( e.g. , the interaction energy of tubulin dimers is 1015 kilocalories / mole ; caplow and fee , 2002 ) , whereas others , such as signaling networks , rely on weak , transient interactions ( e.g. , fractions to a few kilocalories / mole , in the realm of van der waals interactions ) . because weak interactions are numerous and in many cases cooperative ( all surfaces are sticky ! ) , when taken together they have a profound effect on the temporal and spatial distribution of macromolecules in the cell . mcconkey ( 1982 ) coined the term quinary to describe the set of evolutionarily selected weak interactions that are essential to maintaining the functional organization of biological macromolecules in living cells and eloquently pointed out that even the gentlest separation methods may disrupt them . challenge # 1 : to study the roles of weak , quinary interactions ( biological glue ) , we need to interrogate them without significantly perturbing them , which generally requires that we keep the living system intact ( gierasch and gershenson , 2009 ; wirth and gruebele , 2013 ) . therefore we need not only knowledge of the network of weak interactions in which any biomacromolecule participates , but also the time evolution of these interactions , particularly in response to a trigger ( e.g. whereas the physics of weak interactions is a mature science for well - defined molecular systems , the heterogeneity and complexity of cellular assemblies present significant challenges , such as measuring these interactions in vivo without additional perturbations unavoidably generated by introducing fluorescent reporters ( landgraf et al . challenge # 2 : to follow the network of weak interactions over time in response to changing cellular states , we need experimental methods that provide dynamic information about weak interactions . this challenge is driving the development of new optical methods and new biochemical tools to determine interactomes , but these tools are still in their infancy . challenge # 3 : living systems function over a broad range of length , time , and energy scales . to elucidate the physical underpinnings of biology one must decipher the extent to which events at one length , time , or energy scale affect those at others , and , conversely , determine when it is justified to consider only a subset of the length , time , and energetic ranges in analyzing a given process . of importance , interactions at the short range ( angstrom / molecular level ) have long - range implications ( micrometer / cellular level ) when considered in the proper cellular context , as illustrated later by the cellular cell wall biosynthesis machinery . fortunately , we are witnessing impressive progress in several labs that are tackling cell biological questions linked to quinary interactions . these studies are yielding both new methods and new experimental data about weak interactions and their biological roles . in the following vignettes , we briefly describe three examples in which the biological consequences of weak interactions are being explored and revealed . the concentration of proteins in the bacterial cytosol reaches a staggering 250 mg / ml by some estimates ( li et al . , 2014 ) ; factoring in other biological molecules ( e.g. , nucleic acids ) , the concentration of macromolecules within the cellular environment begins to reach 400 mg / ml , resulting in substantial loss of free water . of interest , recent work from the jacobs - wagner lab has shown that under atp - limiting conditions , the bacterial cytoplasm indeed forms a glassy state , where motions of particles are severely restricted ( figure 1a ; parry et al . their work suggests that this fluidization is driven through the action of multiple energy - dependent enzymes and their dynamics , and thus the cell is investing energy to maintain cellular fluidity . this work is in excellent agreement with a prior report from the theriot lab showing that jiggling motions of chromosomal loci in eukaryotic nuclei and in bacterial nucleoids are fueled by atp - dependent processes ( weber et al . for both cases , increases in motion can not be explained by a simple increase in thermal fluctuations due to heat release by energy consumption . instead , these experiments point to a model in which energy - dependent increases in other dynamical processes drive intracellular fluidity . proteins are constantly undergoing conformational changes ( squares / circles ) and breathing ( wavy lines ) . in many cases these dynamics are fueled directly by energy consumption ( e.g. , conformational changes in aaa+ proteins ) or indirectly ( e.g. , client protein remodeling by atp - dependent chaperones ) . ( 2014 ) , in the absence of atp , these interactions may be dampened , freezing proteins into static conformations . at the high solute concentrations in the cell , weak quinary interactions between these proteins can force a phase transition into a more glass - like state . on restoration of atp , motions resume , breaking these weak interactions and fluidizing the entire pool of cellular biomolecules . at low concentrations , weak interactions cooperate to generate a higher - order assembly ( li et al . , 2012 ) . in the case of rna granules , low - complexity regions within multiple proteins produce a dynamic hydrogel that cages rna ( han et al . hydrogels can be disassembled rapidly upon phosphorylation of the proteins ( as shown by yellow circles ) or by changes in temperature and concentration ( kato et al . , 2012 ) . ( c ) transient short - range interactions can tune long - range effects across the cell . at high concentrations ( left ) , there are sufficient cross - linking enzymes to saturate the platforms . if the interaction of cross - linking enzymes with the platform is strong / stable , then a decrease in enzyme levels would result in regions that lack the cross - linking enzyme and thus are unable to add monomers . over a long enough time interval ( t ) by contrast , dynamic weak interactions enable transient association of cross - linking enzymes with platforms and thus buffer changes in enzyme concentration so as to sustain cell - wide synthesis by rapid redistribution of enzymes across many platforms ( lee et al . we suggest that some aspect of this increased motion may arise from changes in quinary interactions upon changes in metabolism . proteins and protein complexes constantly undergo changes in structure that can stem directly or indirectly from atp consumption . for example , in the bacterium caulobacter crescentus , the atp - dependent aaa+ protease clpxp unfolds , remodels , and degrades hundreds of proteins during differentiation and the cell cycle ( bhat et al . , 2013 ) . on depletion of atp , these various direct and indirect dynamics are suppressed , paving the way for weak interactions among multiple unrelated proteins to gradually accrue and locally cage biomolecules , essentially freezing out movement during energy depletion . metabolic restart would increase the supply of atp , fueling protein conformational changes that lead to fluidization of the cytosol . in this light , quinary interactions can be viewed as angstrom - scale frictional forces that hold proteins within micrometer - scale subcellular neighborhoods . inherently weak interactions can produce long - lived associations especially when present in multivalent complexes and suitably high concentrations . similar to the phase transitions seen in any chemical solution close to saturation , the crowded environment of a cytoplasm can foster the abrupt condensation of biomolecules via quinary interactions into stable intracellular clusters . work from the rosen lab recapitulated these phase transitions and showed how they may be controlled by signals such as phosphorylation ( li et al . , 2012 ) . , these granules are critical for spatial and temporal control of translation , and thus their assembly can profoundly alter programming of the entire cell . collections of rna are caged by dynamic protein hydrogels formed in a concentration- , temperature- , and modification - dependent manner by coalescence of weakly interacting , intrinsically unstructured , low - complexity protein regions found within multiple proteins . both of these studies illustrate how short - range weak interactions presented at high enough valency can drive collections of biomolecules to generate large - scale assemblies . macromolecular complexes that perform biosynthetic operations are generally considered to be stable assemblies optimized not only for chemical reactivity , but also for overall stability ( e.g. , the ribosome ) . in contrast to this notion , quinary interactions can promote the formation of transient complexes assembled for specific functions . for decades it has been known that dynamic clustering of enzymes required for sequential steps of a metabolic process into a metabolon promotes substrate reactivity , presumably through a combination of substrate channeling and limiting diffusion of intermediates ( srere , 2000 ; clegg et al . however , these individual enzymes interact only weakly in dilute solution , requiring immobilization or enzyme fusions to capture this enhancement in vitro . recent work from k. c. huang 's lab illustrates how similar assemblies built from transient weak interactions orchestrate bacterial cell wall synthesis ( lee et al . , 2014 ) . formation of the peptidoglycan mesh of proteins and glycans that maintains bacterial cell shape requires multiple enzymes to break the existing mesh , insert new monomers , and cross - link those subunits into the growing matrix . the enzyme responsible for cross - linking newly inserted monomers is required for cell wall integrity , and inhibition of this enzyme blocks cell wall synthesis . these highly dynamic interactions turn out to be beneficial for the cells , as there are only 100 copies of cross - linking enzyme per cell . by contrast , weak binding to the platforms enables the cross - linking enzyme to cross - link the inserted monomers upon binding to a platform at one site and immediately hop to the next unoccupied platform to perform its function there . in this case , substantial decreases in enzyme concentration would be buffered by these dynamics , ensuring robust cell growth . the concentration of proteins in the bacterial cytosol reaches a staggering 250 mg / ml by some estimates ( li et al . , 2014 ) ; factoring in other biological molecules ( e.g. , nucleic acids ) , the concentration of macromolecules within the cellular environment begins to reach 400 mg / ml , resulting in substantial loss of free water . at first glance these concentrated conditions should result in catastrophic aggregation or extremely high viscosity , yet enzyme complexes orchestrate complicated chemistry requiring free diffusion and mobility . of interest , recent work from the jacobs - wagner lab has shown that under atp - limiting conditions , the bacterial cytoplasm indeed forms a glassy state , where motions of particles are severely restricted ( figure 1a ; parry et al . , 2014 ) . their work suggests that this fluidization is driven through the action of multiple energy - dependent enzymes and their dynamics , and thus the cell is investing energy to maintain cellular fluidity . this work is in excellent agreement with a prior report from the theriot lab showing that jiggling motions of chromosomal loci in eukaryotic nuclei and in bacterial nucleoids are fueled by atp - dependent processes ( weber et al . , 2012 ) . proteins are constantly undergoing conformational changes ( squares / circles ) and breathing ( wavy lines ) . in many cases these dynamics are fueled directly by energy consumption ( e.g. , conformational changes in aaa+ proteins ) or indirectly ( e.g. , client protein remodeling by atp - dependent chaperones ) . as illustrated in the work of parry et al . ( 2014 ) , in the absence of atp , these interactions may be dampened , freezing proteins into static conformations . at the high solute concentrations in the cell , weak quinary interactions between these proteins can force a phase transition into a more glass - like state . on restoration of atp , motions resume , breaking these weak interactions and fluidizing the entire pool of cellular biomolecules . ( b ) formation of large - scale biomolecule assemblies can occur with a collection of weakly interacting proteins . at low concentrations , weak interactions cooperate to generate a higher - order assembly ( li et al . , 2012 ) . in the case of rna granules , low - complexity regions within multiple proteins produce a dynamic hydrogel that cages rna ( han et al . , 2012 ) . hydrogels can be disassembled rapidly upon phosphorylation of the proteins ( as shown by yellow circles ) or by changes in temperature and concentration ( kato et al . , 2012 ) . ( c ) transient short - range interactions can tune long - range effects across the cell . proper incorporation of peptidoglycan monomers into bacterial cell envelopes requires cross - linking enzymes ( red ) recruited by a moving assembly platform ( gray ) . at high concentrations ( left ) , there are sufficient cross - linking enzymes to saturate the platforms . over a long enough time interval ( t ) by contrast , dynamic weak interactions enable transient association of cross - linking enzymes with platforms and thus buffer changes in enzyme concentration so as to sustain cell - wide synthesis by rapid redistribution of enzymes across many platforms ( lee et al . , 2014 ) . we suggest that some aspect of this increased motion may arise from changes in quinary interactions upon changes in metabolism . proteins and protein complexes constantly undergo changes in structure that can stem directly or indirectly from atp consumption . for example , in the bacterium caulobacter crescentus , the atp - dependent aaa+ protease clpxp unfolds , remodels , and degrades hundreds of proteins during differentiation and the cell cycle ( bhat et al . , 2013 ) . this oligomeric enzyme undergoes dramatic conformational changes upon atp hydrolysis and also increases protein dynamics more globally due to its actions on its client proteins . on depletion of atp , these various direct and indirect dynamics are suppressed , paving the way for weak interactions among multiple unrelated proteins to gradually accrue and locally cage biomolecules , essentially freezing out movement during energy depletion . in this light , quinary interactions can be viewed as angstrom - scale frictional forces that hold proteins within micrometer - scale subcellular neighborhoods . inherently weak interactions can produce long - lived associations especially when present in multivalent complexes and suitably high concentrations . similar to the phase transitions seen in any chemical solution close to saturation , the crowded environment of a cytoplasm can foster the abrupt condensation of biomolecules via quinary interactions into stable intracellular clusters . work from the rosen lab recapitulated these phase transitions and showed how they may be controlled by signals such as phosphorylation ( li et al . using tandem repeats of weakly interacting peptide ligands and their binding partners , this lab demonstrated how multivalent display can partition proteins into discrete assemblies converting low - affinity interactions into stable complexes ( figure 1b ) . the speed and degree of assembly can be tuned by valency and posttranslational modifications of the binding partners . evidence of these phase transitions has also been seen in heterogeneous biomolecular systems , as shown by the recent characterization of eukaryotic rna granules by the mcknight lab ( han et al . , 2012 ; kato et al . , these granules are critical for spatial and temporal control of translation , and thus their assembly can profoundly alter programming of the entire cell . collections of rna are caged by dynamic protein hydrogels formed in a concentration- , temperature- , and modification - dependent manner by coalescence of weakly interacting , intrinsically unstructured , low - complexity protein regions found within multiple proteins . both of these studies illustrate how short - range weak interactions presented at high enough valency can drive collections of biomolecules to generate large - scale assemblies . macromolecular complexes that perform biosynthetic operations are generally considered to be stable assemblies optimized not only for chemical reactivity , but also for overall stability ( e.g. , the ribosome ) . in contrast to this notion , quinary interactions can promote the formation of transient complexes assembled for specific functions . for decades it has been known that dynamic clustering of enzymes required for sequential steps of a metabolic process into a metabolon promotes substrate reactivity , presumably through a combination of substrate channeling and limiting diffusion of intermediates ( srere , 2000 ; clegg et al . recent work from k. c. huang 's lab illustrates how similar assemblies built from transient weak interactions orchestrate bacterial cell wall synthesis ( lee et al . formation of the peptidoglycan mesh of proteins and glycans that maintains bacterial cell shape requires multiple enzymes to break the existing mesh , insert new monomers , and cross - link those subunits into the growing matrix . the enzyme responsible for cross - linking newly inserted monomers is required for cell wall integrity , and inhibition of this enzyme blocks cell wall synthesis . of interest , single - particle tracking in living escherichia coli shows that the cross - linking enzyme exhibits rapid diffusive motions , in contrast to the slower , circumferential motions of the assembly platform ( lee et al . these highly dynamic interactions turn out to be beneficial for the cells , as there are only 100 copies of cross - linking enzyme per cell . if they were tightly associated with the assembly platform , then small decreases in enzyme concentration would result in empty assembly platforms that lack the ability to properly synthesize peptidoglycan due to the long - lived nature of the existing loaded platforms ( figure 1c ) . by contrast , weak binding to the platforms enables the cross - linking enzyme to cross - link the inserted monomers upon binding to a platform at one site and immediately hop to the next unoccupied platform to perform its function there . in this case , substantial decreases in enzyme concentration would be buffered by these dynamics , ensuring robust cell growth . thus the transient quality of the binding interactions of single proteins at the molecular level is responsible for long - range effects across the entire cell wall . as the foregoing examples show , combinations of powerful new microscopy methods , genetic strategies , and proteomics have revealed the spatial and dynamic organization and reorganization of cellular components during different physiological events . these approaches have pointed out the complexity of the highly concentrated and organized cellular contents . furthermore , these examples have helped the field to realize and appreciate the complexity of biology that arises from collections of weak interactions . these scenarios illustrate the profound concepts that can be more deeply informed by understanding biological glue the panoply of weak quinary interactions . this leaves us with a yearning for the realization of dream experiments : we can imagine the insights that could be gained by knowing the nearest - neighbor distribution for each macromolecule in a cell as a function of time and physiological condition . in what orientation , and with which partners ? is the picture that emerges one of zones at higher interaction density than other zones ? is energy consumed to generate or disrupt these zones ? , 2009 ) ; in situ measurements of pairwise interactions using fragment complementation at a systems level ( remy and michnick , 2004 ; tchekanda et al . , 2014 ) ; cross - linking studies that reveal neighboring proteins both in stable complexes and those that are only transiently interacting ; proximity - induced chemical modifications to capture accumulated snapshots of transient interactions ( slavoff et al . , 2011 ) ; and more that we expect are in development . the outcome of these approaches must be incorporated into robust computational models to frame the results in the proper complex cellular context ( chang et al . , 2013 ; all of these approaches and more will help shed light on the fabric of weak interactions controlling biology .

the aging of the world population is being accompanied by a rise in the prevalence and incidence of alzheimer disease ( ad ) and other neurodegenerative illnesses . in the usa , the number of patients with ad is expected to increase to 13 million by 2050 and in the european union ( eu ) to over 4 million . epidemiologic data indicates that the world population will have grown considerably by 2025 and the percentage of elderly people will be significantly higher . since age is one of the major risk factors in the development of ad , it is expected that the incidence of this disease will also increase . however , experimental evidence detailed in a review by brewer indicates that other factors in addition to age could play a critical role in the development of diseases such as alzheimer and that the contribution of inorganic copper ( cu ) has been underestimated . it is widely known that more than 95% of ad cases are sporadic and only 27% are genetically determined . thus , any environmental factors likely to have an etiopathogenic role in ad should be investigated . though cu is essential to human health , cu overload has been associated with mental decline and particularly with ad development [ 3 , 6 , 7 ] . data from squitti 's group specifically demonstrated that free cu ( also known as ncbc or nonceruloplasmin - bound cu ) is elevated in the blood of ad patients , negatively correlates with cognition , and predicts the rate of loss of cognition [ 810 ] . more recently , our group corroborated these findings in an independent human cohort and demonstrated that increased ncbc has a direct impact on the disease duration . we also proposed the ncbc / ceruloplasmin ratio as a predictive marker of risk for the first - degree relatives of ad patients . sparks and schreurs first demonstrated that cu supplementation in drinking water given to rabbits under a diet with excess cholesterol ( cho ) produced an induction of -amyloid plaques and a learning deficit . in addition , lu et al . reported that trace amounts of cu activate the apoptotic cascade and exacerbate beta amyloid - induced neurotoxicity in cho - fed mice through a tnf - mediated inflammatory pathway [ 13 , 14 ] . very recently , brewer has reviewed the theory of inorganic cu toxicity in alzheimer disease as a causative factor in cognitive loss . however , the question of how inorganic cu might trigger a neurodegenerative process is still a matter of debate . there is little data on cu overload in humans since most of the available evidence ( experimental or epidemiological ) was obtained from animal models . however , the regulatory framework for chronic cu exposure in large human populations indicates that pollution , drinking water , and dietary cu - containing supplements are the main sources of exposure [ 6 , 16 ] . the dietary reference intake for people in the usa , united kingdom , europe , and australia varies from 0.16 to 0.98 ( estimated average requirements ) ear or ( recommended dietary allowance ) rda expressed in mg cu / kg body weight , with considerable variations as a function of age . the ( population reference intake ) pri was reported between 0.3 and 1.5 mg cu / kg body weight ; however , these limits were largely surpassed in many circumstances such as ingestion of fish , bivalves , or contaminated drinking water [ 3 , 17 ] . in accumulated data on 280 samples of household drinking water all across north america , 72% of the samples have cu levels above those enhancing ad in experimental models . the content of inorganic cu in dietary supplements can be as high as 3 mg / pill ( approx . furthermore , we and others have demonstrated significantly higher cu levels in plasma of women using cu - iuds and in blood samples from farmers working with cu - based pesticides [ 1719 ] . most importantly , we found alterations in cu homeostatic biomarkers in neurodegenerative patients and their first - degrees relatives . specifically , cho has been associated with oxidative stress and ad development [ 21 , 22 ] , with most of the experimental evidence emerging from exploration of the role of cho in -amyloid formation . despite the paucity of epidemiological data from human studies , it is reasonable to assume that cho plays at least some role in learning and memory and is associated with ad pathogenesis . of particular interest is evidence showing that cu greatly exacerbates cognitive decline in those people included in the highest quintile of fat ingestion . experimental evidence obtained from rabbit and mouse models suggests that the association of cu and cho can be risk factor for ad development [ 1214 ] . moreover , it was hypothesized that cu could oxidize cho , generating substances toxic to the brain . however , the mechanism of action of cu and cho in ad incidence and development is poorly understood and requires further investigation . thus , the aim of this work was to study the effects of cu and cho association on the two main brain areas affected in ad , cortex and hippocampus , using a model of wistar rats . specifically , we aimed to determine for each nutritional supplement alone or in combination ( i ) the capacity to install oxidative / nitrative damage ; ( ii ) changes in the levels of the main antioxidant molecules ( glutathione and -tocopherol ) ; ( iii ) the possible development of a pro - inflammatory condition by analyzing the concentration of prostaglandins pge2 and pgf2 ; ( iv ) the activities of the two main protease systems associated with programmed cell death , caspase-3 and calpains ( - and m- ) ; and ( v ) possible changes in visuospatial memory as assessed by means of the barnes maze test . our findings could be useful in further investigating the mechanisms underlying the neurodegenerative process and also in localizing putative targets for preventive interventions associated with endogenous and/or exogenous causative factors such as cu and cho , alone or in combination . co. ( buenos aires , argentina or usa ) , merck ( darmstadt , germany ) , and carlo erba ( milan , italy ) . certified pathogen - free male wistar rats were used . the rats were maintained at a controlled temperature ( 25c ) and relative humidity of 60% with forced ventilation , under a normal photoperiod of 12 h darkness and 12 h light . the health of the animals was monitored in accordance with the internationally recommended practices of the ( institute of laboratory animal resources , commission of life sciences , national research council ) ilar . the diets for the experiments were prepared in our laboratory according to the recommendations for wistar rats . the experimental protocol was reviewed and approved by ( bioethics committee of the faculty of medical sciences , unlp ) cobimed under the code # 00382/11 . rats ( 21 days old ) were randomly assigned ( ten animals per group ) to the protocols detailed as follows and treated during eight weeks . the control group ( c ) was maintained on lab - prepared pellets as recommended for normal growth , containing 7 ppm of cu [ 28 , 29 ] . the cu - supplemented experimental group ( cu ) was fed on control pellets and tap water supplemented with 3 mg / l ( or ppm ) of cu in the form of ultrapure cuso4 ( merck , darmstadt , germany ) , the cho - supplemented group ( cho ) was fed on pellets containing 2% ( w / w ) of cho ( 87% pure ) ( obtained from saporiti srl , buenos aires , argentina ) , and the cu + cho - supplemented group ( cucho ) was simultaneously treated with cu in water + cho in food . rats were monitored during the experimental period to observe their behavior , quantify water , and food consumption and determine their body weight gain . total cu concentration in tap water supplemented with cuso4 was determined by means of atomic absorption methodology and was 3.42 0.21 ppm ( means of all daily measurements along the experimental period ) . considering that each animal imbibed between 4.9 0.4 and 15.0 1.1 ml water / day ( at the beginning and the end of the protocol , resp . ) , a maximum of 0.01 to 0.05 mg cu / day was acquired from water ( a dose equivalent to 0.06 and 0.18 mg cu / kg live animal , resp . ) . linear regression curves and anova test for cu content in food demonstrated that there were no significant variations between the 6 preparations used for the experiments ( 7.22 0.31 ppm or mg cu / kg diet ) . fe and zn content ( determined by atomic absorption spectrometry ) were the same in all preparations ( 45.9 1.8 and 66.6 2.0 ppm , resp . ) . ingestion of solid food along the experiments varied from 11.6 0.8 to 29.7 2.8 g / rat , implying that the oral ingestion of cu was in the range of 0.08 to 0.21 mg cu / day / rat ( 0.90 to 1.21 mg cu / kg live animal , a mean of 1.06 0.11 mg cu / kg ) . at the end of the treatments , animals were deeply anesthetized with ketamine ( 70 mg / kg ) and xylazine ( 5 mg / kg ) applied intramuscularly and then sacrificed by decapitation . brains were rapidly taken out and dissected in two zones , cortex and hippocampus , using the atlas of paxinos and watson as a guide for tissue dissection and a binocular stereoscopic arcano ztx-1065 microscope ( instrumental pasteur , buenos aires , argentina ) . both brain regions were washed , weighed , and homogenized using a buffer tris / hcl ( 10 mm ph 7.4 ) with sucrose ( 70 mm ) , mannitol ( 230 mm ) , ethylenediaminetetraacetic acid ( edta ) ( 1 mm ) , and dithiothreitol ( dtt ) ( 1 mm ) . aliquots of sample were digested with a mixture of 4 ml of hno3 ( c ) and 1 ml hclo4 ( aldrich or sigma chem . co. , buenos aires , argentina ) by heating at 120c for 80 min in a mineralization block . the digests were cooled , diluted with ultrapure water ( 18 m cm , carlo erba , milan , italy ) , and ultrafiltered by a 0.22 m millipore membrane ( milli - q purification system , from millipore , ca , usa ) . ultrafiltered dissolutions were directly aspirated into the flame of a perkin - elmer 1100 b spectrophotometer equipped with a perkin - elmer cathode lamp ( perkin - elmer corp . , norwalk , ct , usa ) at a spectral width of 1 nm . cu determinations were calibrated with a standard solution ( 200 ppm ) of cu ( no3)2 in hno3 0.5 n ( tritrisol from merck co. , darmstadt , germany ) . all measurements were carried out in peak height mode ( 324.7 nm line ) . the intra-[(sd / x)100 ] and inter-[(sd/x)100 ] assay coefficients of variation were 15.5 and 6.0% , respectively . we routinely obtained a similar equation for the calibration curve ( ir = 55.10 + 0.048[cu , mg / l ] ) , and the statistical analyses demonstrated a correlation coefficient always between 0.95 and 0.99 . in addition , we explored the so - called matrix effects that could have modified the slopes of the standard regressions . in spiked samples the obtained values varying from 48 to 60.10 were very similar to those of cu standard solutions , indicating that the matrix effect was not significant or was negligible . the mean for recovery and rsd for spiked samples was 99.7% and 3.3% , respectively , and the detection limit was 0.09 mg / l . in order to verify the accuracy of the method , we explored the influence of time after dilution , temperature of acid digestion , and concentration of hno3/hclo4 following the suggestions of terrs - martos et al . . we also checked our results with biological samples ( plasma and homogenates ) against a seronorm trace elements serum ( from sero labs , billingstad , norway ) and found no significant differences between the obtained and the declared ( certified ) concentrations . samples were analyzed by the enzyme conversion of p - phenylenediamine into a blue - colored product which was then measured at 550 nm . reaction proceeded at 37c in buffer glacial acetic / sodium acetate ( 50 mm , ph 5.5 ) directly into flat - bottomed plates , using a microplate reader spectramax m2/m2 model from molecular devices analytical technologies ( sunnyvale , ca , usa ) for 3 min . crp concentrations were calculated by comparison with the reaction rate of pure crp standard ( sigma chem . , we calculated the non - crp - bound cu ( ncbc , or so - called free cu ) as described by brewer by subtracting the amount of cu bound to each mg of crp from data of total cu . this parameter can be easily expressed in percentages using the formula ( ( [ cu]-47.2 [ crp ] ) 100/[cu ] ) , where cu is in mol / l and crp is in g / l . aliquots of this solution were taken to measure total cho by an enzymatic method using a commercial kit from wienner lab ( rosario , argentina ) . to estimate the amount of esterified cho ( echo ) , aliquots of the lipid extracts were seeded in high - resolution pre - coated silica gel plates ( 10 20 cm ) with a concentration zone for thin layer chromatography ( whatman adsorption 60 silica gel hp - tlc plates , ca , usa ) and developed with diethyl ether : hexane : acetic acid ( 90 : 4 : 1 , by vol ) as described elsewhere . authentic standards of cho and cho - esthers ( from avanti polar lipids , ontario , canada ) were run in parallel and revealed by iodine vapors . identified zones were scraped off the plates , eluted with folch reactive , evaporated , dissolved in 50 mm phosphate buffer ( ph 7.4 ) with 1% sodium deoxycholate , and enzymatically analyzed using the commercial kits from wienner lab ( rosario , argentina ) . thiobarbituric acid - reactive substances ( tbars ) were measured in brain homogenates as previously described . tbars ( mainly malondialdehyde , mda ) reacted with 2-thiobarbituric acid ( tba ) to yield tba - mda adducts which were quantified at 532 nm . the concentration of the chromophore was calculated from a calibration curve prepared with fresh tetramethoxypropane ( tmp ) solutions ( tmp was purchased from sigma chem . nitrate and nitrite [ nox ] concentration were measured using the method of griess on samples previously reduced with vanadium chlorohydrate according to miranda et al . . quantification was performed after calibration with standard solutions of sodium nitrate from merck co. ( darmstadt , germany ) . the concentrations of pcs were calculated from a calibration curve prepared with a stock solution of sodium pyruvate ( sigma chem . total glutathione was determined by the glutathione reductase / dithionitrobenzoic ( dtnb ) method that can measure both gsh and gssg . to calculate the gsh / gssg ratio , samples were reanalyzed after derivatization with divinylpyridine ( 3 mm final concentration ) . vitamin e ( -tocopherol ) was measured after extraction with the buttriss and diplock method using the hplc technique of bagnati et al . in a reverse phase / c-18 silica column from alltech associates , the econosil c18 column with a direct - connect cartridge guard column system was operated at a maximum pressure of 500 psi in a hitachi hplc system ( tokyo , japan ) . the amount of vitamin was electronically calculated using internal calibration with pure -tocopherol ( sigma , bs . as . ) , and the results were expressed in m concentration of -tocopherol . the specific activity of the enzyme was calculated for each sample in terms of u / minmg protein ( = 6.22 nmcm for absorbance at 340 nm ) . prostaglandin f2 ( pg f2 ) and prostaglandin e2 ( pge2 ) were measured using the pgf2 eia kit and pge2 express eia kit , respectively ( cayman , migliore laclaustra s.r ) . caspase-3 activity was measured by a colorimetric assay kit ( casp-3-c ) based on the hydrolysis of the synthetic peptide substrate acetyl - asp - glu - val - asp - p - nitroaniline ( ac - devd - pna ) by caspase-3 ( sigma chem . co. , buenos aires , argentina ) . the resulting p - nitroaniline ( p - na ) was monitored at 405 nm . each assay was run in parallel with inhibitor - treated homogenates ( to measure the nonspecific hydrolysis of the substrate ) and caspase-3 positive control ( using commercial caspase-3 , 5 mg / ml provided by the kit manufacturer ) . a calibration curve using a standard solution of p - nitroaniline ( p - na ) was also run for each assay to calculate the activity of the protease expressed as mol p - na released / minmg protein . co. , ca , usa ) by calpain activity and the subsequent detection of trichloroacetic acid ( tca ) soluble peptidic fragments at 280 nm . to select the activity of the calpain isoforms , the level of calcium in the medium was regulated ( 5 mm or 500 m of cacl2 for m- or -calpain , resp . ) . the activity of calpains was calculated considering that a unit of calpain is the amount of enzyme that produces a change of absorbance of 0.01 at 280 nm . beta amyloid peptides ( a ) ( 1 - 40 ) and ( 1 - 42 ) were measured using human / rat amyloid-40 elisa kit wako ii and the human amyloid-42 elisa kit wako high - sensitivity , respectively . the a(1 - 42)/(1 - 40 ) ratio was then calculated from the individual data expressed as picomole of the respective a peptide / mg total protein . the barnes maze is a black acrylic circular platform , 122 cm in diameter and elevated 108 cm off the floor , containing twenty holes around the periphery . the 10 cm diameter holes are uniform in appearance but , one hole is connected to an escape box , consisting of a 38.7 cm long 12.1 cm wide 14.2 depth cm removable box . four proximal visual cues ( 30 cm high , opaque black geometric figures : a cross , a circle , a square , and a triangle ) the escape box remained in a fixed position relative to the cues , to ensure randomization of the hole associated with the escape tunnel . in the center of the platform is a starting chamber ( an opaque , 26 cm diameter , 20 cm high , and white plastic open - ended cylinder ) . a 90 db white - noise generator and a white - light 500 w bulb provided motivation for escaping from the platform . the acquisition session and the probe trial session were performed on the same day . in brief , the experiment consisted of eight acquisition trials ( t1t8 ) followed by a single evaluation trial ( t9 ) . acquisition trials began with the animal inside the starting chamber for 30 seconds in the presence of a constant buzz . the chamber was then raised , the aversive stimulus ( intense bright light ) was switched on , and the rat was allowed to freely explore the maze . if by the end of this period they had not entered the escape box of their own accord , they were gently picked up and placed over the hole above the escape box . the evaluation trial proceeded in the same manner as described above but without the start box . at the end of each trial , the aversive stimulus was switched off , the rat remained on the escape box during 60 s , and the white light was switched off . in order to eliminate any possible olfactory clues from the maze , it was cleaned with 10% ethyl alcohol solution at the beginning of the 15 min intertrial period . an individual hole exploration was defined as being a single downward head deflection toward the inside of the hole . the following parameters were determined : ( i ) first - hole latency time ( in s ) spent by the animal between being released from the start box and exploring a hole in the maze for the first time ; ( ii ) escape - box latency time ( defined in the acquisition and retention test trials as the time ( in s ) spent by the animal between being released from the start box and entering the escape box and , in the case of the preference test and extinction trials , the time elapsed before the first exploration of the escape hole ) ; and ( iii ) nongoal hole exploration ( defined as the number of explorations of holes other than the escape hole , the explorations being considered as errors during the acquisition and probe trials ) . in the case of the evaluation trials , we evaluated the hole exploration frequency ( the number of explorations of each hole during the trial in which the escape hole was numbered as hole 0 for normalized graphical representation purposes , 1 to 10 clockwise , and 1 to 9 counterclockwise ) . the behavioral measurements were recorded using a video camera mounted 110 cm above the platform , linked to a computer . the video performances of each rat were analyzed using the video analysis software kinovea - creative commons attribution ( v 0.7.6 ) . all values represent the mean of 6 rats assayed in triplicate expressed as mean standard deviation ( sd ) . data were analyzed by anova plus tukey test with the aid of spss 11.0.1 software ( spss inc . , chicago , il ) . to analyze the data from the barnes maze test , multiple comparisons were drawn with the control group using two - way anova plus the holm - sidak post hoc test at two levels of significance ( p < 0.05 and 0.01 ) . results were also plotted and analyzed using sigma scientific graphing software ( version 11.0 ) from sigma chem . the statistical significance of differences is indicated by distinct superscript letters ( data with distinct superscript letters are statistically different ( p < 0.01 ) between them ) . table 1 shows the levels of total and free cu ( ncbc or nonceruloplasmin - bound cu ) in plasma and in brain cortex and hippocampus after the experimental treatments . plasma total cu and ncbc exhibited discrete ( but statistically significant ) increases in those groups receiving cu ( cu and cucho ) . total cu also increased in both cortex and hippocampus after cu supplementation ( alone or in combination with cho ) . cortex shows a nonsignificant tendency towards higher ncbc after cu or cucho treatments , whereas hippocampus shows a significant increase in this parameter with respect to the control group . the results of the cho analysis ( free and esterified ) are shown in table 2 , from which it is evident that tissue from the central nervous system has a particular cho metabolism characterized by higher levels of free cho ( 25 times higher than in the case of plasma ) . cu addition to drinking water produced no significant changes in plasma total cho and a slight increase in cortex and hippocampus at the expense of the esterified form ( echo ) . furthermore , cu did not change the ratio between free and echo in plasma samples but did lower this parameter in the brain homogenates . supplementing food with cho caused a higher level of total cho in peripheral plasma ( from 27.0 0.5 in the control group to 33.7 0.6 nmoles / mg protein in cho - supplemented rats ) with a slight but significant decrease in the proportion of free / echo . in brain , all the treatments led to an increase in total ( free + esterified ) cho with respect to control data , though the increase was substantially higher and differentially orchestrated in those groups receiving cho supplementation . in the case of cho - treated rats , this occurred at the expense of free cho ; however , in rats under cucho treatment , it was at the expense of the echo form . under cucho treatment , hippocampus reached even higher values of total cho than cortex ( approx . addition of cu alone significantly elevated the echo in both brain tissues , more noticeably in hippocampus than in cortex . another differential result for the cucho group was a decrease in free cho with respect to control rats in cortex , a phenomenon not observed in hippocampus . the results obtained for the ratios between free and echo strongly suggest that cu addition was able to modify the balance of these two parameters , triggering the accumulation of echo in cortex and hippocampus , perhaps facilitating its esterification or impeding its degradation , or both . this effect is especially noticeable under conditions of simultaneous cu and cho overload and , even more interestingly , was contrary to the effect observed in peripheral plasma samples . in order to detect whether inorganic cu in drinking water and cho in food produced oxidative / nitrative stress , we measured the oxidation end products of lipids ( tbars ) and proteins ( pcs ) and also the levels of nox ( nitrates and nitrites derived from the spontaneous dismutation of nitric oxide ) as biomarkers of damage . levels of the three markers analyzed were higher after cucho treatment both in plasma and in brain cortex and hippocampus ( figures 1(a ) and 1(b ) ) . in plasma , treatment with cu alone increased tbars levels ( figure 1(a ) ) , whereas all three biomarkers were significantly increased after cucho cosupplementation . brain homogenates exhibited similar results , with increased levels of all three biomarkers after cu or cucho supplementation ( with the solo exception of pcs in both brain regions , which increased only with simultaneous exposure to cu and cho ) . we also measured the levels of the two main antioxidant molecules for water- and lipid - soluble cellular compartments ( total glutathione - gsh+gssg- and -tocopherol , resp . ) plasma levels of total glutathione were higher in rats fed on cu plus cho supplements . in the same experimental group ( cucho ) , the -tocopherol concentration was 27% lower than in the control data . in brain cortex , we also observed an increase in total glutathione after cucho treatments and a simultaneous increment in the gssg / gsh ratio as a consequence of a higher level of gssg . concomitantly , the activity of glutathione reductase ( gr ) in cortex was enhanced by cu supplementation and even more so by simultaneous treatment with cu + cho . the level of -tocopherol ( -toc ) decreased significantly only after cucho treatment ( approx . the behavior was very similar , with the exception of the increment in total glutathion content . thus , hippocampus homogenates showed a significant increase in the gssg / gsh ratio , activation of gr in cu- and cucho - treated rats , and a ca . 34% decrease in -toc content compared to control data , with this latter being observed only in the cucho experimental group . in order to evaluate whether simultaneous supplementation with cu and cho also produced an inflammatory condition , we analyzed the levels of two proinflammatory prostaglandins , pge2 and pgf2 ( figure 2 ) . interestingly , cu and cho alone also increased prostaglandins levels with respect to control data , and association of the two supplements produced an additive effect . we also explored whether the prooxidative and proinflammatory environment developed after cucho treatment was able to trigger apoptotic signals , to which end we determined the activities of the two main protease systems involved in programmed cell death , caspase-3 , and calpains ( - and m- ) . caspase-3 activity tends to increase at least in cortex homogenates , but not to a statistically significant degree ( figure 3 ) . both calpain ( milli- and microisoforms ) activities increased after cu treatment and even more so after cucho supplementation in both brain zones ( figure 4 ) . the concentration of the -amyloid peptides ( 1 - 42 and 1 - 40 ) and the a 1 - 42/1 - 40 ratio in cortex and hippocampus are shown in table 4 . the ratio ( which is the main indicator of neurodegenerative process ) was different , depending on the brain zone examined . in cortex , it was found to increase after cu and cho treatments and to further increase after cucho supplementation . in hippocampus , there were no statistically significant changes of the ratio ( 1 - 42)/(1 - 40 ) in peripheral plasma . we also investigated possible alterations in the visuospatial learning capabilities through the barnes maze test . we observed minor ( not statistically different ) changes in latency to the first hole and more spatial preference for the escape region ( holes 1 , 0 , and 1 ) regardless of treatment ( data not shown ) . taken together , these changes demonstrate minor alterations in visuospatial memory suggesting that simultaneous supplementation with cu and cho produces an increment in exploratory activity or a sort of overexcited behavior but with similar final results to those observed in the other experimental groups . supplementation of drinking water with low amounts of inorganic cu such as those used in our experiments was able to modify the basal status of redox biomarkers not only in peripheral plasma but also in the two zones of the central nervous system explored , cortex and hippocampus . because of their short life span , free radicals can not be measured directly , for which reason it is usual to analyze the products arising from reactions caused by reactive species with biomolecules . in line with this , we measured the end - oxidation products of lipids ( tbars ) and proteins ( pcs ) and the production of nox as biomarkers of prooxidative tissue damage . pcs increased only after cucho treatment ; however , tbars and nox increased after a sole exposure to trace amounts of inorganic cu in drinking water . many authors have associated neurodegenerative illnesses with both a local ( brain ) and a systemic ( plasma ) increase in oxidative stress [ 4552 ] . irrespective of whether biomarkers are causative factors or whether they merely constitute phenomenologically associated changes , they are useful tools for evaluating the extent of damage and provide a simple methodology for monitoring large populations subjected to environmental cu pollution or exposed to other risks associated with the development of ad . there is abundant evidence that transition metals in general , and specially cu , are causative factors for oxidative stress and , as we mentioned previously , are strongly associated with the neurodegenerative process [ 5356 ] . the decrease of -tocopherol in plasma and brain zones homogenates and the increased gssg / gsh ratio are both markers of accumulation of reactive species in lipid- and water - soluble cell compartments . the activation of gr can be interpreted as a compensatory mechanism of the enzymatic antioxidant defense system in order to normalize the altered gssg / gsh ratio induced by cotreatment with cu and cho . in agreement with our results , kojima et al . have demonstrated the induction of mrna coding for gr in the brain of mice irradiated with a low dose of -rays . also in line with this , we can speculate that the observed increase in plasma total glutathione levels may be due to a compensatory mechanism under the oxidative insult evoked by the treatment studied here . this explanation is in agreement with the suggestions of other authors and may be the consequence of an induction ( overexpression ) of cysteinyl - synthetase , the enzyme that controls the biosynthesis of glutathione [ 59 , 60 ] . the oxidative stress induced by ncbc could have multiple effects on the signals cascade that depends on the redox state and can also modify the activity of ionic channels , transporters , and enzymes . . observed , in liver of wistar rats treated with thioacetamide , that hmgcoa - reductase overactivation was strictly related to the magnitude of the reactive species accumulated . our results demonstrate that inorganic cu supplementation even at the low levels assayed produced an increase in echo levels in cortex and hippocampus . attributing the increase in cho in brain to oxidative stress - induced hmgcoa - reductase hyperactivity could be oversimplistic , and this intriguing question remains to be resolved on the basis of new experimental evidence . cho in the nervous system ( 10-fold higher than in any other organ ) is mainly unesterified [ 62 , 63 ] . furthermore , most of the cho content in brain depends on the in situ biosynthesis that appears to be regulated by similar mechanisms both outside and inside the brain , with hmgcoa - reductase being the most important regulatory effector . however , the exact extent of cho biosynthesis in neurons and astrocytes in vivo remains unknown , making it difficult to estimate the real effect of the accumulation of reactive species ( ncbc - induced ) on brain cho biosynthesis . interestingly , cho turnover in individual neurons and astrocytes may in fact be very high and reach an estimated 20% per day , depending on the brain zone . unfortunately , the direct effect of higher cho levels in blood on cho concentration in brain is difficult to assess . cho trafficking between brain and peripheral blood implies the participation of the blood - brain barrier ( bbb ) that hinders the direct passage into or out of the central nervous system . however , for reasons not yet understood , this restriction appears to be reduced in ad and other neurodegenerative disorders . also , in certain circumstances , for example , in cases of vascular injury due to oxidative stress , the bbb could be permeable to interaction with the peripheral pool of cho . moreover , recent experimental evidence in a mouse model of ad demonstrated that inflammation is one of the key factors in determining increased bbb vulnerability . thus , from the above , we can assume that peripheral cho might cross the bbb into the brain , in particular when the tissue is subjected to oxidative stress and redox - induced inflammation as observed in our experimental model . despite extensive research in recent years , the role of cho as a risk factor for ad remains controversial , likely due to the still unresolved questions relating to the exact role of peripheral cho in its level in brain . we can speculate that ncbc induces nitrative / oxidative and pro - inflammatory conditions that probably facilitate endothelial damage and indirectly modify bbb properties [ 64 , 65 ] , allowing peripheral cho to enter the central nervous system . obviously , a great deal of experimental work is still required to either confirm or refute this working hypothesis . several unresolved issues raise doubts concerning the beneficial effects of statins in neurodegenerative patients and the notion that high blood cho is associated with brain dysfunction . however , one possibility is that the real risk is the association of hypercholesterolemia with a prooxidative and pro - inflammatory environment induced , for example , by ncbc . brain cho is involved in synaptogenesis , the turnover , maintenance , stabilization , and restoration of synapses . the functionality of synapses requires specific lipid domains in neuronal and axon membranes whose composition is critical for the correct targeting of the major membrane proteins , myelin biogenesis , cellular differentiation , signal transduction , and many other functions that depend on microdomains and specific lipid rafts . the proportion of free cho to echo in these membrane domains is a crucial factor in their biological activities . for example , the enzymes responsible for the processing of the amyloid precursor protein ( app ) to a ( - and -secretases ) reside in cho - rich lipid rafts of plasma membrane . it was suggested that a higher total cho / phospholipid ratio in cellular membranes could affect secretase activities and determine preferential app processing pathways , though in vitro studies suggest that cho might impair the transcription of app and consequently decrease the availability for its conversion to a . however , it appears that this effect has no significant impact on the amount of protein ( that still exceeds the capacity of the secretase system to process it ) and only a slight impact on the levels of the mrna encoding app protein . to date , there are no findings elucidating the exact role of echo and how the ratio of free to esterified forms is able to modify secretase activity and other process associated with the amyloidogenic cascade . . suggest that different sources of oxidative stress , such as ncbc , could trigger the amyloidogenic pathway , which may explain the higher a 1 - 42/1 - 40 ratio we observed in the brain cortex of cu - treated rats and in the group receiving both treatments ( cucho ) . the question as to how cho and cu interact to lower the production of a and enhance oxidative stress and inflammation is difficult to address . experimental evidence obtained in culture cells demonstrates that exposure of macrophages to cuso4 , at a level equivalent to ncbc in humans , induces srebp-2 and consequently the expression of cholesterogenic enzymes , thus tentatively providing a new explanation for the apparently additive effect we observed between cu and cho . however , we were unable to find similar evidence in neuron or astrocyte cultures , or even in experiments conducted on live animals to explore these findings . there is yet another possibility : that the association of the two supplements ( cu and cho ) may affect the clearance of a and facilitate its accumulation . working with rabbits fed on a diet with excess cho and inorganic cu in their drinking water , sparks et al . proposed that cho caused alterations in the bbb associated with an inflammatory condition and a concomitant overproduction of a in the brain . in this model , cu decreased the clearance of a to the blood via inhibition of lrp at the vascular interface . impaired cho metabolism , oxidative stress , and inflammation were all factors associated with the decreased clearance of the a peptide [ 63 , 69 ] . in addition , lu et al . also demonstrated that cu exacerbates a amyloid - induced neurotoxicity through a tnf - mediated inflammatory pathway . though the exact mechanism(s ) underlying all these effects is still unknown , it is nevertheless widely accepted that the ad pathological cascade is likely to be a 2-stage event where deposition of a and neuronal pathology ( tauopathy , neuronal injury , and programmed cell death , or subsequent neurodegeneration with synapse and cognitive loss ) are sequential rather than simultaneous processes [ 70 , 71 ] . our model likely represents a very early step in these successive events since the screening of the damages observed in visuospatial memory revealed only slight modifications . the barnes maze test analysis demonstrated that all groups display almost normal locomotor and exploratory activities and spatial memory retention . the behavioral modifications indicate that the animals of the control and cho groups were fully able to acquire the necessary knowledge for the spatial task through training , whereas the cu and cucho groups were only partially able to do so or presented slight learning difficulties . interestingly , these learning difficulties were more evident during the evaluation trials of cho animals , which explored holes very distant from the escape - hole region . nevertheless , cho and cucho animals both showed a minor degree ( not statistically significant ) of deficit in learning and spatial memory capabilities . plasma levels of a , particularly the a 1 - 40/a 1 - 42 ratio , are well - recognized biomarkers of sporadic ad [ 72 , 73 ] and even indicators of early stages of the pathology . however , apart from its role as a biomarker , accumulation of a peptide in brain is a complex phenomenon with multiple and consecutive ( sometimes unexpected ) consequences . in agreement with this , tamagno et al . demonstrated that oxidative stress induced by a2535 resulted in an early , significant , and time - dependent generation of free hne ( hydroxyl - nonenal ) and h2o2 . also , other authors reported increased levels of oxidative stress biomarkers after a exposure both in vivo and in vitro [ 7779 ] . our results are therefore in agreement with those of other groups reporting that oxidative stress and a are linked to one another . apparently , a can induce oxidative stress [ 80 , 81 ] , and pro - oxidants such as ncbc can increase a production [ 8284 ] in the manner of an autostimulated process . the increased levels of the two main proinflammatory prostaglandins ( e2 and f2 ) are consistent with the inflammatory condition characteristic of ad . this pro - inflammatory and prooxidative environment triggers the activation of calpains , whereas caspase-3 activity was not significantly stimulated under our experimental conditions . in previous papers , we also found such dissociation between the effects of inorganic cu overload on the relative activities of the two protease systems , which at least in vitro experiments appear to depend on the extent and intensity of exposure to cu overload . however , in view of previous experimental evidence , we can not conclude that neuronal death is actually occurring . have reported that the activation of -calpains in ad brain is not necessarily a consequence of the endstage of neuronal degeneration and may reflect a more widespread metabolic alteration that precedes and contributes to neuronal death . in fact , they observed increased activity of -calpains in the cerebellum of ad without any increase in the rate of death neurons . in line with this , nixon established that different factors could lead to calpain activation triggering neurodegeneration in the early stages of ad development . moreover , trinchese et al . also reported that calpains have many substrates that could be affected in ad patients but do not necessarily lead to immediate cell death . they also stated that -calpains are present predominantly in synapses , which is in agreement both with the fact that cu concentration is particularly high ( micromolar ) in the synaptic cleft and with the well - established synaptic pathology in ad [ 90 , 91 ] . nixon suggested that increased activity of calpains during normal aging may also promote the development of neurological disorders and impaired calcium homeostasis , both of which could impact on the role of this cation in the function of cellular membrane receptors and metallosignaling in brain . finally , in discussing the validity and/or limitations of our experimental system , it is necessary to consider the level of the supplementation with cu using oral administration . our experimental conditions were based on previous work [ 13 , 14 ] and resemble the cu levels commonly found as a consequence of involuntary exposure through air , food and water pollution [ 16 , 9294 ] , ingestion of dietary mineral supplements , exposure of professionals engaged in agrochemical activities [ 6 , 11 , 95 ] , neurodegenerative patients and their first - degree relatives , or female users of cu - based intrauterine devices [ 17 , 19 ] . studies performed in rats demonstrated that cu metabolism and homeostasis are essentially identical to those in humans . in terms of dosage , the rats from the groups supplemented with cu received 1.06 0.11 mg cu / kg / day ( including cu acquire from food and water ingestion ) . from the available data in humans , we can assume that general population are receiving 0.16 to 0.98 ear or rda which is equivalent to 0.3 to 1.5 mg cu / kg body weight . very probably , humans are exposed to several types of cu - based compounds of different chemical structures with differences in their physical stabilities , solubility , absorption capacities , life 's times into the organism , and many other particularities related to their excretion or bioaccumulation rates . however , only inorganic cu should be dangerous for its probable role as causative factor for neurodegeneration [ 3 , 6 , 810 ] . thus , we think that there are a lot of questions to be answered before drawing a realistic conclusion about the comparisons between our experimental conditions and the actual human expose to cu . in conclusion , this in vivo study reveals that the association of inorganic cu and cho gives rise to a prooxidative and proinflammatory environment more pronounced than that produced by cu and cho administered alone . as described before , the combination of these two factors is common in many human populations . we suggest that the biochemical changes observed , in particular , oxidative stress , inflammation , and the higher a 1 - 42/1 - 40 ratio in the cortex of rats fed on cu + cho ( cucho ) , could constitute the initial stages of the development of neurodegenerative disease . in view of the abundant evidence of disturbed cu homeostasis in ad [ 7 , 56 , 98 , 99 ] , we strongly recommend more in - depth studies on the mechanism(s ) responsible for the pro - neurodegenerative effect(s ) of the association between ncbc and cho . furthermore , it is recommended that the present experimental evidence be used to promote the investigation of the emerging biomarkers such as those examined in this work to be applied in peripheral plasma as predictive tool(s ) in high - risk populations .

age is one of the main factors involved in the development of neurological illnesses , in particular , alzheimer , and it is widely held that the rapid aging of the world population is accompanied by a rise in the prevalence and incidence of alzheimer disease . however , evidence from recent decades indicates that cu and cho overload are emerging causative factors in neurodegeneration , a hypothesis that has been partially investigated in experimental models . the link between these two variables and the onset of alzheimer disease has opened up interesting new possibilities requiring more in - depth analysis . the aim of the present study was therefore to investigate the effect of the association of cu + cho ( cucho ) as a possible synergistic factor in the development of an alzheimer - like pathology in wistar rats . we measured total- and nonceruloplasmin - bound cu and cho ( free and sterified ) contents in plasma and brain zones ( cortex and hippocampus ) , markers of oxidative stress damage , inflammation , and programmed cell death ( caspase-3 and calpain isoforms ) . the ratio beta - amyloid ( 1 - 42)/(1 - 40 ) was determined in plasma and brain as neurodegenerative biomarker . an evaluation of visuospatial memory ( barnes maze test ) was also performed . the results demonstrate the establishment of a prooxidative and proinflammatory environment after cucho treatment , hallmarked by increased tbars , protein carbonyls , and nitrite plus nitrate levels in plasma and brain zones ( cortex and hippocampus ) with a consequent increase in the activity of calpains and no significant changes in caspase-3 . a simultaneous increase in the plasma a1 - 42/a1 - 40 ratio was found . furthermore , a slight but noticeable change in visuospatial memory was observed in rats treated with cucho . we conclude that our model could reflect an initial stage of neurodegeneration in which cu and cho interact with one another to exacerbate neurological damage .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusions

the aging of the world population is being accompanied by a rise in the prevalence and incidence of alzheimer disease ( ad ) and other neurodegenerative illnesses . epidemiologic data indicates that the world population will have grown considerably by 2025 and the percentage of elderly people will be significantly higher . since age is one of the major risk factors in the development of ad , it is expected that the incidence of this disease will also increase . however , experimental evidence detailed in a review by brewer indicates that other factors in addition to age could play a critical role in the development of diseases such as alzheimer and that the contribution of inorganic copper ( cu ) has been underestimated . data from squitti 's group specifically demonstrated that free cu ( also known as ncbc or nonceruloplasmin - bound cu ) is elevated in the blood of ad patients , negatively correlates with cognition , and predicts the rate of loss of cognition [ 810 ] . very recently , brewer has reviewed the theory of inorganic cu toxicity in alzheimer disease as a causative factor in cognitive loss . however , the regulatory framework for chronic cu exposure in large human populations indicates that pollution , drinking water , and dietary cu - containing supplements are the main sources of exposure [ 6 , 16 ] . in accumulated data on 280 samples of household drinking water all across north america , 72% of the samples have cu levels above those enhancing ad in experimental models . furthermore , we and others have demonstrated significantly higher cu levels in plasma of women using cu - iuds and in blood samples from farmers working with cu - based pesticides [ 1719 ] . experimental evidence obtained from rabbit and mouse models suggests that the association of cu and cho can be risk factor for ad development [ 1214 ] . however , the mechanism of action of cu and cho in ad incidence and development is poorly understood and requires further investigation . thus , the aim of this work was to study the effects of cu and cho association on the two main brain areas affected in ad , cortex and hippocampus , using a model of wistar rats . specifically , we aimed to determine for each nutritional supplement alone or in combination ( i ) the capacity to install oxidative / nitrative damage ; ( ii ) changes in the levels of the main antioxidant molecules ( glutathione and -tocopherol ) ; ( iii ) the possible development of a pro - inflammatory condition by analyzing the concentration of prostaglandins pge2 and pgf2 ; ( iv ) the activities of the two main protease systems associated with programmed cell death , caspase-3 and calpains ( - and m- ) ; and ( v ) possible changes in visuospatial memory as assessed by means of the barnes maze test . our findings could be useful in further investigating the mechanisms underlying the neurodegenerative process and also in localizing putative targets for preventive interventions associated with endogenous and/or exogenous causative factors such as cu and cho , alone or in combination . the cu - supplemented experimental group ( cu ) was fed on control pellets and tap water supplemented with 3 mg / l ( or ppm ) of cu in the form of ultrapure cuso4 ( merck , darmstadt , germany ) , the cho - supplemented group ( cho ) was fed on pellets containing 2% ( w / w ) of cho ( 87% pure ) ( obtained from saporiti srl , buenos aires , argentina ) , and the cu + cho - supplemented group ( cucho ) was simultaneously treated with cu in water + cho in food . ingestion of solid food along the experiments varied from 11.6 0.8 to 29.7 2.8 g / rat , implying that the oral ingestion of cu was in the range of 0.08 to 0.21 mg cu / day / rat ( 0.90 to 1.21 mg cu / kg live animal , a mean of 1.06 0.11 mg cu / kg ) . brains were rapidly taken out and dissected in two zones , cortex and hippocampus , using the atlas of paxinos and watson as a guide for tissue dissection and a binocular stereoscopic arcano ztx-1065 microscope ( instrumental pasteur , buenos aires , argentina ) . both brain regions were washed , weighed , and homogenized using a buffer tris / hcl ( 10 mm ph 7.4 ) with sucrose ( 70 mm ) , mannitol ( 230 mm ) , ethylenediaminetetraacetic acid ( edta ) ( 1 mm ) , and dithiothreitol ( dtt ) ( 1 mm ) . the digests were cooled , diluted with ultrapure water ( 18 m cm , carlo erba , milan , italy ) , and ultrafiltered by a 0.22 m millipore membrane ( milli - q purification system , from millipore , ca , usa ) . we routinely obtained a similar equation for the calibration curve ( ir = 55.10 + 0.048[cu , mg / l ] ) , and the statistical analyses demonstrated a correlation coefficient always between 0.95 and 0.99 . we also checked our results with biological samples ( plasma and homogenates ) against a seronorm trace elements serum ( from sero labs , billingstad , norway ) and found no significant differences between the obtained and the declared ( certified ) concentrations . , we calculated the non - crp - bound cu ( ncbc , or so - called free cu ) as described by brewer by subtracting the amount of cu bound to each mg of crp from data of total cu . to estimate the amount of esterified cho ( echo ) , aliquots of the lipid extracts were seeded in high - resolution pre - coated silica gel plates ( 10 20 cm ) with a concentration zone for thin layer chromatography ( whatman adsorption 60 silica gel hp - tlc plates , ca , usa ) and developed with diethyl ether : hexane : acetic acid ( 90 : 4 : 1 , by vol ) as described elsewhere . a calibration curve using a standard solution of p - nitroaniline ( p - na ) was also run for each assay to calculate the activity of the protease expressed as mol p - na released / minmg protein . to select the activity of the calpain isoforms , the level of calcium in the medium was regulated ( 5 mm or 500 m of cacl2 for m- or -calpain , resp . ) the activity of calpains was calculated considering that a unit of calpain is the amount of enzyme that produces a change of absorbance of 0.01 at 280 nm . beta amyloid peptides ( a ) ( 1 - 40 ) and ( 1 - 42 ) were measured using human / rat amyloid-40 elisa kit wako ii and the human amyloid-42 elisa kit wako high - sensitivity , respectively . the a(1 - 42)/(1 - 40 ) ratio was then calculated from the individual data expressed as picomole of the respective a peptide / mg total protein . four proximal visual cues ( 30 cm high , opaque black geometric figures : a cross , a circle , a square , and a triangle ) the escape box remained in a fixed position relative to the cues , to ensure randomization of the hole associated with the escape tunnel . the chamber was then raised , the aversive stimulus ( intense bright light ) was switched on , and the rat was allowed to freely explore the maze . the following parameters were determined : ( i ) first - hole latency time ( in s ) spent by the animal between being released from the start box and exploring a hole in the maze for the first time ; ( ii ) escape - box latency time ( defined in the acquisition and retention test trials as the time ( in s ) spent by the animal between being released from the start box and entering the escape box and , in the case of the preference test and extinction trials , the time elapsed before the first exploration of the escape hole ) ; and ( iii ) nongoal hole exploration ( defined as the number of explorations of holes other than the escape hole , the explorations being considered as errors during the acquisition and probe trials ) . in the case of the evaluation trials , we evaluated the hole exploration frequency ( the number of explorations of each hole during the trial in which the escape hole was numbered as hole 0 for normalized graphical representation purposes , 1 to 10 clockwise , and 1 to 9 counterclockwise ) . table 1 shows the levels of total and free cu ( ncbc or nonceruloplasmin - bound cu ) in plasma and in brain cortex and hippocampus after the experimental treatments . the results of the cho analysis ( free and esterified ) are shown in table 2 , from which it is evident that tissue from the central nervous system has a particular cho metabolism characterized by higher levels of free cho ( 25 times higher than in the case of plasma ) . cu addition to drinking water produced no significant changes in plasma total cho and a slight increase in cortex and hippocampus at the expense of the esterified form ( echo ) . furthermore , cu did not change the ratio between free and echo in plasma samples but did lower this parameter in the brain homogenates . supplementing food with cho caused a higher level of total cho in peripheral plasma ( from 27.0 0.5 in the control group to 33.7 0.6 nmoles / mg protein in cho - supplemented rats ) with a slight but significant decrease in the proportion of free / echo . in the case of cho - treated rats , this occurred at the expense of free cho ; however , in rats under cucho treatment , it was at the expense of the echo form . the results obtained for the ratios between free and echo strongly suggest that cu addition was able to modify the balance of these two parameters , triggering the accumulation of echo in cortex and hippocampus , perhaps facilitating its esterification or impeding its degradation , or both . this effect is especially noticeable under conditions of simultaneous cu and cho overload and , even more interestingly , was contrary to the effect observed in peripheral plasma samples . in order to detect whether inorganic cu in drinking water and cho in food produced oxidative / nitrative stress , we measured the oxidation end products of lipids ( tbars ) and proteins ( pcs ) and also the levels of nox ( nitrates and nitrites derived from the spontaneous dismutation of nitric oxide ) as biomarkers of damage . levels of the three markers analyzed were higher after cucho treatment both in plasma and in brain cortex and hippocampus ( figures 1(a ) and 1(b ) ) . in plasma , treatment with cu alone increased tbars levels ( figure 1(a ) ) , whereas all three biomarkers were significantly increased after cucho cosupplementation . in the same experimental group ( cucho ) , the -tocopherol concentration was 27% lower than in the control data . in brain cortex , we also observed an increase in total glutathione after cucho treatments and a simultaneous increment in the gssg / gsh ratio as a consequence of a higher level of gssg . concomitantly , the activity of glutathione reductase ( gr ) in cortex was enhanced by cu supplementation and even more so by simultaneous treatment with cu + cho . thus , hippocampus homogenates showed a significant increase in the gssg / gsh ratio , activation of gr in cu- and cucho - treated rats , and a ca . interestingly , cu and cho alone also increased prostaglandins levels with respect to control data , and association of the two supplements produced an additive effect . we also explored whether the prooxidative and proinflammatory environment developed after cucho treatment was able to trigger apoptotic signals , to which end we determined the activities of the two main protease systems involved in programmed cell death , caspase-3 , and calpains ( - and m- ) . both calpain ( milli- and microisoforms ) activities increased after cu treatment and even more so after cucho supplementation in both brain zones ( figure 4 ) . the concentration of the -amyloid peptides ( 1 - 42 and 1 - 40 ) and the a 1 - 42/1 - 40 ratio in cortex and hippocampus are shown in table 4 . in cortex , it was found to increase after cu and cho treatments and to further increase after cucho supplementation . in hippocampus , there were no statistically significant changes of the ratio ( 1 - 42)/(1 - 40 ) in peripheral plasma . we also investigated possible alterations in the visuospatial learning capabilities through the barnes maze test . taken together , these changes demonstrate minor alterations in visuospatial memory suggesting that simultaneous supplementation with cu and cho produces an increment in exploratory activity or a sort of overexcited behavior but with similar final results to those observed in the other experimental groups . supplementation of drinking water with low amounts of inorganic cu such as those used in our experiments was able to modify the basal status of redox biomarkers not only in peripheral plasma but also in the two zones of the central nervous system explored , cortex and hippocampus . pcs increased only after cucho treatment ; however , tbars and nox increased after a sole exposure to trace amounts of inorganic cu in drinking water . irrespective of whether biomarkers are causative factors or whether they merely constitute phenomenologically associated changes , they are useful tools for evaluating the extent of damage and provide a simple methodology for monitoring large populations subjected to environmental cu pollution or exposed to other risks associated with the development of ad . there is abundant evidence that transition metals in general , and specially cu , are causative factors for oxidative stress and , as we mentioned previously , are strongly associated with the neurodegenerative process [ 5356 ] . the decrease of -tocopherol in plasma and brain zones homogenates and the increased gssg / gsh ratio are both markers of accumulation of reactive species in lipid- and water - soluble cell compartments . the activation of gr can be interpreted as a compensatory mechanism of the enzymatic antioxidant defense system in order to normalize the altered gssg / gsh ratio induced by cotreatment with cu and cho . also in line with this , we can speculate that the observed increase in plasma total glutathione levels may be due to a compensatory mechanism under the oxidative insult evoked by the treatment studied here . the oxidative stress induced by ncbc could have multiple effects on the signals cascade that depends on the redox state and can also modify the activity of ionic channels , transporters , and enzymes . observed , in liver of wistar rats treated with thioacetamide , that hmgcoa - reductase overactivation was strictly related to the magnitude of the reactive species accumulated . our results demonstrate that inorganic cu supplementation even at the low levels assayed produced an increase in echo levels in cortex and hippocampus . attributing the increase in cho in brain to oxidative stress - induced hmgcoa - reductase hyperactivity could be oversimplistic , and this intriguing question remains to be resolved on the basis of new experimental evidence . however , the exact extent of cho biosynthesis in neurons and astrocytes in vivo remains unknown , making it difficult to estimate the real effect of the accumulation of reactive species ( ncbc - induced ) on brain cho biosynthesis . moreover , recent experimental evidence in a mouse model of ad demonstrated that inflammation is one of the key factors in determining increased bbb vulnerability . thus , from the above , we can assume that peripheral cho might cross the bbb into the brain , in particular when the tissue is subjected to oxidative stress and redox - induced inflammation as observed in our experimental model . however , one possibility is that the real risk is the association of hypercholesterolemia with a prooxidative and pro - inflammatory environment induced , for example , by ncbc . however , it appears that this effect has no significant impact on the amount of protein ( that still exceeds the capacity of the secretase system to process it ) and only a slight impact on the levels of the mrna encoding app protein . suggest that different sources of oxidative stress , such as ncbc , could trigger the amyloidogenic pathway , which may explain the higher a 1 - 42/1 - 40 ratio we observed in the brain cortex of cu - treated rats and in the group receiving both treatments ( cucho ) . there is yet another possibility : that the association of the two supplements ( cu and cho ) may affect the clearance of a and facilitate its accumulation . impaired cho metabolism , oxidative stress , and inflammation were all factors associated with the decreased clearance of the a peptide [ 63 , 69 ] . though the exact mechanism(s ) underlying all these effects is still unknown , it is nevertheless widely accepted that the ad pathological cascade is likely to be a 2-stage event where deposition of a and neuronal pathology ( tauopathy , neuronal injury , and programmed cell death , or subsequent neurodegeneration with synapse and cognitive loss ) are sequential rather than simultaneous processes [ 70 , 71 ] . our model likely represents a very early step in these successive events since the screening of the damages observed in visuospatial memory revealed only slight modifications . the behavioral modifications indicate that the animals of the control and cho groups were fully able to acquire the necessary knowledge for the spatial task through training , whereas the cu and cucho groups were only partially able to do so or presented slight learning difficulties . plasma levels of a , particularly the a 1 - 40/a 1 - 42 ratio , are well - recognized biomarkers of sporadic ad [ 72 , 73 ] and even indicators of early stages of the pathology . however , apart from its role as a biomarker , accumulation of a peptide in brain is a complex phenomenon with multiple and consecutive ( sometimes unexpected ) consequences . apparently , a can induce oxidative stress [ 80 , 81 ] , and pro - oxidants such as ncbc can increase a production [ 8284 ] in the manner of an autostimulated process . in fact , they observed increased activity of -calpains in the cerebellum of ad without any increase in the rate of death neurons . they also stated that -calpains are present predominantly in synapses , which is in agreement both with the fact that cu concentration is particularly high ( micromolar ) in the synaptic cleft and with the well - established synaptic pathology in ad [ 90 , 91 ] . nixon suggested that increased activity of calpains during normal aging may also promote the development of neurological disorders and impaired calcium homeostasis , both of which could impact on the role of this cation in the function of cellular membrane receptors and metallosignaling in brain . in conclusion , this in vivo study reveals that the association of inorganic cu and cho gives rise to a prooxidative and proinflammatory environment more pronounced than that produced by cu and cho administered alone . we suggest that the biochemical changes observed , in particular , oxidative stress , inflammation , and the higher a 1 - 42/1 - 40 ratio in the cortex of rats fed on cu + cho ( cucho ) , could constitute the initial stages of the development of neurodegenerative disease . in view of the abundant evidence of disturbed cu homeostasis in ad [ 7 , 56 , 98 , 99 ] , we strongly recommend more in - depth studies on the mechanism(s ) responsible for the pro - neurodegenerative effect(s ) of the association between ncbc and cho . furthermore , it is recommended that the present experimental evidence be used to promote the investigation of the emerging biomarkers such as those examined in this work to be applied in peripheral plasma as predictive tool(s ) in high - risk populations .

breast cancer is a malignant disease originated from breast 's cell , where it usually forms from the terminal duct - lobular unit that supplies milk 1 . in 2008 , 460,000 deaths due to breast cancer were reported worldwide 2 . infiltrating ductal carcinoma ( idc ) is the most common and aggressive type of breast cancer 3 . it is originated in the lactiferous duct , where it can break through the duct tube and invade or infiltrate the surrounding breast tissue , it can also spread to other parts of the body through lymph and blood systems 4 . metastasis is defined as the process by which tumor cells spread from primary tumor site to secondary site ( nearby or distant organ ) and proliferate . the extent of lymph node ( n ) metastasis is the major factor influencing staging and prognosis of most malignancies and often determines therapeutic decision 7 . therefore , we aimed to study protein expression changes of n stages of idc breast tissues as classified by the tnm staging system , where emphasis will be given to the up - regulated proteins in cancerous tissues . protein expression profiles for breast cancerous and its adjacent normal tissues were mapped by using proteomics approach . we hoped to identify biomarkers that can be used as diagnostic or therapeutic markers for different stages of breast cancer . tissue collection : human ethical approval was obtained from the human ethical clearance committee of universiti sains malaysia ( usm ) and the ministry of health malaysia . patient consents were obtained from the patients or their next of kin before collection of tissue specimens . thirty - eight pairs of breast tissues comprised of normal and cancerous tissues were collected from penang general hospital ( gh ) after being confirmed as normal and cancerous , respectively by hospital pathologist . there were 13 patients in n0 stage , 13 patients in n1 stage and 12 patients in n2 stage according to the tnm staging system ( table 1 ) . the patients did not receive any preoperative neo - adjuvant chemotherapy or radiotherapy prior to surgery . prior to protein extraction , tissues were thawed to room temperature and then rinsed thoroughly with distilled water . fat tissues were removed from the tissue specimens and the specimens were cut into small pieces and transferred to microcentrifuge tubes . protein extraction : an amount of 250 mg of tissue was subjected to sequential extraction using tris buffer ( tris ) and thiourea lysis buffer ( tlb ) . first , a volume of tris ( 40 mm tris , 1 mm protease inhibitor and 2 mm benzonase ) was added to the tissue in a microcentrifuge tube following a ratio of 1:2 ; tissue ( weight ) : buffer ( volume ) . the lysate was vortex for 30 seconds and centrifuged at 13000 rpm for 15 minutes at 4c . subsequently , 250 l of tris was added to the remaining pellet and vortex briefly . tlb [ 8 m urea , 2 m thiourea , 4% ( w / v ) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate ( chaps ) , 0.4% ( w / v ) carrier ampholyte , 50 mm dtt and 1 mm aebsf ] was added to the remaining pellet following a ratio of 1:1 pellet ( weight ) : buffer ( volume ) . the mixture was mixed thoroughly by brief vortexing and centrifuged at 13000 rpm for 15 minutes at 4c . after centrifugation , the supernatant was collected in a microcentrifuge tube and subjected to protein concentration determination using rc - dc protein assay ( bio - rad , usa ) , two - dimensional polyacrylamide gel electrophoresis ( 2d - page ) : this method was implemented by o'farrell ( 1975 ) and klose ( 1975 ) . 150 ug of lysate in 125 l tlb buffer containing trace amount of bromophenol blue was passively rehydrated unto a 7 cm 4 - 7 immobilized ph gradient ( ipg ) strip ( bio - rad , usa ) for 15 hours , this was followed by isoelectric focusing ( ief ) using an ief cell ( bio - rad , usa ) in a linear ramping mode , starting from 0 to 200 v for 15 minutes , followed by 200 to 4000 v for 2 hours and hold at 4000 v until 7000 v - hr was achieved . after focusing , the ipg strip was treated with equilibration buffer i ( 6 m urea , 0.375 m tris ph 8.8 , 2% sds , 20% glycerol , and 2% dtt ) and subsequently with equilibration ii ( 6 m urea , 0.375 m tris ph 8.8 , 2% sds , 20% glycerol , and 2.5% iodoacetamide ) . the second dimension separation was carried out on 10% sds - page using a constant voltage of 200 v throughout the run . the gel was stained using bio - safe colloidal coomasie blue g-250 stain ( bio - rad , usa ) and destained with deionized water . image analysis : all 2d images were captured using versadoc imaging system ( bio - rad , usa ) and analyzed using pdquest software version 7.3 ( bio - rad , usa ) . match sets consisted of 2d - gel images of protein extracts from normal and cancerous tissues of the same patient were created . the common and differentially expressed protein spots between normal and cancerous tissues were compared and identified . normalization was applied to all the captured gel images , using this device , the intensity of every protein spots were measured as proportion to the total density of valid spots . wilcoxon paired - sample test was used to determine the statistical significance in the difference in intensity between protein spots from normal and cancerous tissues . in - gel digestion : all targeted protein spots were subjected to in - gel digestion according to gam et al . ( 2003 ) . spots containing proteins were excised from gel and rinsed thoroughly with deionized water to remove excess sds . the gel pieces were washed with 100 mm ammonium bicarbonate ( nh4hco3 ) for 10 minutes . the buffer was discarded . acetonitrile ( acn ) was added onto the gel pieces for dehydration purpose and discarded after 5 minutes incubation . then , the gel pieces were subjected to vacuum centrifugation ( eppendorf , germany ) until dried . then , 15 l of 10 ng/l trypsin prepared in digestion buffer ( 50 mm nh4hco3 , 5 mm cacl2 ) was added to the gel pieces and incubated on ice for 45 minutes . the excess digestion buffer containing trypsin was discarded and 10 l of digestion buffer without trypsin was added and incubated at 37c overnight . after centrifugation at 2000 rpm for 2 minutes at room temperature , the supernatant were collected and kept in microcentrifuge tube . subsequently , 15 l of 20 mm nh4hco3 was added to the gel pieces and incubated at room temperature for 10 minutes . then , the remaining peptides were further extracted using four repeated extraction cycles using 5% ( v / v ) formic acid in 7:3 acn : dh2o . in each cycle , gel pieces were incubated for 20 minutes in the buffer and spun down . reverse phase high performance liquid chromatography and tandem mass spectrometry ( lc - ms / ms ) : the peptides were reconstituted in 30 l of 0.1% ( v / v ) formic acid in 85:15 solution of dh2o : acn . the mixture was briefly spun and the supernatant was recovered and transferred into a polypropylene vial insert and placed in autosampler of a hplc system . a sample volume of 15 l was injected into the enrichment column ( zorbax sb c18 , 35 x 0.5 mm , particle size 5 m ) at an isocratic flow rate of 0.05 ml / min using a binary pump hplc 1100 series ( agilent , germany ) . the mobile phase was 0.1% ( v / v ) formic acid in 97:3 dh2o : acn . then , the concentrated peptides were eluted and transferred to a reverse phase column ( zorbax 300sb c18 , 150 x 0.3 mm , particle size 5 m ) by using a capillary pump operated at flow rate of 4 l / min . the peptides were eluted at a linear gradient of 5% b to 95% b in 70 minutes and held constant at 95% b for 5 minutes . mobile phase a was 0.1% ( v / v ) formic acid in deionized water while mobile phase b was 0.1% ( v / v ) formic acid in acn . the ms data was acquired through data dependent analysis method consisted of full scan ms and ms / ms scan . the eluted peptides from hplc were ionized by esi operating in positive ion mode . the two most intense positively charged ions in every ms scan that exceeded the minimum threshold of 3000 counts were isolated and excited to ms / ms scan . in ms / ms scan , the peptide was subjected to collision induced dissociation ( cid ) to produce ms / ms spectrum . the ms parameters used were : dry gas flow rate of 6 l / min , dry gas temperature of 300c , nebulizer gas flow rate of 15 l / min and nebulizer pressure of 15.0 psi . the ms / ms analysis was performed with these parameters : collision energy at 1.15 v , capillary voltage of 3.5 kv , exit capillary voltage of 84.5 v , skimmer 1 voltage of 17.2 v , skimmer 2 voltages at 6.0 v , scan range of 200 - 1800 m / z with a scan time of 1 second , charge state of 2 , and isolation width of 2 m / z . protein identification : the ms / ms data obtained was used to interrogate the identity of protein through mascot protein database ( msdb ) search engine . the parameters used were homo sapiens for taxonomy , trypsin for enzyme , carboxymethyl for fixed modification . 2 da and 0.8 da , respectively . the instrument type was set as esi - trap and only one missed cleavage was allowed . the function and characteristic of proteins were obtained from swiss - prot ( www.expasy.org ) and ncbi ( www.ncbi.nlm.nih.gov ) protein database . the protein name , score , molecular weight ( mw ) , isoelectric point ( pi ) , and sequence coverage ( seq . cov ) were obtained through mascot protein database ( msdb ) search engine ( www.matrixscience.com ) . the grand average of hydropathy ( gravy ) was obtained through expasy proteomic server ( expasy.org ) . gravy is a value calculated by sum of the hydropathy value for each amino acid and dividing by number of residue in protein sequence . it ranged from -4 to + 4 , where the more positive value means the protein is more hydrophobic and vice versa for protein with negative value 8 . the tissue extract was separated using sds - page at constant voltage of 200 v. after sds - page , the gel was soaked in cold transfer buffer [ 25 mm tris , 192 mm glycine , 1.3 mm sds and 20% ( v / v ) methanol ] for 30 minutes while nitrocellulose membrane and blotting papers of 8.0 x 7.4 cm in size ( bio - rad , usa ) were soaked in transfer buffer for 1 hour . semi - dry blotting method ( lauriere , 1993 ) was applied using te semiphor semi - dry transfer unit ( hoefer scientific , germany ) . the transfer of proteins from gel to nitrocellulose membrane was conducted at 134 ma for 1.5 hours . after this , the membrane was rinsed with washing buffer [ 0.1% ( w / v ) bsa , 0.1% ( v / v ) tween 20 in pbs ] and blocked with blocking buffer [ 3% ( w / v ) bsa , 0.1% ( v / v ) tween 20 in pbs ] with gentle agitation for 2 hours at room temperature . the blocking buffer was discarded and the membrane was washed three times with washing buffer . the membrane was then incubated with primary antibody overnight at 4c with gentle agitation . this is followed by incubation with secondary antibody for 2 hours at room temperature after being washed three times with washing buffer . the membrane was incubated with substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate ( bio - rad , usa ) with gentle shaking for 15 minutes in order to visualize the interaction between targeted protein and its antibody ( primary antibody ) . in this study , a total of 38 pairs of normal and cancerous breast tissues were subjected to identical protein extraction and 2d - page procedures . inconsistent expression of a few proteins between patients was observed although a general consistent pattern of proteomes can be identified within normal and cancerous tissues , respectively . hence , only the protein spots that were consistently expressed in greater than 80% of all patients in the same cohort were considered as valid protein spot . using this approach , we hoped to minimize the identification of invalid protein spots that may result from variation between gels or error in the image analysis . the consistently expressed protein is more likely to be involved in the process of transformation of normal cell into cancer cells . a protein spot was categorized as up - regulated when the intensity of the spot in cancerous tissue is at least 2 folds or greater than that of normal tissue . a protein was termed as up - regulated protein when it showed consistent up - regulation characteristic in greater than 65% patients in the cohort . sixty - four valid protein spots on 2d gel images of tlb extracts of breast tissues were detected . the protein spots that showed consistent differentially expressed in > 65% patients were subjected to in - gel digestion , lc - ms / ms analysis and mascot search . figure 1 shows the up- and down - regulated protein spots on 2d gel image of a tlb extract from breast cancerous tissue . an example of ms and ms / ms spectrum of calreticulin are shown in figure 2 . the identities and characteristics of the significant up- and down - regulated proteins are shown in table 2 . twelve proteins were identified as up - regulated proteins and one protein as down - regulated protein in our study ( table 2 ) . the protein name , score , molecular weight ( mw ) , isoelectric point ( pi ) , sequence coverage ( seq . table 3 shows protein expression profiles and average fold change of these proteins in patients suffered from n0 , n1 and n2 stages breast cancer , respectively . there were three up - regulated proteins in n0 stage , namely calreticulin ( 77% ) , tropomyosin alpha-3 chain ( 69% ) and hsp 70 ( 69% ) . a total of ten proteins were up - regulated in n1 stage , these proteins were calreticulin ( 92% ) , 80 k protein h precursor ( 85% ) , tropomyosin alpha-3 chain ( 77% ) , tropomyosin isoform ( 69% ) , pdi ( 77% ) , hsp 90 ( 69.23% ) , tubulin , beta polypeptide ( 69% ) , hsp 60 ( 85% ) , heat shock 70 kda protein 1 ( 69% ) and pdi a3 ( 69% ) . five proteins were up - regulated while one protein was down - regulated in n2 stage . the up - regulated proteins were calreticulin ( 83% ) , tropomyosin alpha-3 chain ( 75% ) , 78 kda glucose - regulated protein ( 75% ) , hsp 60 ( 67% ) and pdi a3 ( 92% ) . the change in protein expression of all these proteins were statistically significant at a 95% confidence level ( p<0.05 ) as analyzed using wilcoxon paired - sample test . the protein identity as identified by lc - ms / ms and mascot database search engine was validated using western blot experiments . all the potential biomarker with commercially available antibodies were subjected to this analysis , these biomarkers included calreticulin , pdi a3 , hsp 60 and hsp 70 , where the identify of all of these proteins were confirmed by western blotting . figure 3 shows the western blot images for calreticulin and pdi a3 in breast normal and cancerous extracts from the same patient while beta - actin was used as an internal control . the higher intensity of antigen- antibody interaction for calreticulin and pdi a3 was observed in cancerous tissue compared to normal tissue , which may indicate the higher expression of these proteins in cancerous tissue . tnm staging is determined according to the outcomes of physical examination , biopsy , and imaging tests 7 . it is useful to guide physicians in treatment option and prognosis of patients 9 . in this study , we focused on the protein expression changes in n0 , n1 and n2 stages of breast cancer . due to limitation in sample size , n3 stage was not included in our study . tlb buffer made up of thiourea , urea and other reagents that favor the extraction of membrane associated or membrane proteins was used to extract proteins from the tissue pellet that was subjected to prewashing with tris buffer for removal of hydrophilic proteins . thirty percent of human proteome is consisted of membrane proteins 9 , these proteins involve in various biological processes of cell , which include signal transduction , immune regulation and transportation 10 . in addition , the nature of these proteins of being membrane associated making them good target for recognition in drug - targeted therapy for treatment of cancers . in general , the differentially expressed proteins identified in this study can be grouped as either common proteins in all stages or predominant protein for each stage . the common proteins can be potentially used as biomarkers for diagnosis or treatment for all n stages . on the other hand , the predominant protein in each stage may be collectively used with other diagnoses for classification of n staging . two common proteins that were up - regulated in all stages were calreticulin and tropomyosin alpha 3 chain . calrecticulin was a very promising biomarker in view of its high expression consistency in most of the patients , where it was up - regulated significantly at 77% , 92% and 83% in n0 , n1 and n2 stages , respectively . it is a ca binding protein that localizes in the peripheral membrane of lumen of er and nuclear envelope . it is important for ca storage , signaling and regulation in er 12 . as reviewed by michalak et al . , ( 1999 ) , calreticulin is also a lectin - like chaperone that involves in the synthesis of a variety of molecules , such as ion channels , surface receptors , intergrins and transporters . it helps to increase the yield of correctly folded proteins and prevents the aggregation and assembly of partially folded proteins . it had been reported as potential biomarker for breast cancer correlated with postoperative metastasis 13 , 14 . tropomyosin alpha 3 chain was up - regulated significantly at 69% , 69% and 75% in n0 , n1 and n2 stages , respectively . tropomyosins ( tms ) are family of microfilament - associated structural proteins 15 - 17 . the expression change of tms was induced by variety of carcinogens including chemical carcinogens , uv radiation , dna and rna tumor viruses during cancer cell transformation . tropomyosin alpha 3 chain ( tm-3 ) is hmw ( high molecular weight with 284 amino acids ) tms . in contrary to our finding , hmw tms was reported down - regulated in breast cancer and bladder cancer although it was up - regulated in cns tumors 15 . the protein that predominantly up - regulated ( 69% ) in n0 stage was hsp 70 , hsp 70 is a member of heat shock family that was induced under stress environment . it is function as atp - dependent molecular chaperone 18 , 19 that interacts with unfolded polypeptide sequences during mrna translation to prevent premature self - association in nascent protein . it also assists in transportation of proteins across cellular membranes and targeting protein for lysosomal degradation 19 . when comparing to other stages , n1 stage has the highest number of predominantly up - regulated proteins , namely 80 k protein h precursor , tropomyosin isoform , pdi , hsp 90 ( or also known as tumor rejection antigen ) , tubulin , beta polypeptide and heat shock 70 kda protein 1 . amongst these , 80 k protein h precursor was the most consistent up - regulated protein in n1 stage , where it was found up - regulated at 85% . this is followed by pdi at 76.9% while the rest of the proteins were up - regulated at 69% . therefore , we foresee the usefulness of 80 k protein h precursor and pdi as promising markers that can be collectively used to indicate n1 stage of breast cancer . 80 k protein h precursor or also known as glucosidase 2 subunit beta , is a substrate for protein kinase c 21 . it involves in the mechanism of elevated fibroblast growth factor-1 ( fgf-1 ) in breast cancer 22 , where fgf-1 is one of the key factors in angiogenesis and lymphangiogenesis in cancer 23 . on the other hand , pd1 is an enzyme that catalyzes the formation , reduction and isomerization of protein disulfide bond in er 24 , 25 , where it functions as molecular chaperone by assisting in protein folding . hence , it plays a critical role in the maintenance of protein structural stability and functional integrity 26 . in addition , pdi is also known as intracellular estrogen - binding protein , which accumulates estrogen in cells and augment the activities of estrogen receptor - mediated transcription 24 . the predominantly up - regulated protein for n2 stage was 78 kda glucose - regulated protein , it was significantly up - regulated at 75% . similar with heat shock family members , it functions as molecular chaperone that assists in folding and assembly of newly synthesized proteins . it is interesting to note the significant up - regulation of pdi a3 was only found in n1 and n2 stages , which represent the breast cancer that had metastasized . in addition , its extent of up - regulation expression in n2 stage is worth mentioning , where it was up - regulated significantly at 91% in n2 stage while only at 69% in n1 stage . the up - regulation of pdi seems to be paralleled with the advancing of n stages ( 51% at n0 stage ) . we believe that pdi a3 may be used in the prognosis of breast cancer , where the presence of this protein may indicate the metastasize potential of breast cancer . the thiol - disulfide exidoreductase activity of pdi a3 catalyzes oxidation , reduction and isomerization of protein disulfide bond . pdi a3 non - covalently interacts with two lectin molecular chaperones known as calnexin and calreticulin . pdi a3 involves in the oxidation folding of newly synthesized glycoproteins 25 , 31 , 32 . it was also reported up - regulated in gastric cancer 33 and pancreatic cancer 34 . in this study , we detected two proteins , namely calreticulin and tropomysin alpha 3 chains as common up - regulated proteins in n0 , n1 and n2 stages of breast cancer , while hsp 70 , 80 k protein h precursor , pdi , and 78 kda glucose - regulated protein were predominantly up - regulated proteins in n0 , n1 and n2 , respectively . on the other hand , the increase up - regulation pattern of pdi a3 was parallel with the advancing n stages of breast cancer . by virtue of cellular location of membrane proteins in cells , their up - regulation expression in cancerous tissues can potentially use as indicators for diagnosis or drug - targeted therapy of breast cancer , nevertheless , before all these potential biomarkers turn to be used in clinical applications , more retrospective and prospective clinical trials are needed to evaluate the overall clinical utility of these indicators . furthermore , the significance of each biomarker for breast cancer can be evaluated by using a greater number of tissue specimens .

breast cancer is the most common cancer among women worldwide . breast cancer metastasis primarily happens through lymphatic system , where the extent of lymph node metastasis is the major factor influencing staging , prognosis and therapeutic decision of the disease . we aimed to study the protein expression changes in different n ( regional lymph nodes ) stages of breast cancer . protein expression profiles of breast cancerous and adjacent normal tissues were mapped by proteomics approach that comprises of two - dimensional polyacrylamide gel electrophoresis ( 2d - page ) and tandem mass spectrometry ( lc - ms / ms ) analysis . calreticulin and tropomyosin alpha 3 chains were the common up - regulated proteins in n0 , n1 and n2 stages of breast cancer . potential biomarker for each n stage was hsp 70 for n0 , 80 k protein h precursor and pdi for n1 stage while 78 kda glucose - regulated protein was found useful for n2 stage . in addition , significant up - regulation of pdi a3 was detected only in the metastasized breast cancer . the up - regulation expression of these proteins in cancerous tissues can potentially use as indicators for diagnosis , treatment and prognosis of different n stages of breast cancer .

Introduction Materials and methods Result Discussion Conclusion

breast cancer is a malignant disease originated from breast 's cell , where it usually forms from the terminal duct - lobular unit that supplies milk 1 . in 2008 , 460,000 deaths due to breast cancer were reported worldwide 2 . infiltrating ductal carcinoma ( idc ) is the most common and aggressive type of breast cancer 3 . it is originated in the lactiferous duct , where it can break through the duct tube and invade or infiltrate the surrounding breast tissue , it can also spread to other parts of the body through lymph and blood systems 4 . metastasis is defined as the process by which tumor cells spread from primary tumor site to secondary site ( nearby or distant organ ) and proliferate . the extent of lymph node ( n ) metastasis is the major factor influencing staging and prognosis of most malignancies and often determines therapeutic decision 7 . therefore , we aimed to study protein expression changes of n stages of idc breast tissues as classified by the tnm staging system , where emphasis will be given to the up - regulated proteins in cancerous tissues . protein expression profiles for breast cancerous and its adjacent normal tissues were mapped by using proteomics approach . we hoped to identify biomarkers that can be used as diagnostic or therapeutic markers for different stages of breast cancer . tissue collection : human ethical approval was obtained from the human ethical clearance committee of universiti sains malaysia ( usm ) and the ministry of health malaysia . patient consents were obtained from the patients or their next of kin before collection of tissue specimens . thirty - eight pairs of breast tissues comprised of normal and cancerous tissues were collected from penang general hospital ( gh ) after being confirmed as normal and cancerous , respectively by hospital pathologist . there were 13 patients in n0 stage , 13 patients in n1 stage and 12 patients in n2 stage according to the tnm staging system ( table 1 ) . the patients did not receive any preoperative neo - adjuvant chemotherapy or radiotherapy prior to surgery . prior to protein extraction , tissues were thawed to room temperature and then rinsed thoroughly with distilled water . fat tissues were removed from the tissue specimens and the specimens were cut into small pieces and transferred to microcentrifuge tubes . protein extraction : an amount of 250 mg of tissue was subjected to sequential extraction using tris buffer ( tris ) and thiourea lysis buffer ( tlb ) . first , a volume of tris ( 40 mm tris , 1 mm protease inhibitor and 2 mm benzonase ) was added to the tissue in a microcentrifuge tube following a ratio of 1:2 ; tissue ( weight ) : buffer ( volume ) . subsequently , 250 l of tris was added to the remaining pellet and vortex briefly . after centrifugation , the supernatant was collected in a microcentrifuge tube and subjected to protein concentration determination using rc - dc protein assay ( bio - rad , usa ) , two - dimensional polyacrylamide gel electrophoresis ( 2d - page ) : this method was implemented by o'farrell ( 1975 ) and klose ( 1975 ) . after focusing , the ipg strip was treated with equilibration buffer i ( 6 m urea , 0.375 m tris ph 8.8 , 2% sds , 20% glycerol , and 2% dtt ) and subsequently with equilibration ii ( 6 m urea , 0.375 m tris ph 8.8 , 2% sds , 20% glycerol , and 2.5% iodoacetamide ) . the second dimension separation was carried out on 10% sds - page using a constant voltage of 200 v throughout the run . the gel was stained using bio - safe colloidal coomasie blue g-250 stain ( bio - rad , usa ) and destained with deionized water . image analysis : all 2d images were captured using versadoc imaging system ( bio - rad , usa ) and analyzed using pdquest software version 7.3 ( bio - rad , usa ) . match sets consisted of 2d - gel images of protein extracts from normal and cancerous tissues of the same patient were created . the common and differentially expressed protein spots between normal and cancerous tissues were compared and identified . wilcoxon paired - sample test was used to determine the statistical significance in the difference in intensity between protein spots from normal and cancerous tissues . in - gel digestion : all targeted protein spots were subjected to in - gel digestion according to gam et al . the gel pieces were washed with 100 mm ammonium bicarbonate ( nh4hco3 ) for 10 minutes . the buffer was discarded . acetonitrile ( acn ) was added onto the gel pieces for dehydration purpose and discarded after 5 minutes incubation . subsequently , 15 l of 20 mm nh4hco3 was added to the gel pieces and incubated at room temperature for 10 minutes . then , the remaining peptides were further extracted using four repeated extraction cycles using 5% ( v / v ) formic acid in 7:3 acn : dh2o . in each cycle , gel pieces were incubated for 20 minutes in the buffer and spun down . reverse phase high performance liquid chromatography and tandem mass spectrometry ( lc - ms / ms ) : the peptides were reconstituted in 30 l of 0.1% ( v / v ) formic acid in 85:15 solution of dh2o : acn . the mixture was briefly spun and the supernatant was recovered and transferred into a polypropylene vial insert and placed in autosampler of a hplc system . a sample volume of 15 l was injected into the enrichment column ( zorbax sb c18 , 35 x 0.5 mm , particle size 5 m ) at an isocratic flow rate of 0.05 ml / min using a binary pump hplc 1100 series ( agilent , germany ) . the peptides were eluted at a linear gradient of 5% b to 95% b in 70 minutes and held constant at 95% b for 5 minutes . the ms data was acquired through data dependent analysis method consisted of full scan ms and ms / ms scan . the two most intense positively charged ions in every ms scan that exceeded the minimum threshold of 3000 counts were isolated and excited to ms / ms scan . in ms / ms scan , the peptide was subjected to collision induced dissociation ( cid ) to produce ms / ms spectrum . the ms / ms analysis was performed with these parameters : collision energy at 1.15 v , capillary voltage of 3.5 kv , exit capillary voltage of 84.5 v , skimmer 1 voltage of 17.2 v , skimmer 2 voltages at 6.0 v , scan range of 200 - 1800 m / z with a scan time of 1 second , charge state of 2 , and isolation width of 2 m / z . protein identification : the ms / ms data obtained was used to interrogate the identity of protein through mascot protein database ( msdb ) search engine . the function and characteristic of proteins were obtained from swiss - prot ( www.expasy.org ) and ncbi ( www.ncbi.nlm.nih.gov ) protein database . the protein name , score , molecular weight ( mw ) , isoelectric point ( pi ) , and sequence coverage ( seq . gravy is a value calculated by sum of the hydropathy value for each amino acid and dividing by number of residue in protein sequence . it ranged from -4 to + 4 , where the more positive value means the protein is more hydrophobic and vice versa for protein with negative value 8 . the tissue extract was separated using sds - page at constant voltage of 200 v. after sds - page , the gel was soaked in cold transfer buffer [ 25 mm tris , 192 mm glycine , 1.3 mm sds and 20% ( v / v ) methanol ] for 30 minutes while nitrocellulose membrane and blotting papers of 8.0 x 7.4 cm in size ( bio - rad , usa ) were soaked in transfer buffer for 1 hour . semi - dry blotting method ( lauriere , 1993 ) was applied using te semiphor semi - dry transfer unit ( hoefer scientific , germany ) . the transfer of proteins from gel to nitrocellulose membrane was conducted at 134 ma for 1.5 hours . after this , the membrane was rinsed with washing buffer [ 0.1% ( w / v ) bsa , 0.1% ( v / v ) tween 20 in pbs ] and blocked with blocking buffer [ 3% ( w / v ) bsa , 0.1% ( v / v ) tween 20 in pbs ] with gentle agitation for 2 hours at room temperature . the membrane was incubated with substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate ( bio - rad , usa ) with gentle shaking for 15 minutes in order to visualize the interaction between targeted protein and its antibody ( primary antibody ) . in this study , a total of 38 pairs of normal and cancerous breast tissues were subjected to identical protein extraction and 2d - page procedures . inconsistent expression of a few proteins between patients was observed although a general consistent pattern of proteomes can be identified within normal and cancerous tissues , respectively . hence , only the protein spots that were consistently expressed in greater than 80% of all patients in the same cohort were considered as valid protein spot . using this approach , we hoped to minimize the identification of invalid protein spots that may result from variation between gels or error in the image analysis . the consistently expressed protein is more likely to be involved in the process of transformation of normal cell into cancer cells . a protein spot was categorized as up - regulated when the intensity of the spot in cancerous tissue is at least 2 folds or greater than that of normal tissue . a protein was termed as up - regulated protein when it showed consistent up - regulation characteristic in greater than 65% patients in the cohort . sixty - four valid protein spots on 2d gel images of tlb extracts of breast tissues were detected . the protein spots that showed consistent differentially expressed in > 65% patients were subjected to in - gel digestion , lc - ms / ms analysis and mascot search . figure 1 shows the up- and down - regulated protein spots on 2d gel image of a tlb extract from breast cancerous tissue . an example of ms and ms / ms spectrum of calreticulin are shown in figure 2 . the identities and characteristics of the significant up- and down - regulated proteins are shown in table 2 . twelve proteins were identified as up - regulated proteins and one protein as down - regulated protein in our study ( table 2 ) . the protein name , score , molecular weight ( mw ) , isoelectric point ( pi ) , sequence coverage ( seq . table 3 shows protein expression profiles and average fold change of these proteins in patients suffered from n0 , n1 and n2 stages breast cancer , respectively . there were three up - regulated proteins in n0 stage , namely calreticulin ( 77% ) , tropomyosin alpha-3 chain ( 69% ) and hsp 70 ( 69% ) . a total of ten proteins were up - regulated in n1 stage , these proteins were calreticulin ( 92% ) , 80 k protein h precursor ( 85% ) , tropomyosin alpha-3 chain ( 77% ) , tropomyosin isoform ( 69% ) , pdi ( 77% ) , hsp 90 ( 69.23% ) , tubulin , beta polypeptide ( 69% ) , hsp 60 ( 85% ) , heat shock 70 kda protein 1 ( 69% ) and pdi a3 ( 69% ) . five proteins were up - regulated while one protein was down - regulated in n2 stage . the up - regulated proteins were calreticulin ( 83% ) , tropomyosin alpha-3 chain ( 75% ) , 78 kda glucose - regulated protein ( 75% ) , hsp 60 ( 67% ) and pdi a3 ( 92% ) . the change in protein expression of all these proteins were statistically significant at a 95% confidence level ( p<0.05 ) as analyzed using wilcoxon paired - sample test . the protein identity as identified by lc - ms / ms and mascot database search engine was validated using western blot experiments . all the potential biomarker with commercially available antibodies were subjected to this analysis , these biomarkers included calreticulin , pdi a3 , hsp 60 and hsp 70 , where the identify of all of these proteins were confirmed by western blotting . figure 3 shows the western blot images for calreticulin and pdi a3 in breast normal and cancerous extracts from the same patient while beta - actin was used as an internal control . the higher intensity of antigen- antibody interaction for calreticulin and pdi a3 was observed in cancerous tissue compared to normal tissue , which may indicate the higher expression of these proteins in cancerous tissue . it is useful to guide physicians in treatment option and prognosis of patients 9 . in this study , we focused on the protein expression changes in n0 , n1 and n2 stages of breast cancer . due to limitation in sample size , n3 stage was not included in our study . tlb buffer made up of thiourea , urea and other reagents that favor the extraction of membrane associated or membrane proteins was used to extract proteins from the tissue pellet that was subjected to prewashing with tris buffer for removal of hydrophilic proteins . thirty percent of human proteome is consisted of membrane proteins 9 , these proteins involve in various biological processes of cell , which include signal transduction , immune regulation and transportation 10 . in addition , the nature of these proteins of being membrane associated making them good target for recognition in drug - targeted therapy for treatment of cancers . in general , the differentially expressed proteins identified in this study can be grouped as either common proteins in all stages or predominant protein for each stage . the common proteins can be potentially used as biomarkers for diagnosis or treatment for all n stages . two common proteins that were up - regulated in all stages were calreticulin and tropomyosin alpha 3 chain . calrecticulin was a very promising biomarker in view of its high expression consistency in most of the patients , where it was up - regulated significantly at 77% , 92% and 83% in n0 , n1 and n2 stages , respectively . it is a ca binding protein that localizes in the peripheral membrane of lumen of er and nuclear envelope . , ( 1999 ) , calreticulin is also a lectin - like chaperone that involves in the synthesis of a variety of molecules , such as ion channels , surface receptors , intergrins and transporters . it had been reported as potential biomarker for breast cancer correlated with postoperative metastasis 13 , 14 . tropomyosin alpha 3 chain was up - regulated significantly at 69% , 69% and 75% in n0 , n1 and n2 stages , respectively . tropomyosins ( tms ) are family of microfilament - associated structural proteins 15 - 17 . tropomyosin alpha 3 chain ( tm-3 ) is hmw ( high molecular weight with 284 amino acids ) tms . in contrary to our finding , hmw tms was reported down - regulated in breast cancer and bladder cancer although it was up - regulated in cns tumors 15 . the protein that predominantly up - regulated ( 69% ) in n0 stage was hsp 70 , hsp 70 is a member of heat shock family that was induced under stress environment . when comparing to other stages , n1 stage has the highest number of predominantly up - regulated proteins , namely 80 k protein h precursor , tropomyosin isoform , pdi , hsp 90 ( or also known as tumor rejection antigen ) , tubulin , beta polypeptide and heat shock 70 kda protein 1 . amongst these , 80 k protein h precursor was the most consistent up - regulated protein in n1 stage , where it was found up - regulated at 85% . this is followed by pdi at 76.9% while the rest of the proteins were up - regulated at 69% . therefore , we foresee the usefulness of 80 k protein h precursor and pdi as promising markers that can be collectively used to indicate n1 stage of breast cancer . 80 k protein h precursor or also known as glucosidase 2 subunit beta , is a substrate for protein kinase c 21 . it involves in the mechanism of elevated fibroblast growth factor-1 ( fgf-1 ) in breast cancer 22 , where fgf-1 is one of the key factors in angiogenesis and lymphangiogenesis in cancer 23 . on the other hand , pd1 is an enzyme that catalyzes the formation , reduction and isomerization of protein disulfide bond in er 24 , 25 , where it functions as molecular chaperone by assisting in protein folding . hence , it plays a critical role in the maintenance of protein structural stability and functional integrity 26 . in addition , pdi is also known as intracellular estrogen - binding protein , which accumulates estrogen in cells and augment the activities of estrogen receptor - mediated transcription 24 . the predominantly up - regulated protein for n2 stage was 78 kda glucose - regulated protein , it was significantly up - regulated at 75% . it is interesting to note the significant up - regulation of pdi a3 was only found in n1 and n2 stages , which represent the breast cancer that had metastasized . in addition , its extent of up - regulation expression in n2 stage is worth mentioning , where it was up - regulated significantly at 91% in n2 stage while only at 69% in n1 stage . the up - regulation of pdi seems to be paralleled with the advancing of n stages ( 51% at n0 stage ) . we believe that pdi a3 may be used in the prognosis of breast cancer , where the presence of this protein may indicate the metastasize potential of breast cancer . the thiol - disulfide exidoreductase activity of pdi a3 catalyzes oxidation , reduction and isomerization of protein disulfide bond . pdi a3 non - covalently interacts with two lectin molecular chaperones known as calnexin and calreticulin . pdi a3 involves in the oxidation folding of newly synthesized glycoproteins 25 , 31 , 32 . it was also reported up - regulated in gastric cancer 33 and pancreatic cancer 34 . in this study , we detected two proteins , namely calreticulin and tropomysin alpha 3 chains as common up - regulated proteins in n0 , n1 and n2 stages of breast cancer , while hsp 70 , 80 k protein h precursor , pdi , and 78 kda glucose - regulated protein were predominantly up - regulated proteins in n0 , n1 and n2 , respectively . on the other hand , the increase up - regulation pattern of pdi a3 was parallel with the advancing n stages of breast cancer . by virtue of cellular location of membrane proteins in cells , their up - regulation expression in cancerous tissues can potentially use as indicators for diagnosis or drug - targeted therapy of breast cancer , nevertheless , before all these potential biomarkers turn to be used in clinical applications , more retrospective and prospective clinical trials are needed to evaluate the overall clinical utility of these indicators . furthermore , the significance of each biomarker for breast cancer can be evaluated by using a greater number of tissue specimens .

the bacillus cereus group is a group of bacteria which includes species that are of significant economic and medical importance , such as b. anthracis , an obligate mammalian pathogen and the causative agent of anthrax , b. cereus , an opportunistic human pathogen causing diverse infections , and b. thuringiensis , an insect pathogen widely used as a biopesticide worldwide . b. cereus group organisms are genetically very closely related at the genetic and genomic levels ( 14 ) . due to the importance of b. cereus group bacteria , the population structure and phylogenetic relationships of the isolates within the group have been extensively studied using diverse typing methods , in order to follow the evolution of strains or identify clones responsible for disease outbreaks . large - scale analyses have been conducted using multilocus enzyme electrophoresis [ mlee ; ( 1,57 ) ] , which is based on electrophoretic mobility of 1020 proteins , fluorescent amplified fragment length polymorphism analysis [ aflp ; ( 810 ) ] , which surveys the genome for length and sequence polymorphisms using restriction enzyme fragmentation and pcr , and more recently multilocus sequence typing [ mlst ; ( 4,1117 ) ] , which compares the sequences of pcr - amplified dna products internal to six or seven housekeeping genes . the separate typing studies have independently revealed that the b. cereus group population is divided into multiple phylogenetic clusters , in which the members of each species , with the exception of the highly clonal b. anthracis , are frequently intermixed . mlst has become the gold standard for bacterial phylogeny , as it is unambiguous and truly electronically portable among laboratories ( 18,19 ) . however , unlike other bacterial species , which are most often typed using a single mlst scheme , five separate schemes have been developed for the b. cereus group , based on different sets of genes . therefore , we recently developed the supercat database , which compiles and integrates mlst data from all the published b. cereus group schemes into a single resource ( 20 ) , as well as provides tools for building a combined phylogenetic tree of the b. cereus group population using supertree techniques ( available at the university of oslo s typing website : http://mlstoslo.uio.no/ ) . while mlst data is currently available for 1430 b. cereus group isolates , an additional 832 isolates have been typed by either aflp or mlee . in order to provide the most comprehensive and truly global view of the b. cereus group population , in addition to phylogenetic supertrees , a clustering algorithm was developed to identify groups of genetically related isolates that share common mlst and/or aflp profiles . the data and analysis tools are available in hypercat , an extension of the supercat database , which provides the b. cereus group research community with an integrated on - line genotyping resource . we describe below the main additions and features in hypercat as well as the procedures for building multi - datatype supertrees and superclusters . integration of mlst , aflp and mlee - based phylogenetic information into a common supertree was possible by taking advantage of the matrix representation by parsimony ( mrp ) technique ( 2022 ) , which consists in merging trees that can be built individually from heterogeneous data . the procedure is similar to that used to build the multi - scheme mlst supertree in supercat . three trees generated from the comparisons of aflp profiles were taken from the studies of ref . 10 ) ( 425 isolates ) , and a phylogeny based on mlee profiles of 316 b. cereus group isolates was from ref . ( 1 ) ( 239 isolates ) and e. helgason ( unpublished ) ( 77 isolates ) . these were combined with the phylogenetic trees built for each of the 26 gene fragments from the five mlst schemes used to reconstruct the mlst supertree in supercat ( 20 ) ( 1400 isolates ) . in the combined mlst+aflp+mlee dataset a total of 2262 isolates were represented , of which 49 were excluded from further analysis because of conflicting data . isolates were excluded because they exhibited incongruent phylogenetic positions in the various trees ( i.e. were located in different clusters of the b. cereus group population depending on the typing method used ) or because the typing sequences or profiles obtained from different collections or databases were inconsistent . a global multi - datatype supertree was then reconstructed by means of the mrp technique ( figure 1 ) , which first consists in recoding each individual tree into a binary matrix representing the branching order ( i.e. phylogenetic groupings ) among the strains . the matrices for all trees are then concatenated into a supermatrix from which a common supertree can be inferred by a maximum parsimony algorithm . for this step the tree analysis using new technology ( tnt ) software [ ( 23,24 ) , http://www.zmuc.dk/public/phylogeny/tnt/ ] was used . tnt is specifically designed for analysis of large datasets and permits ultra - fast supertree building . the speed and algorithms implemented in tnt enable a broader and more efficient exploration of the tree space , thereby allowing the program to find more parsimonious trees , compared to common softwares such as paup and phylip ( 25 ) . here xmult command ( all default options , except a search level of 10 ) , which automatically determines the search parameters for the given dataset and runs a combination of several tree rearrangement algorithms ( sectorial searches , tree - drifting and tree - fusing ) . such combined search strategies have been found to be most appropriate for large datasets [ for details , see ref . the final supertree was taken as the strict consensus of all equally most parsimonious trees . because mlst , aflp and mlee are based on different amounts of genetic information , the supertree procedure was weighted . in the aflp studies of refs ( 8,9 ) , genetic profiles were based on 40 genomic fragments , which can be considered as 40 genomic loci , while the aflp study of ref . ( 10 ) was based on 68 loci , and each mlst gene represents one locus . the actual number of characters analyzed by each typing method is very different ; while aflp relies on restriction sites of 4 or 6 bp , a given mlst locus includes 300600 bp . the mrp supertree technique combines trees , not the raw data used to build them . each aflp tree was based on the combination of 40 or 68 loci , whereas a particular mlst gene tree gives the relationships among isolates at one locus . therefore a weighting scheme based on the number of loci examined by each method was employed . that is , the groupings coming from the aflp trees and from each of the 26 mlst gene trees in the mrp supermatrix were given a weight of 40 [ or 68 for the tree of ref . ( 10 ) ] and 1 , respectively , for the parsimony search ( since 26 gene fragments were used , mlst was thus represented by a total weight of 26 ) . the mlee study relied on 13 enzyme loci , and to take into account the fact that mlee is based on proteins , the weight of the mlee tree was set to 4 ( i.e. 13/3 ) . ccode command in tnt . to reduce the size of the binary supermatrix and the amount of missing data to be treated , as well as to speed up computations , the weighted supertree was built using only one representative ( chosen at random ) from a set of strains having identical typing profiles and was computed in about seven hours ( using a single processor on a linux apache web server ) . to obtain statistical support values for all groupings ( i.e. internal branches ) in the supertree approximate likelihood - ratio tests ( alrts ) for branches were computed using phyml 3.0 with shimodaira - hasegawa - like support values ( 2629 ; http://atgc.lirmm.fr/phyml/ ) . alrts provide a fast way of testing branch support and consist in comparing , for a given branch , the likelihoods of the best tree containing the branch ( i.e. the current supertree ) and the best tree not containing the branch ( keeping the rest of the topology identical ) . if the difference in likelihood is statistically significant , then the branch is supported . for each branch the probability ( or p - value ) of being significant is estimated [ for details , see refs ( 27,2931 ) ] . phyml was run on the mrp supermatrix using a custom substitution model representing binary data and the topology of the supertree obtained by tnt , allowing the branch supports to be computed in 4 h. the procedure described above was also applied to reconstruct a supertree using the data from the dna - based typing methods only ( mlst and aflp , 2143 isolates ) . it contains 97% of the strains included in the database and is more reliable than the mlst+aflp+mlee supertree because mlee , which is based on protein profiles , has a much lower resolution power than mlst and aflp . this is due to the fact that the dna - based methods take every nucleotide difference into account , including those that are silent at the protein level , and also that differences in protein sequences may not necessarily result in visible differences in electrophoretic mobility . thus , mlee does not clearly differentiate some of the major phylogenetic clusters in the b. cereus group population ( 1,5,6 ) . mlst+aflp+mlee supertree is merely provided as a tool for exploring the phylogenetic positions of the 70 isolates that have been typed only by mlee and which are thus not included in the reference figure 1.weighted supertree of 2143 b. cereus group isolates based on phylogenetic information from five mlst schemes and three aflp studies available in hypercat . the supertree was inferred following the mrp strategy , with a weight of 40 or 68 given to the aflp studies and a weight of 1 given to each of 26 mlst genes . for the sake of legibility , ( 10 ) are designated by roman numerals ( i vii ) and their branch support values ( based on alrt probabilities ) are indicated when > 95% . superclusters of genetically closely related isolates ( sharing identical mlst or aflp profiles ) that have been computed independently using a recursive approach and that contain 10 or more isolates have been mapped onto the supertree and are listed on the right ( the supercluster descriptions apply to most isolates of the superclusters , not necessarily all ) . isolates belonging to a given supercluster are drawn in the same color . as can be seen , isolates from the same supercluster are closely related in the tree . one exception is indicated by an asterisk and corresponds to strain b. cereus f4430/73 of supercluster 157 . weighted supertree of 2143 b. cereus group isolates based on phylogenetic information from five mlst schemes and three aflp studies available in hypercat . the supertree was inferred following the mrp strategy , with a weight of 40 or 68 given to the aflp studies and a weight of 1 given to each of 26 mlst genes . for the sake of legibility , isolate names ( 10 ) are designated by roman numerals ( i vii ) and their branch support values ( based on alrt probabilities ) are indicated when > 95% . superclusters of genetically closely related isolates ( sharing identical mlst or aflp profiles ) that have been computed independently using a recursive approach and that contain 10 or more isolates have been mapped onto the supertree and are listed on the right ( the supercluster descriptions apply to most isolates of the superclusters , not necessarily all ) . isolates belonging to a given supercluster are drawn in the same color . as can be seen , isolates from the same supercluster are closely related in the tree . one exception is indicated by an asterisk and corresponds to strain b. cereus f4430/73 of supercluster 157 . even though more than 2200 b. cereus group isolates have been typed by either mlst , aflp , or mlee , there is very little overlap between the data sets investigated by the various methods . a total of 98 strains are common to mlst and aflp datasets , and 34 strains have been characterized by all three methods . nevertheless , the mlst+aflp and for example , mapping onto the supertrees the seven clusters ( denoted i vii ) identified in the aflp analysis of ref . ( 10 ) confirmed that the overall phylogenetic structure of the aflp analysis was retained when combined with phylogenetic information arising from mlst and mlee data , as isolates from a given aflp cluster are all contained within a common subtree in the supertree and the grouping is supported by a high statistical value ( alrt probability > 0.95 ) ( supplementary figure s1 ) . conversely , mapping the lineages defined in the mlst study of ref . ( 11 ) indicated that the phylogenetic signal coming from mlst data was conserved when combined with that coming from aflp and mlee , even though mlst was given a lower weight in the supertree reconstruction ( supplementary figure s3 ) . the integration of multiple types of phylogenetic data demonstrated that even though there is little overlap between the isolate sets analyzed by the various typing methods , the supertrees obtained in the integrated analyses appeared to properly reconstruct the major groupings in the population and to be robust to missing data , a behavior that has been observed in numerous cases and for various datasets in earlier studies ( 3234 ) . overall , a large proportion of the branches ( 70% ) in the supertrees have strong statistical support ( alrt confidence level > 95% ) . the impact of missing data was further tested by including in the supertree four replicates of the 34 strains for which mlst , aflp and mlee data are available , using either all data or data from one of the three typing methods . for all 34 strains , the four replicates were always located in the same phylogenetic cluster and mostly in the same subtree ( i.e. for 28 of the 34 strains the replicates were separated by less than a dozen internal nodes ) , indicating that missing data did not bias the general positioning of the strains ( supplementary figure s4 ) . precise within - clusters branchings may however contain more uncertainty , thus the multi - datatype supertrees should be used primarily as a common framework to explore global relationships among strains . to complement the supertree approach , a tree - independent clustering procedure was applied in order to group isolates that share identical typing data . the procedure is analogous to that of computing clonal complexes of strains as is typically done with mlst data ( 35,36 ) , but with the objective of using information from the multiple b. cereus group mlst schemes as well as results from the multiple aflp studies . for reasons explained in the previous section the mlst+aflp dataset is used as the reference dataset in hypercat , and thus mlee profiles were not included in the supercluster analysis . an algorithm was designed to identify groups of isolates that share identical mlst and/or aflp profiles ( figure 2 ) . for mlst , strains were defined as sharing an identical profile if they shared identical sequences at all loci of a given mlst scheme [ i.e. belong to the same sequence type ( st ) using six or seven loci , depending on the scheme ] . that is , if strain a had a profile identical to strain b based on typing method 1 , and was identical to strain c based on method 2 , then strains a , b and c were grouped . all strains sharing a profile with b and all strains sharing a profile with c in any of the two dna - only typing methods were subsequently added to the supercluster , then all strains identical to the latter strains were added , and so on until no further isolate sharing an identical mlst and/or aflp profile with the strains currently in the group could be found . as a result of this procedure , every isolate of a given supercluster shares identical genotyping data by at least one typing method to at least one other isolate in the group ( figure 2a ) . note also that all superclusters that were built by this procedure are mutually exclusive , i.e. a given isolate belongs to one and only one group . isolates that do not share typing profiles with any other isolate in the database were classified as singletons . in this analysis , a total of 283 superclusters were identified , comprising a total of 1152 isolates , while the remaining 991 isolates were singletons . because of little overlap between the isolate sets analyzed by the different typing schemes , 90% of the superclusters were composed only of isolates that have been typed by the same method or scheme , and most of the clusters ( 85% ) were small ( less than five isolates ) . due to the clustering procedure all strains in a given supercluster may not be identical in any one marker . however , they are likely to be closely related . this was confirmed by mapping the 16 superclusters containing 10 isolates or more onto the mlst+aflp supertree , showing that , with a single exception among 431 isolates ( b. cereus f4430/73 in supercluster 157 ) , isolates belonging to a given supercluster branched in the same subtree or nearby parts of the supertree ( figure 1 ) . the exception could be explained by the fact that b. cereus f4430/73 has been typed by more methods than the other members of supercluster 157 , creating a bias in the analysis due to variable amounts of data . each cluster is reported in a table , listing detailed information about the isolates and the genotying profiles shared . in addition , pressing one of the three buttons above the table allows to view the phylogenetic positions of the isolates in the multi - datatype supertree using archaeopteryx , to extract from the supertree the subtree containing only the isolates forming the supercluster , or to display a network graph showing the specific typing data shared between the isolates of the supercluster . in the latter network , isolates having identical mlst and/or aflp profiles are connected by blue and red lines , respectively . ( b ) graphical overview of all the superclusters resulting from typing information from five mlst schemes and three aflp studies . superclusters were built using a recursive approach , grouping strains that shared identical sequences at all loci of at least one of the mlst schemes and/or have identical aflp profiles . the set of superclusters and singletons can be navigated using an interactive bubble chart generated by means of the ibm / alphaworks many eyes system . each supercluster is represented by a pie proportional to the number of isolates in the supercluster and the pie is divided according to the species or sources of the isolates included in the supercluster . selecting or mousing over a supercluster brings up a window giving information about its content ( shown in the inset indicated by an arrow ) . each cluster is reported in a table , listing detailed information about the isolates and the genotying profiles shared . in addition , pressing one of the three buttons above the table allows to view the phylogenetic positions of the isolates in the multi - datatype supertree using archaeopteryx , to extract from the supertree the subtree containing only the isolates forming the supercluster , or to display a network graph showing the specific typing data shared between the isolates of the supercluster . in the latter network , isolates having identical mlst and/or aflp profiles are connected by blue and red lines , respectively . ( b ) graphical overview of all the superclusters resulting from typing information from five mlst schemes and three aflp studies . superclusters were built using a recursive approach , grouping strains that shared identical sequences at all loci of at least one of the mlst schemes and/or have identical aflp profiles . the set of superclusters and singletons can be navigated using an interactive bubble chart generated by means of the ibm / alphaworks many eyes system . each supercluster is represented by a pie proportional to the number of isolates in the supercluster and the pie is divided according to the species or sources of the isolates included in the supercluster . selecting or mousing over a supercluster brings up a window giving information about its content ( shown in the inset indicated by an arrow ) . hypercat currently contains 2262 isolates covering the diversity of the b. cereus group , including 110 b. anthracis , 841 b. cereus , 511 b. thuringiensis , 54 b. weihenstephanensis , 115 b. mycoides , 8 b. pseudomycoides and 623 unclassified b. cereus group isolates . in hypercat isolates can be selected according to various criteria such as name , source , keyword , typing method , or phylogenetic cluster [ using the classification defined by ref . the database provides all the genotypes that are represented for each typing scheme or method and for combinations of typing methods , with tables listing the sets of isolates that have unique or identical genotyping profiles . the profiles correspond to the sts , aflp types ( ats ) and electrophoretic types ( ets ) for mlst , aflp and mlee , respectively . also , the phylogenetic position of isolates having a particular profile can be highlighted in the mlst+aflp or mlst+aflp+mlee supertree . the supertrees can be browsed interactively by means of the java - based archaeopteryx ( http://www.phylosoft.org/archaeopteryx/ ) and treebolic2 ( http://treebolic.sourceforge.net/ ) viewing applications . archaeopteryx has superceded the atv tool ( 37 ) previously used in supercat . in particular , archaeopteryx includes mouse - driven navigation and can render trees in various styles , e.g. rectangular , circular or unrooted . supertrees can be colored by species or by source of isolate , and subtrees corresponding to selected subsets of strains can be highlighted and extracted . since supertrees contain only one representative from a set of strains having an identical genotype , the representative strain is labeled superprofile_x ( y strains) where x is the genotype i d number and y is the number of strains sharing that genotype . clicking on that label will then load the corresponding table listing all isolates having that genotyping profile using the in addition to archaeopteryx supertrees can be navigated using treebolic which renders trees in 2d hyperbolic space . while the region under focus is displayed in detail , the rest of the tree is shown in a smaller size and the entire tree remains visible at all times . in both archaeopteryx and treebolic views , branches of the supertrees are colored according to the seven main phylogenetic clusters of the b. cereus group population defined by ref . ( 10 ) , which gives a global overview of the phylogenetic classification of the strains . a given isolate was assigned to a particular cluster if it was part of that cluster in the mlst - only , mlst+aflp and mlst+aflp+mlee supertrees . in total , 2144 of 2213 isolates could be consistently classified , demonstrating the congruence between the supertrees . hypercat also provides a number of options for navigating and comparing the scheme - specific mlst - only supertrees ( taken from supercat ) , the aflp - only trees , the mlee phylogeny and the multi - datatype supertrees , for the user who wishes to explore the method - specific trees and/or judge their congruence with the multi - datatype trees . specific pages give information on the strains exhibiting conflicting typing data and allow to analyze their phylogenetic positions . the superclusters of isolates sharing identical mlst and/or aflp profiles can be explored using tabular and graphical displays . tables list the detailed composition of the superclusters , and functions allow to view the phylogenetic position of the isolates in the mlst+aflp supertree and to extract the subtree containing the isolates belonging to a given supercluster . a graphical representation of the supercluster is also provided in the form of a network showing the specific mlst and aflp profiles that are shared between the isolates ( figure 2 a ) . the network is drawn in scalable vector graphics ( svg ) format using the neato program of the at&t graphviz package [ ( 38,39 ) ; http://www.graphviz.org/ ] and can be navigated using the java - based zgrviewer [ see below ; ( 40 ) ; http://zvtm.sourceforge.net/zgrviewer.html ] . in addition , a graphical overview of all the superclusters is given by an interactive bubble chart , in which each cluster or singleton is represented by a pie whose size is proportional to the number of isolates in the cluster ( figure 2b ) . superclusters can be colored according to bacterial species or isolate origin , and sets of clusters containing isolates from a given species or source can be jointly highlighted . the interactive charts were generated using the ibm / alphaworks many eyes data visualization system ( http://manyeyes.alphaworks.ibm.com/manyeyes/ ) . hypercat also offers the possibility to retrieve isolates that share identical mlst , aflp and/or mlee profiles based on user - selected subsets of isolates and typing methods , and to build superclusters following the recursive procedure described in this article . as a further addition to hypercat , the superclusters have been mapped onto the mlst+aflp supertree in order to produce a combined and integrative genetic and phylogenetic overview ( snapshot ) of the b. cereus group population ( figure 3 ) . to generate this view , the isolates belonging to a supercluster have been replaced in the supertree by a randomly chosen isolate selected to represent the supercluster , and the phylogenetic position of the supercluster is given by that isolate . supercluster_x ( y strains) where x is the cluster i d number and y is the number of strains included in that supercluster . this representation allows to see in a single graph the phylogenetic distribution of the superclusters and singleton isolates . zgrviewer ( 40 ) is an advanced graph visualization software based on the zoomable visual transformation machine ( zvtm ) particularly suitable for large networks . the interactive snapshot contains hyperlinks ; middle - button clicking on a supercluster or isolate name will link to an information page with details about that cluster or isolate , and clicking on an internal node will load a new page displaying the subtree including all descendants of that node in the supertree ( figure 3 ) . figure 3.phylogenetic snapshot of the b. cereus group population based on mlst and aflp typing data for 2143 isolates . the supertree is represented as an unrooted network in which the closely related strains belonging to a supercluster have been replaced by a representative isolate . superclusters are drawn as filled lightblue boxes , while singleton isolates are drawn as ellipses colored by bacterial species ( red , b. anthracis ; blue , b. cereus ; green , b. thuringiensis ; brown , b. mycoides ; purple , b. pseudomycoides ; orange , b. weihenstephanensis ; black , uncharacterized b. cereus group isolate ) . clicking on an internal node loads a new page displaying the subtree including all descendants of that node in the supertree . the view in the upper right corner shows the display of the supertree in hyperbolic space using treebolic2 . phylogenetic snapshot of the b. cereus group population based on mlst and aflp typing data for 2143 isolates . the supertree is represented as an unrooted network in which the closely related strains belonging to a supercluster have been replaced by a representative isolate . superclusters are drawn as filled lightblue boxes , while singleton isolates are drawn as ellipses colored by bacterial species ( red , b. anthracis ; blue , b. cereus ; green , b. thuringiensis ; brown , b. mycoides ; purple , b. pseudomycoides ; orange , b. weihenstephanensis ; black , uncharacterized b. cereus group isolate ) . clicking on an internal node loads a new page displaying the subtree including all descendants of that node in the supertree . the view in the upper right corner shows the display of the supertree in hyperbolic space using treebolic2 . in addition to the phylogenetic analysis tools described above , hypercat also contains a number of pages presenting various statistics relating to strain distribution , data content and overlap , as well as descriptions of the supertree and supercluster building procedures . hypercat provides hyperlinks to the integrated microbial database , straininfo [ http://www.straininfo.net/ ; ( 41 ) ] , for additional isolate information and links to database collections . all data in hypercat , including tabular isolate and genotype listings , superclusters , supertrees and networks , can be saved and downloaded freely from the database . in particular , trees can be obtained in the recently developed xml and phyloxml standards ( 42 ) . as mlst is currently the gold standard for bacterial typing , the supercat database devoted to multi - scheme mlst analysis of the b. cereus group , containing 1430 isolates , has been upgraded accordingly . the superclustering tool and associated graphical and tabular displays developed for hypercat have been incorporated into supercat for analysis of mlst data only , for identifying groups of isolates sharing identical mlst profiles in any of the five schemes available . an mlst - based genetic and phylogenetic population snapshot combining supertree and furthermore , for the reconstruction of the multi - scheme mlst supertree by the mrp procedure , the parsimony step , which was originally carried out using the mix program of the phylip package ( 43 ) , is now computed using tnt ( 23,24 ) . for reasons explained earlier , tnt showed an improved accuracy over mix and the use of tnt reduces the computation time from two days to about four hours with the full dataset . finally , the atv tree navigation tool has been replaced by its successor , archaeopteryx ( 37 ) and the hyperbolic viewer treebolic2 has been implemented . in order to serve the b. cereus group research community with the most comprehensive genotyping resource , we have developed at the university of oslo s typing website ( http://mlstoslo.uio.no/ ) a new database , hypercat . this database incorporates analysis tools combining genotyping data not only from multiple mlst schemes , as provided in the supercat database , but also from the altogether different approaches aflp and mlee . while genotyping of the b. cereus group of bacteria is currently essentially done using mlst , hypercat provides a means to combine mlst data with the phylogenetic information from hundreds of strains that have been typed using other methods , and therefore generates a truly global genetic snapshot of the b. cereus group population with moderate to high representativity of all member species . the multi - datatype supertrees and superclusters developed herein provide a common basis for revealing and displaying genotypic relationships among b. cereus group isolates , based on the large amounts of heterogeneous typing data available for this group of bacteria , an effort which would not be straightforward by mere inspection and comparison of results from the two dozens of publications describing mlst , mlee and aflp analyses of the b. cereus group population . this work was supported by grants from the norwegian research council through a strategic university program ( sup ) , the norwegian functional genomics program ( fuge ii channel 3 grant ) , and the consortium for advanced microbial sciences and technologies ( fuge - camst ) . funding for open access charge : norwegian functional genomics ( fuge ii ) platform of the research council of norway and the university of oslo .

the bacillus cereus group of bacteria includes species that are of significant medical and economic importance . we previously developed the supercat database , which integrates data from all five multilocus sequence typing ( mlst ) schemes available to infer the genetic relatedness within this group . since large numbers of isolates have been typed by other techniques , these should be incorporated in order to provide the most comprehensive and truly global view of the b. cereus group population . the supercat system has been extended into a new database , hypercat , with two main additions . first , an extended supertree approach was applied to combine the phylogenetic information available from mlst , amplified fragment length polymorphism and multilocus enzyme electrophoresis . secondly , a tree - independent clustering algorithm was designed to build superclusters of genetically closely related isolates sharing identical genotyping data . the superclusters were then mapped onto the supertree to generate an integrative genetic and phylogenetic snapshot of the b. cereus group population currently incorporating 2143 isolates . hypercat is freely accessible at the university of oslo s typing website , which has also been upgraded with tnt software , allowing improved and ultra - fast supertree reconstructions . in addition , novel and advanced tools have been included for interactive viewing and navigation of trees , clusters and networks.database url : http://mlstoslo.uio.no/

Introduction Multi-dataype supertree reconstruction Multi-datatype supercluster reconstruction HyperCAT database features and manipulation Update of the SuperCAT database Conclusion Supplementary Data Funding

the bacillus cereus group is a group of bacteria which includes species that are of significant economic and medical importance , such as b. anthracis , an obligate mammalian pathogen and the causative agent of anthrax , b. cereus , an opportunistic human pathogen causing diverse infections , and b. thuringiensis , an insect pathogen widely used as a biopesticide worldwide . b. cereus group organisms are genetically very closely related at the genetic and genomic levels ( 14 ) . due to the importance of b. cereus group bacteria , the population structure and phylogenetic relationships of the isolates within the group have been extensively studied using diverse typing methods , in order to follow the evolution of strains or identify clones responsible for disease outbreaks . large - scale analyses have been conducted using multilocus enzyme electrophoresis [ mlee ; ( 1,57 ) ] , which is based on electrophoretic mobility of 1020 proteins , fluorescent amplified fragment length polymorphism analysis [ aflp ; ( 810 ) ] , which surveys the genome for length and sequence polymorphisms using restriction enzyme fragmentation and pcr , and more recently multilocus sequence typing [ mlst ; ( 4,1117 ) ] , which compares the sequences of pcr - amplified dna products internal to six or seven housekeeping genes . the separate typing studies have independently revealed that the b. cereus group population is divided into multiple phylogenetic clusters , in which the members of each species , with the exception of the highly clonal b. anthracis , are frequently intermixed . however , unlike other bacterial species , which are most often typed using a single mlst scheme , five separate schemes have been developed for the b. cereus group , based on different sets of genes . therefore , we recently developed the supercat database , which compiles and integrates mlst data from all the published b. cereus group schemes into a single resource ( 20 ) , as well as provides tools for building a combined phylogenetic tree of the b. cereus group population using supertree techniques ( available at the university of oslo s typing website : http://mlstoslo.uio.no/ ) . while mlst data is currently available for 1430 b. cereus group isolates , an additional 832 isolates have been typed by either aflp or mlee . in order to provide the most comprehensive and truly global view of the b. cereus group population , in addition to phylogenetic supertrees , a clustering algorithm was developed to identify groups of genetically related isolates that share common mlst and/or aflp profiles . the data and analysis tools are available in hypercat , an extension of the supercat database , which provides the b. cereus group research community with an integrated on - line genotyping resource . integration of mlst , aflp and mlee - based phylogenetic information into a common supertree was possible by taking advantage of the matrix representation by parsimony ( mrp ) technique ( 2022 ) , which consists in merging trees that can be built individually from heterogeneous data . 10 ) ( 425 isolates ) , and a phylogeny based on mlee profiles of 316 b. cereus group isolates was from ref . these were combined with the phylogenetic trees built for each of the 26 gene fragments from the five mlst schemes used to reconstruct the mlst supertree in supercat ( 20 ) ( 1400 isolates ) . were located in different clusters of the b. cereus group population depending on the typing method used ) or because the typing sequences or profiles obtained from different collections or databases were inconsistent . a global multi - datatype supertree was then reconstructed by means of the mrp technique ( figure 1 ) , which first consists in recoding each individual tree into a binary matrix representing the branching order ( i.e. tnt is specifically designed for analysis of large datasets and permits ultra - fast supertree building . the speed and algorithms implemented in tnt enable a broader and more efficient exploration of the tree space , thereby allowing the program to find more parsimonious trees , compared to common softwares such as paup and phylip ( 25 ) . here xmult command ( all default options , except a search level of 10 ) , which automatically determines the search parameters for the given dataset and runs a combination of several tree rearrangement algorithms ( sectorial searches , tree - drifting and tree - fusing ) . because mlst , aflp and mlee are based on different amounts of genetic information , the supertree procedure was weighted . the mrp supertree technique combines trees , not the raw data used to build them . phyml was run on the mrp supermatrix using a custom substitution model representing binary data and the topology of the supertree obtained by tnt , allowing the branch supports to be computed in 4 h. the procedure described above was also applied to reconstruct a supertree using the data from the dna - based typing methods only ( mlst and aflp , 2143 isolates ) . it contains 97% of the strains included in the database and is more reliable than the mlst+aflp+mlee supertree because mlee , which is based on protein profiles , has a much lower resolution power than mlst and aflp . this is due to the fact that the dna - based methods take every nucleotide difference into account , including those that are silent at the protein level , and also that differences in protein sequences may not necessarily result in visible differences in electrophoretic mobility . thus , mlee does not clearly differentiate some of the major phylogenetic clusters in the b. cereus group population ( 1,5,6 ) . mlst+aflp+mlee supertree is merely provided as a tool for exploring the phylogenetic positions of the 70 isolates that have been typed only by mlee and which are thus not included in the reference figure 1.weighted supertree of 2143 b. cereus group isolates based on phylogenetic information from five mlst schemes and three aflp studies available in hypercat . the supertree was inferred following the mrp strategy , with a weight of 40 or 68 given to the aflp studies and a weight of 1 given to each of 26 mlst genes . superclusters of genetically closely related isolates ( sharing identical mlst or aflp profiles ) that have been computed independently using a recursive approach and that contain 10 or more isolates have been mapped onto the supertree and are listed on the right ( the supercluster descriptions apply to most isolates of the superclusters , not necessarily all ) . weighted supertree of 2143 b. cereus group isolates based on phylogenetic information from five mlst schemes and three aflp studies available in hypercat . the supertree was inferred following the mrp strategy , with a weight of 40 or 68 given to the aflp studies and a weight of 1 given to each of 26 mlst genes . superclusters of genetically closely related isolates ( sharing identical mlst or aflp profiles ) that have been computed independently using a recursive approach and that contain 10 or more isolates have been mapped onto the supertree and are listed on the right ( the supercluster descriptions apply to most isolates of the superclusters , not necessarily all ) . even though more than 2200 b. cereus group isolates have been typed by either mlst , aflp , or mlee , there is very little overlap between the data sets investigated by the various methods . ( 10 ) confirmed that the overall phylogenetic structure of the aflp analysis was retained when combined with phylogenetic information arising from mlst and mlee data , as isolates from a given aflp cluster are all contained within a common subtree in the supertree and the grouping is supported by a high statistical value ( alrt probability > 0.95 ) ( supplementary figure s1 ) . ( 11 ) indicated that the phylogenetic signal coming from mlst data was conserved when combined with that coming from aflp and mlee , even though mlst was given a lower weight in the supertree reconstruction ( supplementary figure s3 ) . the integration of multiple types of phylogenetic data demonstrated that even though there is little overlap between the isolate sets analyzed by the various typing methods , the supertrees obtained in the integrated analyses appeared to properly reconstruct the major groupings in the population and to be robust to missing data , a behavior that has been observed in numerous cases and for various datasets in earlier studies ( 3234 ) . overall , a large proportion of the branches ( 70% ) in the supertrees have strong statistical support ( alrt confidence level > 95% ) . the impact of missing data was further tested by including in the supertree four replicates of the 34 strains for which mlst , aflp and mlee data are available , using either all data or data from one of the three typing methods . to complement the supertree approach , a tree - independent clustering procedure was applied in order to group isolates that share identical typing data . the procedure is analogous to that of computing clonal complexes of strains as is typically done with mlst data ( 35,36 ) , but with the objective of using information from the multiple b. cereus group mlst schemes as well as results from the multiple aflp studies . an algorithm was designed to identify groups of isolates that share identical mlst and/or aflp profiles ( figure 2 ) . as a result of this procedure , every isolate of a given supercluster shares identical genotyping data by at least one typing method to at least one other isolate in the group ( figure 2a ) . in this analysis , a total of 283 superclusters were identified , comprising a total of 1152 isolates , while the remaining 991 isolates were singletons . because of little overlap between the isolate sets analyzed by the different typing schemes , 90% of the superclusters were composed only of isolates that have been typed by the same method or scheme , and most of the clusters ( 85% ) were small ( less than five isolates ) . this was confirmed by mapping the 16 superclusters containing 10 isolates or more onto the mlst+aflp supertree , showing that , with a single exception among 431 isolates ( b. cereus f4430/73 in supercluster 157 ) , isolates belonging to a given supercluster branched in the same subtree or nearby parts of the supertree ( figure 1 ) . the exception could be explained by the fact that b. cereus f4430/73 has been typed by more methods than the other members of supercluster 157 , creating a bias in the analysis due to variable amounts of data . in addition , pressing one of the three buttons above the table allows to view the phylogenetic positions of the isolates in the multi - datatype supertree using archaeopteryx , to extract from the supertree the subtree containing only the isolates forming the supercluster , or to display a network graph showing the specific typing data shared between the isolates of the supercluster . superclusters were built using a recursive approach , grouping strains that shared identical sequences at all loci of at least one of the mlst schemes and/or have identical aflp profiles . each supercluster is represented by a pie proportional to the number of isolates in the supercluster and the pie is divided according to the species or sources of the isolates included in the supercluster . in addition , pressing one of the three buttons above the table allows to view the phylogenetic positions of the isolates in the multi - datatype supertree using archaeopteryx , to extract from the supertree the subtree containing only the isolates forming the supercluster , or to display a network graph showing the specific typing data shared between the isolates of the supercluster . superclusters were built using a recursive approach , grouping strains that shared identical sequences at all loci of at least one of the mlst schemes and/or have identical aflp profiles . each supercluster is represented by a pie proportional to the number of isolates in the supercluster and the pie is divided according to the species or sources of the isolates included in the supercluster . hypercat currently contains 2262 isolates covering the diversity of the b. cereus group , including 110 b. anthracis , 841 b. cereus , 511 b. thuringiensis , 54 b. weihenstephanensis , 115 b. mycoides , 8 b. pseudomycoides and 623 unclassified b. cereus group isolates . the database provides all the genotypes that are represented for each typing scheme or method and for combinations of typing methods , with tables listing the sets of isolates that have unique or identical genotyping profiles . also , the phylogenetic position of isolates having a particular profile can be highlighted in the mlst+aflp or mlst+aflp+mlee supertree . in both archaeopteryx and treebolic views , branches of the supertrees are colored according to the seven main phylogenetic clusters of the b. cereus group population defined by ref . ( 10 ) , which gives a global overview of the phylogenetic classification of the strains . the superclusters of isolates sharing identical mlst and/or aflp profiles can be explored using tabular and graphical displays . tables list the detailed composition of the superclusters , and functions allow to view the phylogenetic position of the isolates in the mlst+aflp supertree and to extract the subtree containing the isolates belonging to a given supercluster . a graphical representation of the supercluster is also provided in the form of a network showing the specific mlst and aflp profiles that are shared between the isolates ( figure 2 a ) . in addition , a graphical overview of all the superclusters is given by an interactive bubble chart , in which each cluster or singleton is represented by a pie whose size is proportional to the number of isolates in the cluster ( figure 2b ) . hypercat also offers the possibility to retrieve isolates that share identical mlst , aflp and/or mlee profiles based on user - selected subsets of isolates and typing methods , and to build superclusters following the recursive procedure described in this article . as a further addition to hypercat , the superclusters have been mapped onto the mlst+aflp supertree in order to produce a combined and integrative genetic and phylogenetic overview ( snapshot ) of the b. cereus group population ( figure 3 ) . to generate this view , the isolates belonging to a supercluster have been replaced in the supertree by a randomly chosen isolate selected to represent the supercluster , and the phylogenetic position of the supercluster is given by that isolate . this representation allows to see in a single graph the phylogenetic distribution of the superclusters and singleton isolates . the interactive snapshot contains hyperlinks ; middle - button clicking on a supercluster or isolate name will link to an information page with details about that cluster or isolate , and clicking on an internal node will load a new page displaying the subtree including all descendants of that node in the supertree ( figure 3 ) . figure 3.phylogenetic snapshot of the b. cereus group population based on mlst and aflp typing data for 2143 isolates . the supertree is represented as an unrooted network in which the closely related strains belonging to a supercluster have been replaced by a representative isolate . superclusters are drawn as filled lightblue boxes , while singleton isolates are drawn as ellipses colored by bacterial species ( red , b. anthracis ; blue , b. cereus ; green , b. thuringiensis ; brown , b. mycoides ; purple , b. pseudomycoides ; orange , b. weihenstephanensis ; black , uncharacterized b. cereus group isolate ) . clicking on an internal node loads a new page displaying the subtree including all descendants of that node in the supertree . phylogenetic snapshot of the b. cereus group population based on mlst and aflp typing data for 2143 isolates . the supertree is represented as an unrooted network in which the closely related strains belonging to a supercluster have been replaced by a representative isolate . superclusters are drawn as filled lightblue boxes , while singleton isolates are drawn as ellipses colored by bacterial species ( red , b. anthracis ; blue , b. cereus ; green , b. thuringiensis ; brown , b. mycoides ; purple , b. pseudomycoides ; orange , b. weihenstephanensis ; black , uncharacterized b. cereus group isolate ) . clicking on an internal node loads a new page displaying the subtree including all descendants of that node in the supertree . the view in the upper right corner shows the display of the supertree in hyperbolic space using treebolic2 . in addition to the phylogenetic analysis tools described above , hypercat also contains a number of pages presenting various statistics relating to strain distribution , data content and overlap , as well as descriptions of the supertree and supercluster building procedures . all data in hypercat , including tabular isolate and genotype listings , superclusters , supertrees and networks , can be saved and downloaded freely from the database . as mlst is currently the gold standard for bacterial typing , the supercat database devoted to multi - scheme mlst analysis of the b. cereus group , containing 1430 isolates , has been upgraded accordingly . the superclustering tool and associated graphical and tabular displays developed for hypercat have been incorporated into supercat for analysis of mlst data only , for identifying groups of isolates sharing identical mlst profiles in any of the five schemes available . an mlst - based genetic and phylogenetic population snapshot combining supertree and furthermore , for the reconstruction of the multi - scheme mlst supertree by the mrp procedure , the parsimony step , which was originally carried out using the mix program of the phylip package ( 43 ) , is now computed using tnt ( 23,24 ) . in order to serve the b. cereus group research community with the most comprehensive genotyping resource , we have developed at the university of oslo s typing website ( http://mlstoslo.uio.no/ ) a new database , hypercat . this database incorporates analysis tools combining genotyping data not only from multiple mlst schemes , as provided in the supercat database , but also from the altogether different approaches aflp and mlee . while genotyping of the b. cereus group of bacteria is currently essentially done using mlst , hypercat provides a means to combine mlst data with the phylogenetic information from hundreds of strains that have been typed using other methods , and therefore generates a truly global genetic snapshot of the b. cereus group population with moderate to high representativity of all member species . the multi - datatype supertrees and superclusters developed herein provide a common basis for revealing and displaying genotypic relationships among b. cereus group isolates , based on the large amounts of heterogeneous typing data available for this group of bacteria , an effort which would not be straightforward by mere inspection and comparison of results from the two dozens of publications describing mlst , mlee and aflp analyses of the b. cereus group population . funding for open access charge : norwegian functional genomics ( fuge ii ) platform of the research council of norway and the university of oslo .

the application of genomic technologies to cancers has revealed that patients with tumors that appear indistinguishable to the clinician may have completely different causes at the molecular level [ 1 , 2 ] resulting in very different prognoses and responses to possible treatments . nonetheless , most human cancers seem to follow a relatively small number of progression pathways [ 1 , 2 , 5 ] , each characterized by an approximately equivalent sequence of mutations . this observation is key to the success of targeted therapeutics , a groundbreaking approach to cancer treatment in which drugs are developed to treat specific molecular abnormalities shared by large subgroups of patients . by identifying common progression pathways and characterizing their conserved features , it is hoped that we can find new subgroups of patients who will respond to a common treatment , identify the specific abnormalities that will provide effective therapeutic targets for those subgroups , and develop clinically useful diagnostic tests to identify new patients in those subgroups . one of the significant challenges to identifying and characterizing progression pathways is the heterogeneity of cancers both within and between patients . any two patients , even with a common progression pathway , will exhibit many differences in the details of the causal mutations along that pathway , as well as in the assortment of random passenger mutations distinct to each patient that do not contribute to their pathology . even within a single patient , a tumor will generally be highly heterogeneous , with genetically distinct cell populations corresponding to different stages along the progression of their tumor and possibly even different branches along those progression pathways within a single tumor . this heterogeneity is problematic for methods for profiling tumor states , since there is at present no technology to determine the genetic states of single cells at a genomic scale . genome - wide methods for tumor profiling such as expression microarrays , rna - seq , or array comparative genomic hybridization ( acgh)necessarily mix contributions from many discrete cell types . this mixing would be expected to result in a conflation of distinct states along a progression pathway , obscuring characteristics of individual subpopulations of cells and hiding the discrete steps in progression that may provide clinically valuable markers of early stages in progression or important clues to major decision points in a tumor 's evolution . this heterogeneity is particularly challenging to phylogenetic approaches to inferring tumor progression , which depend on our ability to at least approximately identify discrete steps in tumor evolution and can benefit greatly from information about ancestral states and the combinations of states present in distinct tumor samples . one approach is to use alternative technologies designed to profile single cells as a way of directly observing discrete states within tumors . this approach has been successfully used for tumor phylogeny inference from single cell fluorescent in situ hybridization ( fish ) data [ 12 , 13 ] . using single - cell assays has substantial drawbacks , however , because single - cell technologies can profile only a few preselected markers per cell . an alternative is to separate cells into approximately homogeneous populations prior to applying genomic methods , as was done recently by , who used a combination of microdissection and post - dissection cell sorting to separate discrete sub - populations of cells prior to whole - genome dna copy number profiling by acgh . a third alternative , used in the present work , is to apply genomic technologies to heterogeneous samples but attempt to computationally separate distinct cell populations from the outputs of these samples . such computational unmixing methods have been previously used in tumor analysis to correct for stromal contamination of tumor cells and have been useful to similar applications of evolutionary inference from heterogeneous samples , such as in reconstructing evolutionary steps in viral quasispecies . in previous work , we proposed the use of such unmixing methods for identifying cell states for phylogeny inference and demonstrated their ability to separate biologically meaningful tumor cell populations from expression microarray data and acgh data . in this paper , we build on that prior work by developing a pipeline for converting inferred cell profiles into phylogenetic trees describing likely stages of tumor progression and common progression pathways by which they evolve . the first applies our prior unmixing model to infer profiles of major progression steps from heterogeneous tumor data . the second step uses a novel statistical test to identify amplified genomic regions that can serve as markers of progression . the third step then uses a second statistical approach to call these markers as amplified or nonamplified in individual inferred cell states , creating a matrix of phylogenetic states suitable for character - based phylogenetic inference . the fourth step then applies maximum parsimony phylogeny inference to the resulting data to identify likely progression trees , labeled by changes in the marker set inferred in step two . these progression trees establish a model of tumor evolution identifying discrete steps of progression among these markers and possible ancestral stages of tumor progression not directly apparent from the identified components . validation on simulated data demonstrates the effectiveness of the method at identifying markers , assigning them to progression states , and inferring trees from those states . application to real breast cancer cgh data results in a phylogeny that recapitulates key features of our current understanding of major breast cancer progression pathways while elaborating in several potentially significant ways . the work represents , to our knowledge , the first use of character - based phylogenetic inference for similar whole - genome tumor profiles , providing advantages over prior distance - based approaches in identifying likely markers and describing specific mutations that may underlie key steps in tumor progression . in the remainder of this paper , we present our method and an application to a publicly available acgh data set . in section 2.1 , we describe our overall phylogenetic inference pipeline and the novel computational and statistical methods developed for it . in section 2.2 , we provide details on specific use of the methods developed here and their application to the analysis of the breast tumor acgh data of . in section 3 , we present the results , identifying a set of phylogenetic markers and a resulting tumor phylogeny . in section 3 , we also discuss the biological significance of the results , examining both their concordance with prior literature and interesting novel predictions of the methods . at a high level , our method consists of an analysis pipeline to convert raw data on profiles of heterogeneous tumor samples into phylogenetic inferences on computationally inferred profiles of discrete cell states . while the method can in principle work with any technology for profiling tumor state , we assume in the present work that we are specifically using acgh data describing dna copy numbers at a discrete genome - wide probe set . the data are assumed to be in the form of copy numbers of n probes in m tumors or tumor sections . these data are assumed to be raw or baseline normalized raw input , rather than the conventional log ratios . the pipeline consists of the following steps : computational unmixing of raw acgh data to infer acgh profiles of well - populated tumor states , identification of significantly amplified marker regions of the genome from the component acgh data , assignment of marker states to components , and phylogenetic inference on cell states to produce an inferred progression tree . we initially accomplished this assignment using an unmixing method previously developed by our group based on an interpretation of the problem as that of fitting a simplex to an observed set of data points , where simplex vertices will then correspond to inferred components of the mixture . the method is based on prior work by ehrlich and full adapted to better handle the high dimension and noise level characteristic of genomic data . we have since updated that method to use nonnegative matrix factorization ( nnmf ) to eliminate the possibility of negative copy number values and other artifacts that can induce in the code . we first preprocess the data by applying l1-l1 total variation denoising to the raw acgh profiles . in the initial method , we then use principal components analysis ( pca ) to convert acgh profiles of tumor samples to points in a low - dimensional space . the acgh profiles are then explained as mixtures drawn from a set of common cell types by fitting a simplex to the point set , with some allowance for noise in the data . any point in the simplex can then be explained as a linear combination of the vertices of the simplex . these vertex points are interpreted as the cell types from which each tumor sample is generated and can be projected back into the original dimension of the acgh array to construct virtual acgh profiles of the inferred cell types . the outputs of the method are an inferred set of mixture components , identifying a projected copy number of each cell type at each probe , and a set of mixture fractions , explaining each observed tumor sample as a sum of fractional contributions of cell types . the mixture components can be represented as a matrix c in which each entry cij describes the inferred copy number of component or cell type i at acgh probe j. for the present pipeline , we use only the component matrix c and discard the mixture fractions . space does not permit a detailed description of the method , so we refer the reader to for a more thorough description of our general unmixing strategy for tumor phylogenetics and to for a detailed discussion of the specific noise - tolerant unmixing algorithm used in our primary results here . our most recent algorithm functions identically except that initially dimensionality reduction is accomplished by nnmf rather than pca and an additional nonnegativity constraint is imposed during the optimization of components c. the primary results below are based on components previously determined in tolliver et al . by the pca - based method , although the improved method is applied to develop components from simulated data and from a secondary breast cancer data set to provide additional points of comparison . once we have the inferred components , it is next necessary to identify markers for tracking phylogenetic state . for acgh data , unmixing is performed in the linear , rather than log , domain , and a deletion represents only a small linear change in copy number , so we expect the method to have poor sensitivity to deletions . given the high variability from probe to probe in the data , it is necessary to use a statistically robust test for amplification . to accomplish this , we developed a test designed to test for significant amplification of a window of w contiguous probes across the m components . we assume gaussian noise in the data , thus modeling each individual probe as drawn from a gaussian distribution with mean 1 ( corresponding to diploid dna ) . the variance is assumed to be the empirically measured variance , , across all probes in all components . we then seek to reject the hypothesis that the collection of w m probes under consideration were drawn from the corresponding gaussian . for this purpose , we take as our statistic the sum of squares of z - scores of the probe values : ( 1)xk=i=1mj = kk+w1(cij1)2 , where k is the index of the first probe in the window . under the null hypothesis , this statistic would be expected to be chi - square distributed with w m degrees of freedom . we thus test for significant amplification with a one - sided chi - square significance test for the appropriate degrees of freedom . we apply this test to sliding windows of probes of fixed width w across the genome . after identification of discrete amplified windows , we apply a postprocessing step to collapse any overlapping amplified windows into a single larger window and treated the union of probes in all overlapping significant windows as the marker for subsequent analysis . we would normally expect the detected regions to be a subset of those one would find by performing a comparable statistical test on the raw acgh measurements rather than the inferred components , as we would expect that features that are not robust to a significant fraction of samples will be interpreted as noise and suppressed at the unmixing step . the scan for significant windows was done through custom matlab code using the chi2cdf function for chi - square significance testing . after identifying a set of markers , we next need to determine the states of those markers in each inferred cell component . for this purpose , we again treat the problem as that of attempting to reject the hypothesis that the individual copy numbers are drawn from a gaussian of mean 1 and variance corresponding to the empirically measured variance across all probes . for each component i and marker j , we compute the mean copy number over all probes in the given marker for the given component : ( 2)ij=1bjaj+1k = ajbjcik , where aj is the leftmost probe index and bj the rightmost probe index for marker j. we then evaluate the single - sided p value for the hypothesis that ij is drawn from a gaussian with mean 1 and variance /(bj aj + 1 ) , where is again the empirical variance across all probes in all components . we implicitly build in a prior probability that any given marker is not amplified in any given component by using a p value cutoff of 0.001 for calling a probe amplified . the result of this analysis is an assigned state ( amplified or not amplified ) for each component at each phylogenetic marker . these values can be represented as an m + 1 k matrix p of phylogenetic markers , where element pij is a binary value indicating whether marker j is amplified or not amplified in component i. custom matlab code was used to assign phylogenetic states to each component at each marker using the normcdf function . the matrix of phylogenetic marker states p produced in the previous step serves as the input to a character - based phylogenetic inference . given the lack of any sound empirical basis for setting parameters for a bayesian or maximum likelihood method , we favor use of a simpler parsimony method and therefore treat tumor phylogeny inference as the problem of finding a maximum parsimony steiner tree in which the observed components are leaves of the tree . for similar reasons , we do not weight markers , treating gain or loss of any marker as equally likely and seeking a minimum weight steiner tree capable of explaining the data . the actual phylogeny construction is accomplished with paup ( portable version 4.0b10 for unix ) . the program was run with the maximum parsimony optimality criterion using heuristic search for 10 repetitions , random sequence addition , and the tree bisection reconnection option for swapping . , we applied our methods to a set of simulated acgh data to specifically test the effectiveness of our method at identifying markers , grouping them into components , and properly placing the components in a phylogenetic tree . we simulated data for a single hypothetical chromosome of 1000 probes , assuming cell states evolve according to a binary tree from an initially diploid root state . we then assumed each of the edges would contribute a single mutation , represented as a segment of 11 consecutive probes with amplification level 20 placed uniformly at random on the simulated chromosome , rejecting placements that would place segments less than 10 probes away from another segment . we then drew 200 simulated tumor samples from this tree of components by choosing a single node at random from the tree and using all nodes on the path from the root to chosen node as the mixture components of that sample . we chose mixture fractions for the components in each simulated tumor sample by choosing uniform random weights for each component assigned to the sample and normalizing by the sum of these weights to derive fractional contributions of each component to each mixture fraction . finally , we add simulated gaussian noise to each probe value for each simulated tumor sample with mean zero and standard deviation set to 0.05 , 0.10 , 0.15 , and 0.20 in separate experiments . we then applied the nnmf - based unmixing algorithm with regularization parameter 100 and the analysis pipeline described above using a p value cutoff of 10 and window size of 5 for marker identification . we measured accuracy based on amplified segments correctly identified , components correctly identified , and tree edges correctly identified . we first assessed the fraction of the amplified segments correctly identified during marker selection for each scenario . next , we computed the fraction of components correctly identified , with an assignment judged correct if it was assigned the same state as the true component for all markers that were correctly identified in the previous step . finally , we assessed the fraction of tree edges correctly identified among those subdividing nodes correctly identified in the previous step . a tree edge was considered correct if it subdivided the node set identically in the inferred tree and in the true tree when collapsed to the subset of nodes identified correctly during the marker assignment step . all three analyses were repeated for k = 47 components for each of the four noise levels . our primary analysis consisted of application of our method to a set of previously identified mixture components derived in using a publicly available set of acgh data from sectioned primary ductal breast tumors . this dataset was selected because the sectioning and cell sorting approach developed by navin et al . was specifically chosen to facilitate phylogenetic inference and provides additional data on intratumor heterogeneity useful in validating the methods . the raw data comprises 87 tumor sectors obtained from 14 ductal breast cancer tumors run on a high - density roma platform with 83,055 probes . we confined our analysis to the twenty - two autosomal chromosomes , reducing the dataset to 78,874 probes . the raw acgh data was preprocessed and unmixed as described in our prior work . as before , data was converted from log to linear domain , denoised with a total variation denoising , and unmixed to generate components . six components were chosen , as described in the prior work , based on an analysis of the eigen - decomposition of the data . the resulting components are the same as those described in that prior paper and we refer the reader there for detailed information on the unmixing method and its application to this data set . we used a window size of w = 20 for the initial sliding - window scan of the genome . the p value threshold for each window in isolation was set to 10 to account for bonferroni correction for the 78,855 sliding windows of size 20 possible for the 78,874 probes . this threshold corresponds to a corrected p value threshold of 7.9 10 . after collapsing overlapping windows , we found a total of 27 phylogenetic marker regions significantly amplified across samples . in order to investigate the possible biological significance of these markers , we identified all genes overlapping the probe set for each marker region using the ucsc genome browser applied to the human reference genome build 17 ( ncbi35 ) . we use ncbi build 35 , rather than a more recent build , to conform to the acgh platform specifications . we further attempted to identify any genes with a known association with cancer by manually examining online mendelian inheritance in man ( omim ) entries for all genes overlapping the probes , specifically noting those with a prior association with cancers in general or breast cancers specifically . as a secondary validation of our approach , we applied it to a second set of mixture components derived from a second publicly available second breast cancer acgh dataset consisting of 44 predominantly advanced primary breast tumors and 10 breast cancer cell lines . the dataset consists of 59 samples and 6691 probes each corresponding to a single gene , making it substantially lower in resolution than the navin et al . we ran our recent nnmf - based unmixing method with tv denoising regularization parameter 6 and , unmixing parameters k = 6 components and = 100 regularization , with window size 20 and bonferroni corrected p - value cutoff 1.7 10 . while the lower resolution of the data prevented direct comparison to the navin et al . results , we evaluated the method based on its ability to identify four markers ( on 1q , 8q , 17q and 20q ) specifically cited by the authors of the study as well as others that showed up as important markers in the analysis of the navin et al . surprisingly , marker - level accuracy generally improves with increasing component numbers but appears relatively insensitive to noise level over the ranges examined here . component - level accuracy shows a more complicated profile , with generally worse performance for larger numbers of components at any given noise level . analysis of specific identified components suggests a common error is the identification of more than one inferred component closely corresponding to a single true component , leading to other true components getting omitted from the data . the accuracy of tree edges in partitioning the identified components is 100% across most noise levels and component numbers , except for 20% noise and 15% noise for k = 6 components and 20% noise for k = 7 components . it is important to note , though , that we defined these error measures so that the method would not be penalized for failed marker detection in assessing component or tree edge detection nor be penalized for failed component detection in assessing tree edge detection . this decision was motivated by a desire to assess the accuracy of each step independent of the others . the reported accuracies would appear more pessimistic if we counted components correct only if all markers were detected or counted tree edges incorrect if the components they separate were not detected . application of our analysis to the data yielded six components corresponding to inferred cell states , in addition to a seventh normal cell type added to root the subsequent tree . the components themselves and a detailed analysis of those components and the associated mixture fractions are provided in our prior work and we therefore refer the reader to that prior literature for a detailed discussion of the mixture components by themselves . the analysis yielded a total of 27 nonoverlapping regions at which the components collectively showed significant amplification . in addition , we provide a list of genes overlapping the regions that have some known association to cancers . most of the regions contain at least one gene known to have some prior association with cancers , including several genes specifically associated with breast cancers ( cd55 , mdm4 , wnt2 , erbb2 , grb7 , bcas , ccne , cttn , aurka , bcl2 , myc , tnfrsf11a , znf217 , cyp24a1 ) . in several other cases , a region lacking known cancer - associated genes is found adjacent to one with a known association and might be presumed to be part of a common amplicon ( e.g. , 18q22.2 - 18q22.3 ) . these regions overlap a total of 343 genes , of which 56 ( 16.3% ) were manually found to be associated with cancers in omim . it is difficult to rigorously establish a global frequency with which genes are cancer related , but we can derive an estimate by reference to the work bajdik et al . , who used a text - mining approach to determine that 1,943 genes as of the time of their work were annotated as cancer - related in omim . comparing this number to the number of refseq transcripts , 27,704 ( ncbi genome build 35 ) , provides an estimate that 7.01% of all genes are annotated as cancer - associated in omim . the comparison suggests that the marker regions identified by our study are strongly enriched for known cancer - related genes . a chi - squared statistical test shows this difference in frequencies to be highly significant ( chi - square score 43.2 , p value < 0.0001 ) . we would expect the unmixing to screen out amplifications that occur in only a small fraction of samples , leading to the discovery of fewer but more robust markers than would be found from the raw acgh data . to test that assumption this process yielded 47 marker regions , including 24 of the 27 found from the unmixed data . three markers ( markers 6 , 22 , and 23 ) , we do not provide the complete list of markers obtained from the raw data . we next assigned states to each of the identified marker regions in each component . we further manually examined the copy number profiles for the predicted components in each marker region . figure 4 provides two illustrative examples , showing the inferred copy number data for the six components and identifying those components determined to be amplified versus nonamplified . figure 4(a ) shows the inferred profile for marker 1 , corresponding to locus 1q32.1 - 1q32.2 . c1 , c3 , c4 , and c5 are determined to be amplified , which appears to provide a good correspondence to those with copy numbers significantly above one . it is worth noting , however , that there is a finer resolution of amplification apparent in the figure 4(a ) : c1 shows broad but low amplification across the region , c3 shows a more specific amplification of the subregion approximately from probes 5250 to 5300 , and c4 shows a distinct pattern of multiple amplicons across the region . these observations suggest the marker - identification method is performing well at a coarse resolution but that there is considerable finer - scale structure that could in principle exploited by a more sophisticated marker selection strategy , particularly where contiguous regions show distinct patterns of amplification . figure 4(b ) shows a second example , the inferred copy number profile for marker 20 , corresponding to an amplicon at 17q12 - 17q21.2 . we would expect this site to be picked up as a marker and to show high amplification , since it is the site of the her-2 locus . the region again shows a strong but selective amplification , with c5 and c6 highly amplified ( although with distinct fine - scale structures ) , c4 slightly amplified , and others showing no amplification . the result again confirms that the method produces correct answers at a coarse resolution , although there may be a finer - scale structure that could exploited by a more sophisticated method . figure 5 shows the phylogenetic tree produced from the six inferred progression components and the additional normal component manually added to the analysis . the majority of markers are gained at a unique point in the tree and never subsequently lost . marker 9 ( 8q12.1 ) is lost in the tree in the transition to component c4 . in addition , some markers are inferred to be gained more than once in the tree . most notable of these is the collection of 17q markers , which are gained separately in the subtree leading to component 6 and that leading to steiner node 8 and then to components 4 and 5 . application to a second component set derived from the lower - resolution data of pollack et al . provides a secondary validation of the reproducibility of the results on distinct datasets , acgh platforms , and unmixing methods for a common tumor type . the lower resolution of the data leads to substantially more possible genes per amplicon than were found with the navin et al . we therefore must compare the two results more indirectly based on markers reported by pollack et al . in their own analysis of their data as well as known breast cancer markers found in the primary analysis of the navin et al . described finding 1q , 8q , 17q , and 20q as predominantly amplified regions in the data , and our method did find sizeable amplicons on each of these regions . other amplicons appear to correspond to several important tumor markers , including the her2 , ccnd1 , c - myc , and ccne1 loci noted in the analysis of the navin et al . data as well as the fgfr1 locus that is conspicuously absent from our analysis of the navin et al . data . of note , the ccne1 locus is found as a significant marker when analyzing the unmixed components but is not detected by a similar marker analysis of the raw data without unmixing . all other markers found in the unmixed data are also found in the raw data , as was observed with the navin et al . figure 6 shows the inferred phylogenetic tree . for these data , it was not necessary to add a normal root component c0 , as was done with the navin et al . data , because the method directly inferred component c1 to be nonamplified at all markers and thus to serve as the expected normal root . analysis on simulated data shows the method to have generally good accuracy at identifying amplified markers , identifying complete components with defined patterns of marker amplification , and grouping these components into phylogenies . the dependence of accuracy on various model parameters is difficult to analyze , with generally better marker - level accuracy but worse component - level and tree - edge - level accuracy as greater numbers of components are modeled . examination of different noise levels , chosen to roughly approximate noise levels observed on the real data , shows no strong dependence within a range of 520% noise . overall , the results suggest that methods show good although far from perfect performance , picking out 79.2% of true markers and greater than 72.8% of true components in most scenarios and correctly identifying 94.8% of tree edges dividing the identified components . the high specificity of the marker assignment , with no false positives observed in any of the tests , suggests that there may be room to tune the methods to improve accuracy by trading off sensitivity for a somewhat higher rate of false positives . while simulated data provides some assessment of the effectiveness of the method , however , there are many features of tumor evolution that are not yet well enough understood to permit a faithful simulation of real tumor data . in assessing our methods , we must therefore rely primarily on more indirect validation on real data . there is no closely comparable method to ours of which we are aware that we could use as a basis for comparison and we therefore validate the results on the navin et al . one could in principle validate our results against recent work of navin et al . using single - cell analysis of the subsections of tumor 10 analyzed here . navin 's phylogenetic approach , however , leads to progression trees dominated by changes in overall ploidy , which is not examined in our trees and precludes any direct comparison . as noted previously , a majority of the markers we find correspond to some genes with known cancer associations . these include well - characterized breast cancer amplicons at 17q , 11q , and 20q [ 2830 ] . the most notable absence among well - known breast cancer markers would be the 8p locus associated with the gene fgfr1 . a majority of the markers ( 16 of 27 ) include genes with some annotated relationship with cancers , although only 7 of those ( markers 1 , 12 , 13 , 20 , 22 , 24 , and 26 ) are annotated in omim as specifically associated with breast cancers . of those markers lacking an annotated association with breast cancers , many are in close proximity to and inherited with breast - cancer - associated markers and might plausibly be assumed to contain distinct portions of common amplicons . table 4 identifies those proximal markers that are coinherited in the tree and likely reflect common amplicons . for example , 17q is interpreted as three distinct markers ( markers 1921 ) , and although only marker 20 contains genes with an annotated breast cancer association ( erbb2/her-2/neu , stat5 , and grb7 ) , all are inherited together apparently as a common amplicon . similar explanations can account for markers 2 on 1q , which is coinherited with marker 1 ( mdm4 ) ; markers 10 and 11 on 8q , which are coinherited with marker 12 ( myc ) ; marker 25 and 27 on 20q , which are coinherited with marker 26 ( znf217 , cyp24a1 , bcas1 , and aurka ) . in other cases , however , we observe coinherited markers for which no specific explanation is available for any of the markers . it is impossible to say purely from a computational analysis whether these represent false positives , discoveries not annotated specifically in omim , or even novel but significant associations with breast cancer progression . examining the phylogeny itself allows us to further examine the possible biological significance of the data and its concordance with current knowledge about breast cancer progression . in this regard , it is helpful to interpret the tree as a set of possible progression pathways from the healthy root cell type ( c0 ) . as the tree implies , however , different progression pathways do not function in isolation but rather may share some common features in early progression . the first internal node , steiner node 12 , is inferred to be identical to the root , but diverges at the top level into two pathways . the first such progression pathway ( c0 12 c2 ) describes a short terminal progression pathway isolated from the rest of the tree . the progression pathway is resolved only to a single step of mutation corresponding to amplification of 11q14.111q13.4 , 18q21.32 - 18q22.2 , 18q22.218q22.3 . 11q is a known breast cancer amplicon [ 29 , 30 ] and harbors ccnd1 , which has been found to be amplified in breast cancers ; fgf3 and fgf4 , which are known oncogenes ; and cttn , which is frequently overexpressed in breast cancers . the region also contains other genes , such as npat , with functions in cell cycle regulation that might be considered candidates for an oncogenic function . 18q21.32 - 18q22.2 harbors the oncogene bcl2 , which is involved in the myc pathway and tnfrsf11a , which is frequently expressed in late - stage breast cancers [ 35 , 36 ] . 18q22.218q22.3 does not carry any currently known cancer - related genes but may be gained due to proximity to 18q21.32 - 18q22.2 as part of a common amplicon . together , these abnormalities appear to define a distinct subclass of breast tumor cells with early divergence from all other cell types . within the subbranch rooted at steiner node 11 , one branch leads directly to a terminal node characterizing a second progression pathway ( c0 11 c6 ) . this progression pathway is characterized by amplification of 5q21.1 - 5q21.3 , 5q22.3 - 5q23.1 , 11q23.3 , 15q26.3 , and 19q12 and is one of two subtrees characterized by amplification of 17q11.2 , 17q12 - 17q21.2 , and 17q21.33 . the 17q region is a well - established breast cancer hotspot [ 28 , 30 ] , including genes erbb2 ( her-2/neu ) , grb7 , and stat5 . 19q12 contains ccne1 , an important prognostic marker for breast cancer progression [ 37 , 38 ] . ccne1 amplification has been specifically associated with basal - like breast cancers , but has been previously identified as coassociated with particularly aggressive her-2 positive breast tumors . our phylogeny is consistent with the notion that 17q/19q coamplification defines a distinct subtype of her-2 positive tumors . region 15q26.3 has no genes specifically noted to be breast - cancer associated in omim , although amplification of the locus was identified as predictive of recurrence in systematic breast cancers and the region contains igf1r , an antiapoptotic gene broadly amplified in cancers . the biological significance of the 5q amplicon is not apparent . while 5q22.3 - 5q23.1 has several genes associated with cancers ( e.g. , atg12 , tnfaip8 , sema6a , which are associated with lung cancer ) , they are predominantly tumor suppressors . likewise , there is no obvious relevance to the 15q amplicon , although it is close to other known 15q markers . the next major division in the tree corresponds to the branch from steiner nodes 11 to 10 , characterized by gains in 1q32.1 - 1q32.2 , 1q44 , 8q12.1 , 8q12.3 - 8q13.2 , 8q13.2 - 8q13.3 , and 8q21.11 - 8q24.3 . 1q32.1 includes the breast cancer associated gene mdm4 , a putative oncogene involved in apoptosis regulation of p53 activity , in addition to various genes associated with cancers more generally . we can suggest , then , that the 11 10 branch corresponds to a specific subset of progression pathways characterized by myc amplification and suppression of apoptosis . a third progression pathway can be identified within this branch through progression into c1 ( c0 12 11 10 c1 ) . the final step on this pathway is characterized by amplifications on 12p11.22 - 12p11.21 and 19q12 . 19q12 is the locus of ccne1 suggesting a generic connection to cell cycle control on this pathway . 12p11.22 - 12p11.21 has no known cancer - related genes but carries the apoptosis - related gene dnm1l and the telomerase - related gene ddx11 . further progression pathways diverge from steiner node 10 through steiner node 9 with gains on 5p15.33 - 5p14.2 , 20q13.12 , 20q13.2 - 20q13.32 , and 20q13.33 . the 5p amplicon contains two genes with known cancer associations , cdh18 and papd7 , although neither appears to have a known role in breast cancers specifically . 20q13.2 - 20q13.32 contains several genes associated with breast cancers , including znf217 , cyp24a1 , bcas1 , and aurka , making it difficult to ascribe a particular mechanism to this branch . within the steiner node 9 subtree , we can characterize a fourth progression pathway terminating in c3 ( c0 11 10 9 c3 ) . the final step on this progression pathway corresponds to gains on 2p12 , 3q25.1 - 3q25.2 , and 7q31.31 - 7q31.32 . the 7q31.32 marker contains the wnt2 gene associated with many cancer types , including breast cancer . 7q31.31 has no known cancer - related genes and is perhaps gained due to its proximity to 7q31.2 . 3q25.1 - 3q25.2 has been previously detected as an amplicon in fraction of breast cancers , although we can offer no mechanistic explanation for its presence . we are not aware of any prior suggestion of an association between 2p12 and cancers . the remaining two terminal nodes of the tree , c4 and c5 , appear likely to represent two steps on a common progression pathway . both branchs from steiner node 9 through 8 by acquisition of 17q11.2 , 17q12 - 17q21.2 , 17q21.33 ( the her-2 locus ) along with 11q13.2 . this subtree might thus be characterized primarily as a second her-2 positive progression group associated with gain of ccnd1 , distinct from the her-2 positive progression group terminating at c6 and associated with gain of ccne1 . c5 branches from steiner node 8 with no changes , indicating a single progression pathway corresponding to c0 11 10 9 c5 c4 . the final step in this pathway is then characterized by a series of amplifications on 5q21.1 , 5q22.3 , 12p11.22 , 15q25.2 , 15q26.3 , and loss of 8q12.1 . we would not expect loss of a previously gained marker , and can suggest that this apparent loss might be better explained as a miscall of the state of that marker . most of these loci have no annotated association with any cancers , with the only specific annotated breast cancer association being to 11q13.2 , described above . this lack of associations may again represent false positive inferences specifically associated with this component . we can suggest , however , that such markers might be have been missed if they are specific only to late progression of one subtype of her-2 positive breast tumor . summarizing across the tree , we can note that there is clear support in the prior literature for many of the specific markers , although there is little evidence one way or the other supporting the specific sequences of mutations suggested by our phylogeny analysis . chief among these would be the identification of two apparently distinct pathways to her-2/neu amplification that separate relatively early in progression and exhibit distinct sets of co - occurring amplifications . the tree suggests several distinct patterns of coamplification that may be useful in identifying or classifying novel subtypes , particularly with respect to her-2 amplifying tumors . of particular interest are the observation of two distinct her-2 amplifying subtrees , one showing coamplification with ccnd1 and c - myc and the other with ccne1 . have previously reported separate cyclin - d amplified and cyclin - e amplified subgroups of breast cancer following separate pathways of oncogenesis , with her-2/neu overexpression and c - myc amplification accompanying both subgroups . coamplification of her-2 , ccnd1 , and c - myc is supported by additional literature , with this particular coamplification associated with later or more advanced stages of breast cancer [ 40 , 49 , 50 ] . , however , do suggest that c - myc amplification should occur late in this sequence , a finding not supported by our phylogeny . other more recent work has supported the idea of her-2 and ccne1 coamplification in breast cancers [ 51 , 52 ] with scaltriti et al . specifically suggesting this coamplification as a possible mechanism for herceptin resistance in her+ breast tumors . other patterns of complication are apparent in the tree although not to our knowledge supported by prior literature or any obvious functional interpretation , for example , the observation of coamplification of loci on 5q and 15q in both her-2 amplifying subtrees provides little additional insight into breast tumor development , although it does provide some independent validation of our method . while the lower resolution of those data prevents an analysis of specific amplified breast tumor genes comparable to that done with the navin et al . data , we can nonetheless observe that the method is effective at picking out those amplicons noted by the authors of that study . furthermore , the additional markers it detects beyond those four include several of those also inferred to be important progression markers on the navin et al . data and supported by extensive prior literature , most prominently the loci of her-2 , ccnd1 , and ccne1 . these results show that the method can robustly find at least some prominent known tumor markers across two distinct sets of tumor samples using very different acgh platforms and distinct unmixing methods . the tree itself provides no obvious new insights into breast tumor progression , as the method detected only four components that were actually distinct at the level of assigned markers , with three components determined to be amplified at all markers . it is notable that the tree implies amplification of most of the identified markers in a majority of components , perhaps because of the late clinical stages of the tumor samples and the presence of cell lines that would provide reasonably homogeneous representations of advanced states of breast tumor progression . in this paper , we have developed a computational pipeline for tumor phylogeny inference from genome - scale profiles of tumor state , specifically to test the feasibility of using computational unmixing methods to circumvent the problem of cell type heterogeneity in tumor phylogeny inference . we have developed a set of statistical tests to allow us to analyze computationally inferred mixture components representing inferred profiles of well - populated cell types from which heterogeneous tumor samples can be explained to identify phylogenetic markers , assign them to specific inferred cell types , and use them in phylogenetic inference of tumor progression . we have demonstrated the approach with specific application to acgh dna copy number data , applied to a breast cancer data set , showing that the method is effective at locating biologically meaningful markers of tumor progression and assembling a biologically plausible model of breast tumor progression pathways . the inferred progression pathways provide several novel suggestions about possible steps in tumor evolution and key molecular abnormalities associated with progression . these inferences may provide useful guidance into the basic biology of tumor development as well as suggestions of possible targets for future diagnostics and therapeutics . further application to a secondary lower - resolution breast tumor data set and to a series of simulated acgh data sets provides additional evidence for the effectiveness of the method at identifying markers of tumor progression , grouping them correctly into well - represented progression states , and accurately placing these states in phylogenetic trees . validation remains a challenge for tumor phylogeny inference , as there is no alternative method by which we can determine progression pathways with certainty for any real tumor data set . simulated data can lend some confidence that the method works effectively relative to a model of the real data , as has been done here , but real tumor progression mechanisms are likely to be far more complex than our simulation models can capture . comparison to single - cell approaches like fish [ 13 , 49 , 53 ] and single - cell sequencing efforts can help to verify the pure cell states determined by the unmixing within a single sample and potentially validate some ancestral states predicted by the phylogenetic inference . fish data provides only a few markers per cell , making it infeasible for a comprehensive validation of the results of our method , but could be used prospectively on targeted markers selected from an inferred phylogeny . single - cell sequencing approaches could in principle eventually overcome this limitation given sufficient volumes and quality of data . other sources of data in which more information is available about the true pathways of progression might also be useful . while we know of no such data currently available , one might in principle construct such a data set by , for example , studying discrete passages of cell lines or through the use of animal models in which one can monitor tumor development and progression over time . while gathering such a data set would be beyond the scope of the present work , it could in principle provide a basis for a more thorough future assessment of the accuracy of the pipeline implemented here or other methods for the problem of tumor phylogeny inference . while this pilot study was intended to establish the feasibility of an unmixing approach to tumor phylogenetics , there are many ways by which the work might be advanced in the future . it will be important to further establish the reproducibility of the specific markers and phylogenetic pathways in additional breast tumor datasets . novel markers found to be robustly predictive of particular progression pathways will ultimately need to be experimentally verified . in addition , it will be important to establish that the approach is applicable to other forms of tumors . the approach developed here depends on use of an unmixing method for identifying progression states , a problem which itself might benefit from improvements in the model and algorithms to more precisely fit the kind of sparse noisy data characteristic of tumor data sets . adapting the methods to more reliable data types , such as next generation sequencing data , may also prove valuable in that regard . the results on marker detection suggest there is room for improvement in more precisely determining the fine - scale structure of specific amplicons , especially when contiguous regions show distinct patterns of amplification across components . likewise , there would appear to be room for improvement in better discriminating between normal and slightly elevated copy numbers . it is a weakness of the general approach that , because the unmixing models must work in linear rather than log space , they have difficulty distinguishing the relatively small linear change between normal and deleted regions . improving sensitivity for deletions , or for subtler variations among amplification levels , may provide additional data for phylogeny construction . finally , the phylogeny construction itself used a standard parsimony method not specifically tailored to tumor progression . this parsimony model has advantages in not requiring parameters for which there is currently no empirical basis and in allowing us to test for unexpected behavior , such as loss of previously amplified regions , that can help to validate the method . nonetheless , there is now sufficient data that one might in principle learn more sophisticated probabilistic models of cancer progression or of the behavior of particular amplicons and build these models into the phylogeny inference .

tumorigenesis can in principle result from many combinations of mutations , but only a few roughly equivalent sequences of mutations , or progression pathways , seem to account for most human tumors . phylogenetics provides a promising way to identify common progression pathways and markers of those pathways . this approach , however , can be confounded by the high heterogeneity within and between tumors , which makes it difficult to identify conserved progression stages or organize them into robust progression pathways . to tackle this problem , we previously developed methods for inferring progression stages from heterogeneous tumor profiles through computational unmixing . in this paper , we develop a novel pipeline for building trees of tumor evolution from the unmixed tumor data . the pipeline implements a statistical approach for identifying robust progression markers from unmixed tumor data and calling those markers in inferred cell states . the result is a set of phylogenetic characters and their assignments in progression states to which we apply maximum parsimony phylogenetic inference to infer tumor progression pathways . we demonstrate the full pipeline on simulated and real comparative genomic hybridization ( cgh ) data , validating its effectiveness and making novel predictions of major progression pathways and ancestral cell states in breast cancers .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusion

nonetheless , most human cancers seem to follow a relatively small number of progression pathways [ 1 , 2 , 5 ] , each characterized by an approximately equivalent sequence of mutations . by identifying common progression pathways and characterizing their conserved features , it is hoped that we can find new subgroups of patients who will respond to a common treatment , identify the specific abnormalities that will provide effective therapeutic targets for those subgroups , and develop clinically useful diagnostic tests to identify new patients in those subgroups . one of the significant challenges to identifying and characterizing progression pathways is the heterogeneity of cancers both within and between patients . genome - wide methods for tumor profiling such as expression microarrays , rna - seq , or array comparative genomic hybridization ( acgh)necessarily mix contributions from many discrete cell types . this mixing would be expected to result in a conflation of distinct states along a progression pathway , obscuring characteristics of individual subpopulations of cells and hiding the discrete steps in progression that may provide clinically valuable markers of early stages in progression or important clues to major decision points in a tumor 's evolution . this heterogeneity is particularly challenging to phylogenetic approaches to inferring tumor progression , which depend on our ability to at least approximately identify discrete steps in tumor evolution and can benefit greatly from information about ancestral states and the combinations of states present in distinct tumor samples . this approach has been successfully used for tumor phylogeny inference from single cell fluorescent in situ hybridization ( fish ) data [ 12 , 13 ] . using single - cell assays has substantial drawbacks , however , because single - cell technologies can profile only a few preselected markers per cell . such computational unmixing methods have been previously used in tumor analysis to correct for stromal contamination of tumor cells and have been useful to similar applications of evolutionary inference from heterogeneous samples , such as in reconstructing evolutionary steps in viral quasispecies . in previous work , we proposed the use of such unmixing methods for identifying cell states for phylogeny inference and demonstrated their ability to separate biologically meaningful tumor cell populations from expression microarray data and acgh data . in this paper , we build on that prior work by developing a pipeline for converting inferred cell profiles into phylogenetic trees describing likely stages of tumor progression and common progression pathways by which they evolve . the first applies our prior unmixing model to infer profiles of major progression steps from heterogeneous tumor data . the second step uses a novel statistical test to identify amplified genomic regions that can serve as markers of progression . the third step then uses a second statistical approach to call these markers as amplified or nonamplified in individual inferred cell states , creating a matrix of phylogenetic states suitable for character - based phylogenetic inference . the fourth step then applies maximum parsimony phylogeny inference to the resulting data to identify likely progression trees , labeled by changes in the marker set inferred in step two . these progression trees establish a model of tumor evolution identifying discrete steps of progression among these markers and possible ancestral stages of tumor progression not directly apparent from the identified components . validation on simulated data demonstrates the effectiveness of the method at identifying markers , assigning them to progression states , and inferring trees from those states . application to real breast cancer cgh data results in a phylogeny that recapitulates key features of our current understanding of major breast cancer progression pathways while elaborating in several potentially significant ways . the work represents , to our knowledge , the first use of character - based phylogenetic inference for similar whole - genome tumor profiles , providing advantages over prior distance - based approaches in identifying likely markers and describing specific mutations that may underlie key steps in tumor progression . in the remainder of this paper , we present our method and an application to a publicly available acgh data set . in section 2.1 , we describe our overall phylogenetic inference pipeline and the novel computational and statistical methods developed for it . in section 2.2 , we provide details on specific use of the methods developed here and their application to the analysis of the breast tumor acgh data of . in section 3 , we present the results , identifying a set of phylogenetic markers and a resulting tumor phylogeny . in section 3 , we also discuss the biological significance of the results , examining both their concordance with prior literature and interesting novel predictions of the methods . at a high level , our method consists of an analysis pipeline to convert raw data on profiles of heterogeneous tumor samples into phylogenetic inferences on computationally inferred profiles of discrete cell states . while the method can in principle work with any technology for profiling tumor state , we assume in the present work that we are specifically using acgh data describing dna copy numbers at a discrete genome - wide probe set . the pipeline consists of the following steps : computational unmixing of raw acgh data to infer acgh profiles of well - populated tumor states , identification of significantly amplified marker regions of the genome from the component acgh data , assignment of marker states to components , and phylogenetic inference on cell states to produce an inferred progression tree . we initially accomplished this assignment using an unmixing method previously developed by our group based on an interpretation of the problem as that of fitting a simplex to an observed set of data points , where simplex vertices will then correspond to inferred components of the mixture . in the initial method , we then use principal components analysis ( pca ) to convert acgh profiles of tumor samples to points in a low - dimensional space . the acgh profiles are then explained as mixtures drawn from a set of common cell types by fitting a simplex to the point set , with some allowance for noise in the data . these vertex points are interpreted as the cell types from which each tumor sample is generated and can be projected back into the original dimension of the acgh array to construct virtual acgh profiles of the inferred cell types . the outputs of the method are an inferred set of mixture components , identifying a projected copy number of each cell type at each probe , and a set of mixture fractions , explaining each observed tumor sample as a sum of fractional contributions of cell types . the mixture components can be represented as a matrix c in which each entry cij describes the inferred copy number of component or cell type i at acgh probe j. for the present pipeline , we use only the component matrix c and discard the mixture fractions . by the pca - based method , although the improved method is applied to develop components from simulated data and from a secondary breast cancer data set to provide additional points of comparison . for acgh data , unmixing is performed in the linear , rather than log , domain , and a deletion represents only a small linear change in copy number , so we expect the method to have poor sensitivity to deletions . given the high variability from probe to probe in the data , it is necessary to use a statistically robust test for amplification . after identification of discrete amplified windows , we apply a postprocessing step to collapse any overlapping amplified windows into a single larger window and treated the union of probes in all overlapping significant windows as the marker for subsequent analysis . we would normally expect the detected regions to be a subset of those one would find by performing a comparable statistical test on the raw acgh measurements rather than the inferred components , as we would expect that features that are not robust to a significant fraction of samples will be interpreted as noise and suppressed at the unmixing step . after identifying a set of markers , we next need to determine the states of those markers in each inferred cell component . these values can be represented as an m + 1 k matrix p of phylogenetic markers , where element pij is a binary value indicating whether marker j is amplified or not amplified in component i. custom matlab code was used to assign phylogenetic states to each component at each marker using the normcdf function . the matrix of phylogenetic marker states p produced in the previous step serves as the input to a character - based phylogenetic inference . given the lack of any sound empirical basis for setting parameters for a bayesian or maximum likelihood method , we favor use of a simpler parsimony method and therefore treat tumor phylogeny inference as the problem of finding a maximum parsimony steiner tree in which the observed components are leaves of the tree . , we applied our methods to a set of simulated acgh data to specifically test the effectiveness of our method at identifying markers , grouping them into components , and properly placing the components in a phylogenetic tree . our primary analysis consisted of application of our method to a set of previously identified mixture components derived in using a publicly available set of acgh data from sectioned primary ductal breast tumors . was specifically chosen to facilitate phylogenetic inference and provides additional data on intratumor heterogeneity useful in validating the methods . the p value threshold for each window in isolation was set to 10 to account for bonferroni correction for the 78,855 sliding windows of size 20 possible for the 78,874 probes . we further attempted to identify any genes with a known association with cancer by manually examining online mendelian inheritance in man ( omim ) entries for all genes overlapping the probes , specifically noting those with a prior association with cancers in general or breast cancers specifically . as a secondary validation of our approach , we applied it to a second set of mixture components derived from a second publicly available second breast cancer acgh dataset consisting of 44 predominantly advanced primary breast tumors and 10 breast cancer cell lines . results , we evaluated the method based on its ability to identify four markers ( on 1q , 8q , 17q and 20q ) specifically cited by the authors of the study as well as others that showed up as important markers in the analysis of the navin et al . application of our analysis to the data yielded six components corresponding to inferred cell states , in addition to a seventh normal cell type added to root the subsequent tree . the components themselves and a detailed analysis of those components and the associated mixture fractions are provided in our prior work and we therefore refer the reader to that prior literature for a detailed discussion of the mixture components by themselves . most of the regions contain at least one gene known to have some prior association with cancers , including several genes specifically associated with breast cancers ( cd55 , mdm4 , wnt2 , erbb2 , grb7 , bcas , ccne , cttn , aurka , bcl2 , myc , tnfrsf11a , znf217 , cyp24a1 ) . it is difficult to rigorously establish a global frequency with which genes are cancer related , but we can derive an estimate by reference to the work bajdik et al . we would expect the unmixing to screen out amplifications that occur in only a small fraction of samples , leading to the discovery of fewer but more robust markers than would be found from the raw acgh data . to test that assumption this process yielded 47 marker regions , including 24 of the 27 found from the unmixed data . three markers ( markers 6 , 22 , and 23 ) , we do not provide the complete list of markers obtained from the raw data . it is worth noting , however , that there is a finer resolution of amplification apparent in the figure 4(a ) : c1 shows broad but low amplification across the region , c3 shows a more specific amplification of the subregion approximately from probes 5250 to 5300 , and c4 shows a distinct pattern of multiple amplicons across the region . provides a secondary validation of the reproducibility of the results on distinct datasets , acgh platforms , and unmixing methods for a common tumor type . described finding 1q , 8q , 17q , and 20q as predominantly amplified regions in the data , and our method did find sizeable amplicons on each of these regions . all other markers found in the unmixed data are also found in the raw data , as was observed with the navin et al . the dependence of accuracy on various model parameters is difficult to analyze , with generally better marker - level accuracy but worse component - level and tree - edge - level accuracy as greater numbers of components are modeled . the high specificity of the marker assignment , with no false positives observed in any of the tests , suggests that there may be room to tune the methods to improve accuracy by trading off sensitivity for a somewhat higher rate of false positives . while simulated data provides some assessment of the effectiveness of the method , however , there are many features of tumor evolution that are not yet well enough understood to permit a faithful simulation of real tumor data . navin 's phylogenetic approach , however , leads to progression trees dominated by changes in overall ploidy , which is not examined in our trees and precludes any direct comparison . a majority of the markers ( 16 of 27 ) include genes with some annotated relationship with cancers , although only 7 of those ( markers 1 , 12 , 13 , 20 , 22 , 24 , and 26 ) are annotated in omim as specifically associated with breast cancers . of those markers lacking an annotated association with breast cancers , many are in close proximity to and inherited with breast - cancer - associated markers and might plausibly be assumed to contain distinct portions of common amplicons . similar explanations can account for markers 2 on 1q , which is coinherited with marker 1 ( mdm4 ) ; markers 10 and 11 on 8q , which are coinherited with marker 12 ( myc ) ; marker 25 and 27 on 20q , which are coinherited with marker 26 ( znf217 , cyp24a1 , bcas1 , and aurka ) . in other cases , however , we observe coinherited markers for which no specific explanation is available for any of the markers . in this regard , it is helpful to interpret the tree as a set of possible progression pathways from the healthy root cell type ( c0 ) . as the tree implies , however , different progression pathways do not function in isolation but rather may share some common features in early progression . the first such progression pathway ( c0 12 c2 ) describes a short terminal progression pathway isolated from the rest of the tree . 11q is a known breast cancer amplicon [ 29 , 30 ] and harbors ccnd1 , which has been found to be amplified in breast cancers ; fgf3 and fgf4 , which are known oncogenes ; and cttn , which is frequently overexpressed in breast cancers . 18q21.32 - 18q22.2 harbors the oncogene bcl2 , which is involved in the myc pathway and tnfrsf11a , which is frequently expressed in late - stage breast cancers [ 35 , 36 ] . ccne1 amplification has been specifically associated with basal - like breast cancers , but has been previously identified as coassociated with particularly aggressive her-2 positive breast tumors . the 5p amplicon contains two genes with known cancer associations , cdh18 and papd7 , although neither appears to have a known role in breast cancers specifically . 20q13.2 - 20q13.32 contains several genes associated with breast cancers , including znf217 , cyp24a1 , bcas1 , and aurka , making it difficult to ascribe a particular mechanism to this branch . this subtree might thus be characterized primarily as a second her-2 positive progression group associated with gain of ccnd1 , distinct from the her-2 positive progression group terminating at c6 and associated with gain of ccne1 . we can suggest , however , that such markers might be have been missed if they are specific only to late progression of one subtype of her-2 positive breast tumor . summarizing across the tree , we can note that there is clear support in the prior literature for many of the specific markers , although there is little evidence one way or the other supporting the specific sequences of mutations suggested by our phylogeny analysis . chief among these would be the identification of two apparently distinct pathways to her-2/neu amplification that separate relatively early in progression and exhibit distinct sets of co - occurring amplifications . , however , do suggest that c - myc amplification should occur late in this sequence , a finding not supported by our phylogeny . other more recent work has supported the idea of her-2 and ccne1 coamplification in breast cancers [ 51 , 52 ] with scaltriti et al . data , we can nonetheless observe that the method is effective at picking out those amplicons noted by the authors of that study . furthermore , the additional markers it detects beyond those four include several of those also inferred to be important progression markers on the navin et al . the tree itself provides no obvious new insights into breast tumor progression , as the method detected only four components that were actually distinct at the level of assigned markers , with three components determined to be amplified at all markers . it is notable that the tree implies amplification of most of the identified markers in a majority of components , perhaps because of the late clinical stages of the tumor samples and the presence of cell lines that would provide reasonably homogeneous representations of advanced states of breast tumor progression . in this paper , we have developed a computational pipeline for tumor phylogeny inference from genome - scale profiles of tumor state , specifically to test the feasibility of using computational unmixing methods to circumvent the problem of cell type heterogeneity in tumor phylogeny inference . we have developed a set of statistical tests to allow us to analyze computationally inferred mixture components representing inferred profiles of well - populated cell types from which heterogeneous tumor samples can be explained to identify phylogenetic markers , assign them to specific inferred cell types , and use them in phylogenetic inference of tumor progression . we have demonstrated the approach with specific application to acgh dna copy number data , applied to a breast cancer data set , showing that the method is effective at locating biologically meaningful markers of tumor progression and assembling a biologically plausible model of breast tumor progression pathways . the inferred progression pathways provide several novel suggestions about possible steps in tumor evolution and key molecular abnormalities associated with progression . further application to a secondary lower - resolution breast tumor data set and to a series of simulated acgh data sets provides additional evidence for the effectiveness of the method at identifying markers of tumor progression , grouping them correctly into well - represented progression states , and accurately placing these states in phylogenetic trees . validation remains a challenge for tumor phylogeny inference , as there is no alternative method by which we can determine progression pathways with certainty for any real tumor data set . simulated data can lend some confidence that the method works effectively relative to a model of the real data , as has been done here , but real tumor progression mechanisms are likely to be far more complex than our simulation models can capture . comparison to single - cell approaches like fish [ 13 , 49 , 53 ] and single - cell sequencing efforts can help to verify the pure cell states determined by the unmixing within a single sample and potentially validate some ancestral states predicted by the phylogenetic inference . fish data provides only a few markers per cell , making it infeasible for a comprehensive validation of the results of our method , but could be used prospectively on targeted markers selected from an inferred phylogeny . while gathering such a data set would be beyond the scope of the present work , it could in principle provide a basis for a more thorough future assessment of the accuracy of the pipeline implemented here or other methods for the problem of tumor phylogeny inference . the approach developed here depends on use of an unmixing method for identifying progression states , a problem which itself might benefit from improvements in the model and algorithms to more precisely fit the kind of sparse noisy data characteristic of tumor data sets . improving sensitivity for deletions , or for subtler variations among amplification levels , may provide additional data for phylogeny construction . nonetheless , there is now sufficient data that one might in principle learn more sophisticated probabilistic models of cancer progression or of the behavior of particular amplicons and build these models into the phylogeny inference .

biodegradable polymers made from renewable resources such as agricultural wastes , corn , cassava , tapioca , whey , and so forth , do not lead to depletion of finite resources . the most studied of the biodegradable polymers include polyesters , polylactides , aliphatic polyesters , polysaccharides , and various copolymers . these biopolymers have many of the desirable physical and chemical properties of conventional synthetic polymers . the focus of this project was production of the biopolymer polyhydroxyalkanoate ( pha ) at a low cost . pha is actually a term used to describe a diverse family of polymers that are composed of 3-hydroxy fatty acid monomers . the carboxyl group of one monomer forms an ester bond with hydroxyl group of the neighboring monomer . phb has good oxygen impermeability , moisture resistance , water insolubility , and optical purity [ 4 , 5 ] . young 's modulus and tensile strength of phb are similar to polypropylene , but elongation at break is 6% as opposed to that of 400% for polypropylene . the oxygen permeability is very low , making phb a suitable material for use in packaging oxygen - sensitive products . phb has low water vapor permeability compared to other bio - based polymers but higher than most standard polyolefins and synthetic polyesters [ 5 , 8 ] . since phb is toxicologically safe , it can be used for articles which come into contact with skin , feed , or food . in the food industry , pha has a wide application as edible packaging material , coating agent , flavor delivery agent , and as dairy cream substitute [ 10 , 11 ] . it can also be used for making bottles , cosmetics , containers , pens , golf tees , films , adhesives and nonwoven fabrics , toner , and developer compositions , ion - conducting polymers , and as latex for paper coating applications [ 12 , 13 ] . the degradation products of phb are found in large concentrations in human - blood plasma , so is not toxic for human use . hence cells are first grown to high density , after which a key nutrient is limited to trigger pha synthesis . because of this dual phase process , pha production lends itself to fed - batch , as well as , continuous operation . this follows pontryagin 's maximum principle , which is an optimal feeding strategy for fed - batch fermentation . . the maximum growth rate ( max ) should be initially maintained , then switched to the critical growth rate ( c ) at tc to maximize the specific product production rate ( max ) . since growth rate is affected by carbon : nitrogen ( c : n ) ratio , it should also be changed at tc . a variety of carbon sources have been used for production of pha using different fermentation strategies . carbohydrates , oils , alcohols , fatty acids , and hydrocarbons are potential carbon sources for pha production . ethanol byproducts , cane and beet molasses , cheese whey , plant oils , hydrolysates of corn , cellulose , hemicellulose , palm oil , soybean oil , tallow , corn steep liquor , casamino acids , and food scraps had been used as substrates to produce pha using different organisms [ 1621 ] . the substrate evaluated in this study for the growth phase of r. eutropha was condensed corn solubles ( ccs ) , which is a low - value byproduct of the dry - mill , corn ethanol industry . in the dry mill process , the whole corn is milled , mixed with water , and enzymatically hydrolyzed to convert starch to glucose , which is converted to ethanol by fermentation . after distillation to remove ethanol , the larger corn particles are recovered by centrifugation as distiller 's wet grains . corn - milling byproducts are typically marketed as animal feed because of their protein content . however , these byproducts may also contain residual carbohydrates , which might be utilized by microbial fermentation to produce industrial biopolymers . the objective of this study was to determine the effects of adding artificial rumen fluid ( arf ) on cell viability , growth as well as pha production to a 48 h culture of r. eutropha . different strategies of feeding simulated arf into the bioreactors were assessed to maximize pha production . the culture was routinely transferred to nutrient broth and incubated on a reciprocating shaker ( 250 rpm ) at 30c for 24 h. for short - term maintenance , the culture was stored on tryptic soy agar ( tsa ) slants covered with mineral oil and stored in the refrigerator . inoculum for all trials was prepared in a stepwise manner , by transferring the culture from tsa plates into 100 ml of the ccs medium ( described below ) , then incubating for 24 h on a rotary shaker ( 250 rpm ) at 30c . the inoculum rate for all bioreactor trials was 1% ( v v ) from a 24 h grown culture to an average od of 1.04 . a low - cost medium based on ccs was developed in a prior study . this medium , containing 240 g ccs l , with a c : n ratio of 50 : 1 was the best medium for the growth of r. eutropha . the medium was prepared by mixing 1,370 ml ccs with 4,630 ml deionized water , adjusting the ph to 6.5 using 10 m sodium hydroxide ( naoh ) , then centrifuging at 11,000 rpm for 7 min at 1525c . a filter sterilized 178 g l ammonium bicarbonate ( nh4hco3 ) stock solution was prepared and then 20.4 ml of this solution was added to each liter of ccs medium to adjust the c : n ratio to 50 : 1 . the ph was further adjusted to 7.0 by adding 10 n sulfuric acid ( h2so4 ) before inoculation . experimental trials were conducted in 5 l new brunswick , bioflo iii , edison , nj bioreactor that contained 4 l of ccs medium . filter sterilized air ( 1 l l min ) was sparged into the bioreactor , and 2 - 3 ml of antifoam ( cognis clerol fba 5059 , cognis , cincinnati , oh ) were added to the medium before inoculation . fermentation medium was incubated at 30c and 500 rpm for 48 h , since prior research had shown r. eutropha to reach its maximum population by this time / temperature combination . at 48 h we began feeding the fermenter a mixed short - chain fatty acid ( scfa ) solution , using any of the three different strategies . this scfa solution , referred to as arf , contained 10 parts acetic , 2 parts butyric , 15 parts lactic , and 20 parts propionic acids ( v v ) . the composition of arf was based on a separate study evaluating fermentation of biomass with rumen consortia . a total volume of 372 ml of arf was fed to the culture over a period of 48 h. in the first feeding strategy ( 24 h feeding ) , 124 ml of arf was added at 48 , 72 , and 96 h. in the second feeding strategy ( 3 h feeding ) , 20 ml arf was added at 48 h and then every 3 h until 109 h. in the third feeding strategy ( continuous feeding ) , arf was continuously added from 48 to 96 h at a rate of 7.75 ml h. all incubations were continued until 144 h. two replications were performed for each feeding strategy to determine the effect of mixed fatty acids on cell viability , acid utilization , and pha production . samples were collected every 12 h and viable cell counts were done with tsa . at 72 , 96 , and 144 h , 50 ml samples were collected to determine cell dry weights . samples were centrifuged and the precipitate was dried in the hot air oven at 80c for 2 days . ph was measured using an acumet 950 ph meter ( thermo fisher scientific of waltham , ma ) . samples were also analyzed via a waters hplc system ( milford , ma ) for sugars , organic acids and glycerol . these samples were first filtered through a nonsterile 0.2 m filter to remove solids , and then frozen until analysis . an aminex hpx 87h column ( bio - rad laboratories , hercules , ca ) , operated at 65c with a helium - degassed , 4 mm h2so4 mobile phase at a flow rate of 0.6 ml min was used . standard solutions of maltose , glucose , lactic acid , butyric acid , acetic acid , propionic acid , succinic acid , and glycerol ( at 3 and 30 samples collected at 0 , 72 , and 120 h were also tested for ammonia and phosphate using hach ammonium and phosphate unicell tests ( hach company , loveland , colorado ) . to measure pha , 50 ml samples of broth were collected at 72 , 96 , and 144 h and centrifuged . the method developed by braunegg et al . was used to simultaneously extract and derivatize pha to the 3-hydroxyalkanoate methyl esters of the monomers . in this method , 2030 mg of ground cells were digested by adding 5 ml of digest solution and incubating at 90100c for 4 h. the digest solution contained 50% chloroform , 42.5% methanol , 7.5% h2so4 ( v v ) . after cooling , sample was washed with 2 ml of water , and the bottom layer ( containing the chloroform and methyl esters of pha ) was collected and placed in a gas chromatography ( gc ) vial with anhydrous sodium sulfate ( to remove residual water ) . pha was quantified using a hewlett - packard 5890 series ii ( palo alto , ca ) gc with a flame ionization detector ( gc - fid ) [ 24 , 25 ] . split injection was used onto a supelco ssp-2380 ( park bellefonte , pa ) capillary column ( 30 m 0.25 mm i.d . with 0.20 um film ) . the inlet head pressure was maintained at 28 psi , and the temperature program started at 50c for 4 min , then increased by 3c min to a final temperature of 146c for 4 min . purified poly ( 3-hydroxybutyric acid co-3-hydroxyvaleric acid ) ( p(hb - hv ) ) obtained from sigma - aldrich was used for a standard calibration . the copolymer consisted of 88% 3-hydroxybutyric acid ( 3-hb ) and 12% 3-hydroxyvaleric acid ( 3-hv ) . copolymer concentrations of 210 mg ml were digested as above , and then analyzed by gc - fid . retention times were 14.9 min for methylated 3-hb , and 17.8 min for methylated 3-hv . various fermentation parameters ( maximum cell populations , scfa utilization rates , pha productivity , etc . ) were analyzed to determine least significant differences between treatments using randomized complete block design . fermentation efficiency ( fe ) was calculated as the percentage of substrate supplied to that was consumed . pha productivity was calculated as the concentration of pha produced per hour whereas pha content was determined as a percentage of pha concentration over cell dry weight . data were analyzed using the proc glm procedure of sas software to determine f values and least squares ( ls ) means . exponential regression equations were used to determine growth rates and acid utilization rates for each replication , from which means were calculated . in all bioreactor trials , the organism was incubated under similar conditions ( 30c , 500 rpm , and aeration at 1 l l min ) for the first 48 h , and this data was relatively uniform . figure 1 and table 2 show the average cell population , growth rate , acid utilization , and ammonia and phosphate uptake rates during this growth period . compared to shake flasks trials ( table 3 ) , the maximum cell population in bioreactor trials was almost 10-fold higher ( 2.3 10 cfu ml ) . likewise , the growth rate of r. eutropha was also higher in the bioreactor ( 0.20 h ) compared to the aerated shake flasks ( 0.13 h ) . in the shake flask trials , lactic acid was consumed at the fastest rate followed by acetic , butyric , succinic , and propionic acids . in the bioreactors , the organic acid utilization rates in the growth phase were generally similar to shake flask trials except for lactic acid . overall acid utilization rates were higher in the bioreactors than in shake flasks probably due to the higher cell population . percentage acid consumptions were also higher in the bioreactor with the exception of lactic acid . ammonia and phosphate usage rates were higher in bioreactor trials again due to the higher cell population . figures 2 , 3 , and 4 show that arf feeding resulted in a slight increase in cell populations . the effects of different feeding strategies on maximum cell population , and ammonia and phosphate utilization rates are shown in table 4 . the maximum cell populations were not significantly different between the three treatments . in all cases , the cell population continued to rise after arf additions began , suggesting that the acid levels were not inhibitory . as expected , the 24 h addition method ( figure 2 ) resulted in the highest spikes in scfa concentration , with acid levels then falling as r. eutropha metabolized the acids to pha . there was no apparent accumulation of lactic , butyric , or succinic acids for the 24 h feeding strategy . however , acetic acid ( 2.3 g l ) , and to a lesser extent propionic acid ( 0.5 g l ) , had accumulated over time . all the scfas were completely utilized by 144 h for the 3h feeding strategy . for the continuous - feeding strategy , all the scfas were consumed by 144 h with the exception of propionic acid ( 0.5 g l ) . utilization rates of the individual scfas during the final 96 h of fermentation ( 48 to 144 h ) , along with the combined acid utilization rates , are shown in table 4 . the combined utilization rate also included consumption of succinic acid that was already present in the ccs medium . the highest combined acid utilization rates were observed in the 3 h and continuous feeding strategies , with the lowest rate in the 24 h feeding method . the trend of higher acid utilization rates with the 3 h and continuous feeding methods also evident for the individual acids . propionic and lactic acids were used most rapidly followed by acetic and butyric acid . table 4 also shows the fes of the four scfas , along with the combined fe . the 3 h and continuous - feeding strategies resulted in the highest combined fe , while the lowest occurred with the 24 h feeding method . this is consistent with the lower acid utilization rates observed with the 24 h feeding strategy . in comparing the individual acids , lactic acid was consumed completely , with greater than 95% utilization of propionic and butyric acids . fes were higher for all the acids in the bioreactor trials compared to the prior shake - flask trials ( table 5 ) . cell dry weights and pha production parameters for the different arf - feeding strategies are provided in table 4 . the 3 h feeding strategy resulted in the highest cell dry weight , pha concentration , and pha content , but the values were not significantly different from the other feeding strategies . cell dry weight and pha production were much higher than that obtained during the shake - flask trials . the highest pha concentration in the cells in the shake - flask trials was 4.6 g l ( table 5 ) , whereas in the fermenter trials , the pha concentration of the cells was about 1.82 times higher . typically , pha production occurs during stationary phase . hence cells are first grown through exponential phase in a balanced medium to maximize growth rate . this medium is formulated to run out of a key nutrient when the maximum cell population is achieved , then additional carbon is added by fed - batch or continuous mode to maximize pha production . in this study , nitrogen - supplemented ccs medium resulted in better growth of r. eutropha in the bioreactors as compared to the shake flasks due to the improved aeration and agitation provided in the bioreactor , along with more consistent ph control . the organism continued to grow after the scfas were added at 48 h. this growth was supported by the residual ammonia and phosphorus present in the medium . the organism reached its stationary phase by 96 h when most of the ammonia and phosphorous present in the medium were consumed . ideally , one or both of these nutrients becomes limiting at the end of exponential phase , to trigger the shift from reproductive metabolism to pha synthesis . because nitrogen and phosphate were not depleted until 72 h , this could have contributed to the continued increase in cell numbers observed after 48 h. it is likely that at least some of the scfas fed at 48 h were utilized for growth , until the point at which nitrogen became limiting . researchers have found that the complete lack of nitrogen may suppress enzyme activity in pha synthesis . thus , a small amount of ammonia in the media might be necessary to trigger pha synthesis . the utilization rate of lactic acid in the initial 48 h was lower than that of acetic acid probably due to higher concentration of lactic acid ( 1.6 g l ) in the bioreactor media compared to the shake - flask media ( 1 g l ) . this variation was due to the different batches of ccs obtained from the ethanol plant . in this study , fed - batch ( 24 h feeding ) and continuous - feeding strategies were chosen to determine their effect on pha production . the lowest acid utilization rates and fes were observed for the 24 h feeding strategy due to the periodic spikes in scfa concentrations , that might have disrupted the acid utilization and decreased cell activity . at high scfa concentration the ph of the medium can reach below the pkas for scfas ( lactic 3.86 , acetic 4.76 , butyric 4.83 , and propionic 4.87 ) . at low phs the undissociated form predominates , and the scfas readily cross the cell membrane . once inside , they rapidly dissociate and acidify the cytoplasm . as a result , the proton gradient can not be maintained as desired , and energy generation and transport systems dependent on proton gradient are disrupted . this can also cause an increase in osmotic pressure due to the accumulation of anions . at ph levels closer to the optimum for r eutropha ( ~7.0 ) , scfas would be in the dissociated form in the medium . while the anions would n't be transported as readily , once inside the cell they would not cause the adverse effects of the undissociated form . therefore , scfas can only be effective carbon sources when ph and scfa concentrations are carefully regulated . acid utilization rates might have also been reduced by depletion of certain acids at the end of each 24 h phase . for the continuous strategy , small volumes of the scfas were fed continuously . though fes of the scfas were higher than that of the 24 h feeding strategy it was thus necessary to develop an optimal feeding strategy which could potentially increase acid utilizations and fes . since the scfas were added in smaller doses at shorter intervals , ph of the medium was more efficiently regulated , which resulted in better utilization and 100% fe of the organic acids . in comparison to the prior aerated shake - flasks trials ( table 5 ) , which were fed with individual scfas , the addition of mixed acids to the bioreactor resulted in more rapid uptake of propionic acid and slower utilization of butyric acid . propionate utilization rate rose from 0.046 g l h in aerated shake flasks to approximately 0.08 g l h when added as a part of the arf mixture in the bioreactor trials . this was likely due to the higher cell populations achieved in the bioreactor trials coupled with the fact that proprionate utilization by r. eutropha is energetically favorable . moreover , addition of propionic acid in small doses might have controlled the change in ph , and thus resulted in better utilization . the decline in the utilization rate of butyric acid , from 0.041 g l h when fed individually in aerated shake flasks to 0.011 g l h in bioreactor trials , may have been due to additional atp needed to transport this acid [ 32 , 33 ] . thus utilization of other acids was preferred over butyric acid when fed as a mixture for growth . the high fes for lactic and propionic acids were consistent with the metabolic preference of r. eutropha , especially considering that the arf contained 15 and 20 the high fe of butyric acid may be due to the fact that arf contained only 2 g l of this acid . while conducting the shake - flask trials , we had previously noted that r. eutropha did not prefer acetic acid , therefore its lower fe was not surprising . thus , it can be concluded that in the 24 h strategy the sudden rise in the scfa concentrations must have lowered the ph by accumulation of acid . though the dosages of acids were small for the continuous feeding , continuous addition might have lowered the efficiency of the process . the smaller dosage and the fed - batch mode of the 3 h feeding strategy might have resulted in better control of ph and of catabolite repression . so the optimized growth conditions ( as discussed before ) for the organism and 3h feeding strategy of arf addition was considered the best to obtain optimum pha production by the organism . yu et al . suggested in their study that an average yield of pha was 0.39 g g of carbon sources ( acetic , butyric , and propionic acids ) . in the current study the highest yield of about 0.2 g g was observed when the 3 h feeding strategy was used . investigators have reported that inexpensive carbon sources lead to low pha productivity due to inefficient utilization of nutrients by organisms [ 3436 ] . a majority of carbon sources was probably utilized to generate energy for cell maintenance . wild - type strain of r. eutropha used in this study was not able to use glucose and did not completely utilize the high amount of glycerol present in the ccs medium . pha biosynthesis genes can be inserted in organisms that have wider range of utilizable substrates to increase pha productivity and pha content . for example , the recombinant strains of r. eutropha ( h16 ) containing glucose - utilizing genes of escherichia coli ( e. coli ) , and e. coli harboring the r. eutropha genes had higher pha productivity and concentration as compared to the wild - type strain [ 14 , 34 ] . since the carbon source is a major contributor to pha cost , inexpensive sources of carbon are important . from an economical point of view , the use of purified media to increase pha yield will significantly increase the production cost . this study showed that r. eutropha is capable of growing in ccs medium , a low - value byproduct of ethanol industry . we also concluded that mixture of scfas in the same compositional ratios of arf was not inhibitory when added at stationary phase of growth . scfas can be diverted toward pha production by r. eutropha . as the use of purified scfas is cost prohibitive , developing a mixed culture system to produce a mixture of scfas from another ethanol industry byproduct is highly cost - effective . thus utilization of inexpensive carbon sources may lead to economically viable pha production in future when superior genetics and fermentation strategies are applied together .

the research described in this present study was part of a larger effort focused on developing a dual substrate , dual fermentation process to produce polyhydroxyalkanoate ( pha ) . the focus of this study was developing and optimizing a strategy for feeding a mixture of scfas ( simulated arf ) and maximizing pha production in a cost - effective way . three different feeding strategies were examined in this study . the substrate evaluated in this study for the growth phase of r. eutropha was condensed corn solubles , a low - value byproduct of the dry - mill , corn ethanol industry . the culture was grown to high cell densities in nitrogen - supplemented condensed corn solubles media in 5 l bioreactors . the overall growth rate of r. eutropha was 0.2 h1 . the 20 ml arf feeding every 3 h from 48 to 109 h strategy gave the best results in terms of pha production . pha productivity ( 0.0697 g l1 h1 ) , pha concentration ( 8.37 g l1 ) , and pha content ( 39.52% ) were the highest when arf was fed every 3 h for 61 h. this study proved that condensed corn solubles can be potentially used as a growth medium to boost pha production by r. eutropha thus reducing the overall cost of biopolymer production .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

biodegradable polymers made from renewable resources such as agricultural wastes , corn , cassava , tapioca , whey , and so forth , do not lead to depletion of finite resources . the most studied of the biodegradable polymers include polyesters , polylactides , aliphatic polyesters , polysaccharides , and various copolymers . these biopolymers have many of the desirable physical and chemical properties of conventional synthetic polymers . the focus of this project was production of the biopolymer polyhydroxyalkanoate ( pha ) at a low cost . the carboxyl group of one monomer forms an ester bond with hydroxyl group of the neighboring monomer . phb has good oxygen impermeability , moisture resistance , water insolubility , and optical purity [ 4 , 5 ] . since phb is toxicologically safe , it can be used for articles which come into contact with skin , feed , or food . in the food industry , pha has a wide application as edible packaging material , coating agent , flavor delivery agent , and as dairy cream substitute [ 10 , 11 ] . it can also be used for making bottles , cosmetics , containers , pens , golf tees , films , adhesives and nonwoven fabrics , toner , and developer compositions , ion - conducting polymers , and as latex for paper coating applications [ 12 , 13 ] . hence cells are first grown to high density , after which a key nutrient is limited to trigger pha synthesis . because of this dual phase process , pha production lends itself to fed - batch , as well as , continuous operation . this follows pontryagin 's maximum principle , which is an optimal feeding strategy for fed - batch fermentation . the maximum growth rate ( max ) should be initially maintained , then switched to the critical growth rate ( c ) at tc to maximize the specific product production rate ( max ) . since growth rate is affected by carbon : nitrogen ( c : n ) ratio , it should also be changed at tc . a variety of carbon sources have been used for production of pha using different fermentation strategies . carbohydrates , oils , alcohols , fatty acids , and hydrocarbons are potential carbon sources for pha production . ethanol byproducts , cane and beet molasses , cheese whey , plant oils , hydrolysates of corn , cellulose , hemicellulose , palm oil , soybean oil , tallow , corn steep liquor , casamino acids , and food scraps had been used as substrates to produce pha using different organisms [ 1621 ] . the substrate evaluated in this study for the growth phase of r. eutropha was condensed corn solubles ( ccs ) , which is a low - value byproduct of the dry - mill , corn ethanol industry . in the dry mill process , the whole corn is milled , mixed with water , and enzymatically hydrolyzed to convert starch to glucose , which is converted to ethanol by fermentation . however , these byproducts may also contain residual carbohydrates , which might be utilized by microbial fermentation to produce industrial biopolymers . the objective of this study was to determine the effects of adding artificial rumen fluid ( arf ) on cell viability , growth as well as pha production to a 48 h culture of r. eutropha . different strategies of feeding simulated arf into the bioreactors were assessed to maximize pha production . the culture was routinely transferred to nutrient broth and incubated on a reciprocating shaker ( 250 rpm ) at 30c for 24 h. for short - term maintenance , the culture was stored on tryptic soy agar ( tsa ) slants covered with mineral oil and stored in the refrigerator . inoculum for all trials was prepared in a stepwise manner , by transferring the culture from tsa plates into 100 ml of the ccs medium ( described below ) , then incubating for 24 h on a rotary shaker ( 250 rpm ) at 30c . a low - cost medium based on ccs was developed in a prior study . this medium , containing 240 g ccs l , with a c : n ratio of 50 : 1 was the best medium for the growth of r. eutropha . the medium was prepared by mixing 1,370 ml ccs with 4,630 ml deionized water , adjusting the ph to 6.5 using 10 m sodium hydroxide ( naoh ) , then centrifuging at 11,000 rpm for 7 min at 1525c . a filter sterilized 178 g l ammonium bicarbonate ( nh4hco3 ) stock solution was prepared and then 20.4 ml of this solution was added to each liter of ccs medium to adjust the c : n ratio to 50 : 1 . experimental trials were conducted in 5 l new brunswick , bioflo iii , edison , nj bioreactor that contained 4 l of ccs medium . filter sterilized air ( 1 l l min ) was sparged into the bioreactor , and 2 - 3 ml of antifoam ( cognis clerol fba 5059 , cognis , cincinnati , oh ) were added to the medium before inoculation . fermentation medium was incubated at 30c and 500 rpm for 48 h , since prior research had shown r. eutropha to reach its maximum population by this time / temperature combination . at 48 h we began feeding the fermenter a mixed short - chain fatty acid ( scfa ) solution , using any of the three different strategies . this scfa solution , referred to as arf , contained 10 parts acetic , 2 parts butyric , 15 parts lactic , and 20 parts propionic acids ( v v ) . the composition of arf was based on a separate study evaluating fermentation of biomass with rumen consortia . a total volume of 372 ml of arf was fed to the culture over a period of 48 h. in the first feeding strategy ( 24 h feeding ) , 124 ml of arf was added at 48 , 72 , and 96 h. in the second feeding strategy ( 3 h feeding ) , 20 ml arf was added at 48 h and then every 3 h until 109 h. in the third feeding strategy ( continuous feeding ) , arf was continuously added from 48 to 96 h at a rate of 7.75 ml h. all incubations were continued until 144 h. two replications were performed for each feeding strategy to determine the effect of mixed fatty acids on cell viability , acid utilization , and pha production . at 72 , 96 , and 144 h , 50 ml samples were collected to determine cell dry weights . these samples were first filtered through a nonsterile 0.2 m filter to remove solids , and then frozen until analysis . an aminex hpx 87h column ( bio - rad laboratories , hercules , ca ) , operated at 65c with a helium - degassed , 4 mm h2so4 mobile phase at a flow rate of 0.6 ml min was used . standard solutions of maltose , glucose , lactic acid , butyric acid , acetic acid , propionic acid , succinic acid , and glycerol ( at 3 and 30 samples collected at 0 , 72 , and 120 h were also tested for ammonia and phosphate using hach ammonium and phosphate unicell tests ( hach company , loveland , colorado ) . to measure pha , 50 ml samples of broth were collected at 72 , 96 , and 144 h and centrifuged . was used to simultaneously extract and derivatize pha to the 3-hydroxyalkanoate methyl esters of the monomers . in this method , 2030 mg of ground cells were digested by adding 5 ml of digest solution and incubating at 90100c for 4 h. the digest solution contained 50% chloroform , 42.5% methanol , 7.5% h2so4 ( v v ) . after cooling , sample was washed with 2 ml of water , and the bottom layer ( containing the chloroform and methyl esters of pha ) was collected and placed in a gas chromatography ( gc ) vial with anhydrous sodium sulfate ( to remove residual water ) . the inlet head pressure was maintained at 28 psi , and the temperature program started at 50c for 4 min , then increased by 3c min to a final temperature of 146c for 4 min . the copolymer consisted of 88% 3-hydroxybutyric acid ( 3-hb ) and 12% 3-hydroxyvaleric acid ( 3-hv ) . copolymer concentrations of 210 mg ml were digested as above , and then analyzed by gc - fid . retention times were 14.9 min for methylated 3-hb , and 17.8 min for methylated 3-hv . various fermentation parameters ( maximum cell populations , scfa utilization rates , pha productivity , etc . ) pha productivity was calculated as the concentration of pha produced per hour whereas pha content was determined as a percentage of pha concentration over cell dry weight . in all bioreactor trials , the organism was incubated under similar conditions ( 30c , 500 rpm , and aeration at 1 l l min ) for the first 48 h , and this data was relatively uniform . figure 1 and table 2 show the average cell population , growth rate , acid utilization , and ammonia and phosphate uptake rates during this growth period . compared to shake flasks trials ( table 3 ) , the maximum cell population in bioreactor trials was almost 10-fold higher ( 2.3 10 cfu ml ) . likewise , the growth rate of r. eutropha was also higher in the bioreactor ( 0.20 h ) compared to the aerated shake flasks ( 0.13 h ) . in the shake flask trials , lactic acid was consumed at the fastest rate followed by acetic , butyric , succinic , and propionic acids . in the bioreactors , the organic acid utilization rates in the growth phase were generally similar to shake flask trials except for lactic acid . figures 2 , 3 , and 4 show that arf feeding resulted in a slight increase in cell populations . the effects of different feeding strategies on maximum cell population , and ammonia and phosphate utilization rates are shown in table 4 . as expected , the 24 h addition method ( figure 2 ) resulted in the highest spikes in scfa concentration , with acid levels then falling as r. eutropha metabolized the acids to pha . there was no apparent accumulation of lactic , butyric , or succinic acids for the 24 h feeding strategy . however , acetic acid ( 2.3 g l ) , and to a lesser extent propionic acid ( 0.5 g l ) , had accumulated over time . all the scfas were completely utilized by 144 h for the 3h feeding strategy . for the continuous - feeding strategy , all the scfas were consumed by 144 h with the exception of propionic acid ( 0.5 g l ) . utilization rates of the individual scfas during the final 96 h of fermentation ( 48 to 144 h ) , along with the combined acid utilization rates , are shown in table 4 . the highest combined acid utilization rates were observed in the 3 h and continuous feeding strategies , with the lowest rate in the 24 h feeding method . the trend of higher acid utilization rates with the 3 h and continuous feeding methods also evident for the individual acids . table 4 also shows the fes of the four scfas , along with the combined fe . the 3 h and continuous - feeding strategies resulted in the highest combined fe , while the lowest occurred with the 24 h feeding method . cell dry weights and pha production parameters for the different arf - feeding strategies are provided in table 4 . the 3 h feeding strategy resulted in the highest cell dry weight , pha concentration , and pha content , but the values were not significantly different from the other feeding strategies . cell dry weight and pha production were much higher than that obtained during the shake - flask trials . the highest pha concentration in the cells in the shake - flask trials was 4.6 g l ( table 5 ) , whereas in the fermenter trials , the pha concentration of the cells was about 1.82 times higher . typically , pha production occurs during stationary phase . hence cells are first grown through exponential phase in a balanced medium to maximize growth rate . this medium is formulated to run out of a key nutrient when the maximum cell population is achieved , then additional carbon is added by fed - batch or continuous mode to maximize pha production . in this study , nitrogen - supplemented ccs medium resulted in better growth of r. eutropha in the bioreactors as compared to the shake flasks due to the improved aeration and agitation provided in the bioreactor , along with more consistent ph control . the organism continued to grow after the scfas were added at 48 h. this growth was supported by the residual ammonia and phosphorus present in the medium . the organism reached its stationary phase by 96 h when most of the ammonia and phosphorous present in the medium were consumed . because nitrogen and phosphate were not depleted until 72 h , this could have contributed to the continued increase in cell numbers observed after 48 h. it is likely that at least some of the scfas fed at 48 h were utilized for growth , until the point at which nitrogen became limiting . thus , a small amount of ammonia in the media might be necessary to trigger pha synthesis . the utilization rate of lactic acid in the initial 48 h was lower than that of acetic acid probably due to higher concentration of lactic acid ( 1.6 g l ) in the bioreactor media compared to the shake - flask media ( 1 g l ) . in this study , fed - batch ( 24 h feeding ) and continuous - feeding strategies were chosen to determine their effect on pha production . the lowest acid utilization rates and fes were observed for the 24 h feeding strategy due to the periodic spikes in scfa concentrations , that might have disrupted the acid utilization and decreased cell activity . at high scfa concentration the ph of the medium can reach below the pkas for scfas ( lactic 3.86 , acetic 4.76 , butyric 4.83 , and propionic 4.87 ) . at low phs the undissociated form predominates , and the scfas readily cross the cell membrane . as a result , the proton gradient can not be maintained as desired , and energy generation and transport systems dependent on proton gradient are disrupted . at ph levels closer to the optimum for r eutropha ( ~7.0 ) , scfas would be in the dissociated form in the medium . while the anions would n't be transported as readily , once inside the cell they would not cause the adverse effects of the undissociated form . for the continuous strategy , small volumes of the scfas were fed continuously . though fes of the scfas were higher than that of the 24 h feeding strategy it was thus necessary to develop an optimal feeding strategy which could potentially increase acid utilizations and fes . since the scfas were added in smaller doses at shorter intervals , ph of the medium was more efficiently regulated , which resulted in better utilization and 100% fe of the organic acids . in comparison to the prior aerated shake - flasks trials ( table 5 ) , which were fed with individual scfas , the addition of mixed acids to the bioreactor resulted in more rapid uptake of propionic acid and slower utilization of butyric acid . propionate utilization rate rose from 0.046 g l h in aerated shake flasks to approximately 0.08 g l h when added as a part of the arf mixture in the bioreactor trials . this was likely due to the higher cell populations achieved in the bioreactor trials coupled with the fact that proprionate utilization by r. eutropha is energetically favorable . moreover , addition of propionic acid in small doses might have controlled the change in ph , and thus resulted in better utilization . the decline in the utilization rate of butyric acid , from 0.041 g l h when fed individually in aerated shake flasks to 0.011 g l h in bioreactor trials , may have been due to additional atp needed to transport this acid [ 32 , 33 ] . thus utilization of other acids was preferred over butyric acid when fed as a mixture for growth . the high fes for lactic and propionic acids were consistent with the metabolic preference of r. eutropha , especially considering that the arf contained 15 and 20 the high fe of butyric acid may be due to the fact that arf contained only 2 g l of this acid . while conducting the shake - flask trials , we had previously noted that r. eutropha did not prefer acetic acid , therefore its lower fe was not surprising . thus , it can be concluded that in the 24 h strategy the sudden rise in the scfa concentrations must have lowered the ph by accumulation of acid . though the dosages of acids were small for the continuous feeding , continuous addition might have lowered the efficiency of the process . the smaller dosage and the fed - batch mode of the 3 h feeding strategy might have resulted in better control of ph and of catabolite repression . so the optimized growth conditions ( as discussed before ) for the organism and 3h feeding strategy of arf addition was considered the best to obtain optimum pha production by the organism . suggested in their study that an average yield of pha was 0.39 g g of carbon sources ( acetic , butyric , and propionic acids ) . in the current study the highest yield of about 0.2 g g was observed when the 3 h feeding strategy was used . investigators have reported that inexpensive carbon sources lead to low pha productivity due to inefficient utilization of nutrients by organisms [ 3436 ] . wild - type strain of r. eutropha used in this study was not able to use glucose and did not completely utilize the high amount of glycerol present in the ccs medium . pha biosynthesis genes can be inserted in organisms that have wider range of utilizable substrates to increase pha productivity and pha content . for example , the recombinant strains of r. eutropha ( h16 ) containing glucose - utilizing genes of escherichia coli ( e. coli ) , and e. coli harboring the r. eutropha genes had higher pha productivity and concentration as compared to the wild - type strain [ 14 , 34 ] . this study showed that r. eutropha is capable of growing in ccs medium , a low - value byproduct of ethanol industry . we also concluded that mixture of scfas in the same compositional ratios of arf was not inhibitory when added at stationary phase of growth . scfas can be diverted toward pha production by r. eutropha . as the use of purified scfas is cost prohibitive , developing a mixed culture system to produce a mixture of scfas from another ethanol industry byproduct is highly cost - effective . thus utilization of inexpensive carbon sources may lead to economically viable pha production in future when superior genetics and fermentation strategies are applied together .

the toranomon hospital health management center study included a cohort comprising mainly apparently healthy government employees who underwent an annual health screening in tokyo , japan . routine health checkups are common in japan because the japanese government and companies encourage people to receive periodic examinations . among the 41,931 individuals , this cross - sectional study included 41,700 individuals for whom data on sex and menopausal status were available ( 6,458 premenopausal women , 5,701 postmenopausal women , and 29,541 men ) . among those 41,700 individuals , we excluded 1,027 women who did not report a cause for menopause ( natural , surgical , or other ) . after the exclusions , 40,673 individuals ( 6,458 premenopausal women , 3,630 women in natural menopause , 943 women in surgical menopause , 101 postmenopausal women by other causes , and 29,541 men ) were available for analysis . we excluded 81 women aged 65 years who had been in the premenopausal category because their persistent vaginal bleeding after the age 65 was not likely a result of menses but of pathologic processes ( 12 ) . we also excluded individuals with missing data on characteristics of lifestyle habits or clinical measures . subsequently , 40,067 individuals ( 6,308 premenopausal women , 3,552 women in natural menopause , 1,018 women in surgical menopause or another cause , and 29,189 men ) were included in the current analysis . with regard to women with missing data on age at menopause ( n = 154 ) , we excluded them only for the analysis of the relationship between age at menopause and dysglycemia . the study protocol followed the japanese government s ethical guidelines regarding epidemiological studies in accordance with the declaration of helsinki and was reviewed by the institutional review board at toranomon hospital . diagnosis of type 2 diabetes was made according to american diabetes association criteria ( 22 ) of a fasting plasma glucose ( fpg ) level 7.0 mmol / l ( 126 mg / dl ) , self - reported clinician - diagnosed diabetes or the use of hypoglycemic agents or insulin , or hba1c 6.5% ( 48 mmol / mol ) . prediabetes was indicated by an fpg of 5.66.9 mmol / l ( 100125 mg / dl ) or an hba1c of 5.76.4% ( 3946 mmol / mol ) ( 22 ) without type 2 diabetes . we assessed the menopausal status of women with a self - report questionnaire at the time of the examination . if so , they were asked to indicate the reason for menopause ( natural , surgical , or other ) and the age at which menopause occurred ( 39 , 4044 , 4549 , or 50 years ) . parental history of diabetes , smoking habit ( never , former , or current ) , physical activity habit ( any physical activity for 2030 min or longer at least once weekly ) , and self - reported history of medical treatment for hypertension or diabetes were also assessed by the questionnaire for both men and women . blood samples were collected after an overnight fast ( 12 h ) , and measurements were made with an automatic clinical chemistry analyzer . blood glucose concentrations were measured by enzymatic methods , and hba1c was assessed by high - performance liquid chromatography . the value for hba1c ( % ) was estimated as the national glycohemoglobin standardization program value ( % ) calculated by eq . 1 : hba1c ( % ) = hba1c ( japan diabetes society ) ( % ) 1.02 + 0.25% ( 23 ) . logistic regression analysis was performed to calculate odds ratios ( ors ) and 95% cis . we initially investigated whether there was a difference in the association of dysglycemia between men and women and then calculated ors for dysglycemia for postmenopausal women ( regardless of cause ) and for men , with premenopausal women as the reference group . after that , we assessed whether there was a difference in the association according to cause of menopause . because few women reported an age at menopause of < 39 years , we categorized age at menopause into three groups ( < 45 , 4549 , and 50 years ) for the analysis . to investigate effect modifications , we performed logistic regression analysis with adjustment for age ( model 1 ) ; age and other demographic factors ( bmi , parental history of diabetes , physical activity habit , and smoking habit ) ( model 2 ) ; and age , demographic , and metabolic factors ( hypertension indicated by systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or medical treatment and hdl cholesterol and log - transformed triglyceride levels ) ( model 3 ) . we also examined whether a significant association existed between menopause and prediabetic hyperglycemia among nondiabetic individuals after excluding those with type 2 diabetes . in an additional analysis , we stratified women according age at the time of examination ( < 50 or 50 years ) because the mean age of menopause has been considered to be 50 years ( 9,11,14,21,24,25 ) . a combined effect of older age at the time of the examination and the postmenopausal condition on the presence of dysglycemia ( either prediabetes or type 2 diabetes ) was assessed , with premenopausal women aged < 50 years as the reference group . we then conducted a stratified analysis based on age at the time of examination ( < 50 or 50 years ) and calculated ors for dysglycemia across categories of age at menopause , with the premenopausal state as the reference group for women aged < 50 or 50 years . analysis was performed with ibm spss statistics version 19 ( ibm , armonk , ny ) . diagnosis of type 2 diabetes was made according to american diabetes association criteria ( 22 ) of a fasting plasma glucose ( fpg ) level 7.0 mmol / l ( 126 mg / dl ) , self - reported clinician - diagnosed diabetes or the use of hypoglycemic agents or insulin , or hba1c 6.5% ( 48 mmol / mol ) . prediabetes was indicated by an fpg of 5.66.9 mmol / l ( 100125 mg / dl ) or an hba1c of 5.76.4% ( 3946 mmol / mol ) ( 22 ) without type 2 diabetes . we assessed the menopausal status of women with a self - report questionnaire at the time of the examination . if so , they were asked to indicate the reason for menopause ( natural , surgical , or other ) and the age at which menopause occurred ( 39 , 4044 , 4549 , or 50 years ) . parental history of diabetes , smoking habit ( never , former , or current ) , physical activity habit ( any physical activity for 2030 min or longer at least once weekly ) , and self - reported history of medical treatment for hypertension or diabetes were also assessed by the questionnaire for both men and women . blood samples were collected after an overnight fast ( 12 h ) , and measurements were made with an automatic clinical chemistry analyzer . blood glucose concentrations were measured by enzymatic methods , and hba1c was assessed by high - performance liquid chromatography . the value for hba1c ( % ) was estimated as the national glycohemoglobin standardization program value ( % ) calculated by eq . 1 : hba1c ( % ) = hba1c ( japan diabetes society ) ( % ) 1.02 + 0.25% ( 23 ) . logistic regression analysis was performed to calculate odds ratios ( ors ) and 95% cis . we initially investigated whether there was a difference in the association of dysglycemia between men and women and then calculated ors for dysglycemia for postmenopausal women ( regardless of cause ) and for men , with premenopausal women as the reference group . after that , we assessed whether there was a difference in the association according to cause of menopause . because few women reported an age at menopause of < 39 years , we categorized age at menopause into three groups ( < 45 , 4549 , and 50 years ) for the analysis . to investigate effect modifications , we performed logistic regression analysis with adjustment for age ( model 1 ) ; age and other demographic factors ( bmi , parental history of diabetes , physical activity habit , and smoking habit ) ( model 2 ) ; and age , demographic , and metabolic factors ( hypertension indicated by systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or medical treatment and hdl cholesterol and log - transformed triglyceride levels ) ( model 3 ) . we also examined whether a significant association existed between menopause and prediabetic hyperglycemia among nondiabetic individuals after excluding those with type 2 diabetes . in an additional analysis , we stratified women according age at the time of examination ( < 50 or 50 years ) because the mean age of menopause has been considered to be 50 years ( 9,11,14,21,24,25 ) . a combined effect of older age at the time of the examination and the postmenopausal condition on the presence of dysglycemia ( either prediabetes or type 2 diabetes ) was assessed , with premenopausal women aged < 50 years as the reference group . we then conducted a stratified analysis based on age at the time of examination ( < 50 or 50 years ) and calculated ors for dysglycemia across categories of age at menopause , with the premenopausal state as the reference group for women aged < 50 or 50 years . analysis was performed with ibm spss statistics version 19 ( ibm , armonk , ny ) . mean ( sd ) age was 48.4 ( 9.9 ) years among the 10,878 women studied and 48.0 ( 9.7 ) years among the 29,189 men studied ( table 1 ) . of the 10,878 women , 2,340 ( 21.5% ) had prediabetes and 246 ( 2.3% ) had type 2 diabetes . premenopausal women were younger ( 42.1 [ 6.6 ] years ) compared with postmenopausal women . we did not observe a marked difference in bmi between premenopausal and postmenopausal women . among the premenopausal women , only 69 ( 1.1% ) had type 2 diabetes , whereas the prevalence rate was high at 3.8% in women after natural menopause and 4.0% after surgical menopause or menopause from other causes . had either prediabetes or type 2 diabetes . characteristics of total women , premenopausal women , postmenopausal women ( by cause ) , and men table 2 shows ors for type 2 diabetes and prediabetes among men and among women by menopausal status . men were 2.10 ( 95% ci 1.812.45 ) times more likely to have type 2 diabetes than the total number of women studied according to multivariate model 3 , which included age and demographic and metabolic factors . postmenopausal women had a significant association with type 2 diabetes ( 1.36 [ 1.011.82 ] ) compared with premenopausal women that was independent of age , bmi , smoking habit , physical activity habit , and parental history of diabetes ( model 2 ) , although the or was not as high as that in men ( 2.87 [ 2.233.69 ] ) . after adjustment for lipid measurements and hypertension ( model 3 ) , the or for the postmenopausal women was attenuated ( 1.17 [ 0.881.58 ] ) , and a significant association with the presence of type 2 diabetes remained only among the men ( 2.35 [ 1.823.03 ] ) . among the women , we did not find an obvious difference in the association of type 2 diabetes and menopausal status regardless of the cause of menopause . the association of prediabetes with menopause among individuals without type 2 diabetes showed that postmenopausal women had a significantly elevated or for prediabetes in model 3 ( 1.33 [ 1.201.48 ] ) compared with premenopausal women . additionally , the men had a significantly elevated or for prediabetes compared with postmenopausal women ( 1.93 [ 1.772.10 ] ) . regardless of age or demographic or metabolic factors , women with natural menopause or other causes of menopause had similarly elevated ors for prediabetes ( 1.36 [ 1.221.52 ] and 1.22 [ 1.041.43 ] , respectively ) . we observed that early age at menopause ( < 45 years ) was significantly associated with an elevated or for diabetes after adjustment for age ( 1.89 [ 1.212.96 ] ) or for age and demographic factors ( 1.73 [ 1.102.73 ] ) . this association was not significant after adjustment for hypertension and lipid measurements ( model 3 ) . we did not observe an association of early menopause with an increased probability of having prediabetes among individuals without type 2 diabetes . ors ( 95% ci ) for type 2 diabetes or prediabetes between women and men ( a ) , among pre- or postmenopausal women and men ( b and c ) , or among pre- or postmenopausal women according to age at menopause and men ( d ) figure 1 shows the combined effect of age at examination and menopausal status on the presence of dysglycemia ( either prediabetes or type 2 diabetes ) . although older age alone at the time of the examination ( 50 years ) was significantly associated with dysglycemia ( or 2.21 [ 95% ci 1.852.65 ] ) , postmenopausal status alone was also significantly associated with an elevated or for dysglycemia ( 1.50 [ 1.181.91 ] ) . the postmenopausal condition and older age additively influenced an elevated or because postmenopausal women aged 50 years had a markedly elevated or ( 3.69 [ 3.344.08 ] ) for dysglycemia . we stratified women by age at the time of the examination and investigated whether there was an association of age at menopause with the presence of dysglycemia ( table 3 ) . compared with premenopausal women , postmenopausal women who underwent menopause at < 45 or 4549 years had a 1.41 ( 0.982.02 ) and 1.59 ( 1.152.20 ) times increased or for dysglycemia , respectively , even among women aged < 50 years at the time of examination ( n = 5,991 ) . adjustment for demographic and metabolic factors ( multivariate model 2 ) attenuated the ors ( 1.18 [ 0.801.74 ] and 1.29 [ 0.911.82 ] , respectively ) . among women aged 50 years , the postmenopausal state was significantly associated with the presence of dysglycemia , regardless of age at which menopause occurred . in multivariate model 2 , postmenopausal women had a similarly elevated or for dysglycemia to premenopausal women , regardless of age at menopause ( < 45 years of age 1.58 [ 1.222.04 ] , 4549 years of age 1.62 [ 1.312.00 ] , 50 years of age 1.65 [ 1.371.99 ] ) . probability of having dysglycemia ( either prediabetes or type 2 diabetes ) through a combination of age at the time of examination and menopausal status . association of dysglycemia ( either prediabetes or type 2 diabetes ) and age at menopause among women aged < 50 or 50 years at the time of examination we found that older age and a postmenopausal state independently and additively influenced the high prevalence of dysglycemia in japanese women . even among women aged < 50 years at the time of examination , menopause was associated with the presence of dysglycemia . the study is unique because it compared the probability of dysglycemia among women across menopausal states with that among mainly middle - aged men who underwent health screening in japan . although the or for postmenopausal women was not high compared with that of the men , their ors for type 2 diabetes and prediabetes were significantly elevated independently of age compared with those in premenopausal women . whether menopausal status would influence the occurrence of diabetes independently of age and other confounding factors remains controversial because it is difficult to conduct studies to separate the effects of normal aging from the menopausal transition . a few studies showed a significant positive association of hyperglycemia with menopausal status after adjustment for age and other risk factors for diabetes ( 7,13,19 ) , but multivariate analyses in other studies showed no such associations ( 4,11,14,18,26 ) . in a cross - sectional multicenter study of italian women from outpatient menopausal clinics , those with natural menopause had a 1.38 times higher multivariate - adjusted or for diabetes than premenopausal women ( 13 ) . on the other hand , the researchers did not find a significant association in women with surgical menopause and diabetes ( 13 ) . a cross - sectional study of korean women suggested that in postmenopausal women , there is a significant association with the presence of hyperglycemia ( fasting glucose level 110 mg / dl or antidiabetes medications ) compared with premenopausal women that is independent of age and bmi ( 7 ) . in women at high risk for diabetes who participated in the diabetes prevention program , no association was found between natural menopause or bilateral oophorectomy and increased risk of developing diabetes after adjustment for age ( 12 ) . another influence of the controversy might be that assessment of the menopausal state usually is based on self - reported responses or interviews and that participant characteristics vary among studies . the present study shows a significant positive association between postmenopause ( regardless of cause ) and the presence of type 2 diabetes independently of normal aging compared with premenopause . however , we did not include oral glucose tolerance test ( ogtt ) data in the diagnosis of diabetes . diabetes and impaired fasting glycemia are reported to be more common in men than in women 3069 years of age , whereas the prevalence of isolated postload hyperglycemia , particularly impaired glucose tolerance , is reportedly higher in women than in men , especially in individuals > 70 years of age ( 27 ) . additionally , impaired glucose tolerance is more prevalent than impaired fasting glycemia in asian populations for all age - groups ( 28 ) . the lack of data on ogtt for the diagnosis of dysglycemia might lead to an underestimation of the associations between menopause and the prevalence of diabetes . the significant association of type 2 diabetes and postmenopausal status was particularly attenuated after adjustments for hypertension and blood lipid measurements , suggesting that when we consider the association of menopause with diabetes , we also should consider the influence of related metabolic factors . because the transition from the premenopausal to the postmenopausal state is associated with changes in body composition ( increased body fat mass , increased abdominal fat , and decreased lean body mass ) ( 3 ) and substantial metabolic changes , features of metabolic syndrome would occur in many women ( 17 ) . more recent research , however , suggested that postmenopausal women have higher levels of adiposity , but the association was predominantly a result of aging ( 29 ) . another recent study raised the possibility that even if body composition changes with the menopausal transition , these changes are not accompanied by cardiometabolic deterioration during a relatively short follow - up period ( 30 ) . because data on body composition or visceral fat were not available for the current study , we could not assess whether differences in body composition across the menopausal state might have influenced the presence of dysglycemia , even if bmis across the menopausal state were relatively low . asians are more likely to have a higher percentage of fat or visceral adipose tissue at a given bmi than europeans ( 31 ) . this ethnic difference regarding the obese phenotype might increase insulin resistance , leading to impaired glucose metabolism . although we did not have data on reproductive hormone concentrations and can not explain the mechanism for the current observations , it was shown that natural menopause is characterized by increased relative androgenicity , which was reported to be associated with glucose metabolism ( 15 ) ; furthermore , reproductive hormone concentrations can vary by ethnicity and were shown to be confounded by ethnic disparities in body mass ( 32 ) . further studies should investigate mechanisms that link menopause and diabetes with detailed assessments of body composition , insulin sensitivity , insulin secretion , and reproductive hormone concentrations in perimenopausal women across various ethnic groups to confirm the current findings . the current results show that among individuals without diabetes , prediabetic hyperglycemia is significantly associated with postmenopausal status independently of age and demographic and metabolic parameters . in a cross - sectional study of japanese women without diabetes , stepwise regression analysis showed natural menopause rather than age as a significant determinant of fpg concentrations ( 19 ) . on the other hand , among middle - aged women living in north taiwan , no significant difference in fpg , insulin levels , homeostasis model assessment of insulin resistance , and prevalence of hyperglycemia between premenopausal and postmenopausal women was shown ( 6 ) . the current results show that prediabetic hyperglycemia and the postmenopausal state are positively associated , suggesting that postmenopausal women might be at high risk for diabetes . in a prospective case - cohort study that included only postmenopausal women , earlier age at menopause the hazard ratio for diabetes was 32% higher in women who entered menopause before 40 years of age compared with those experiencing menopause at age 5054 ( 20 ) . a study in chinese postmenopausal women , however , showed no association between age at menopause and diabetes ( 21 ) . the results of the current cross - sectional investigation suggest that early age at menopause ( < 45 years ) might be more strongly associated with type 2 diabetes than menopause at 50 years . nonetheless , regardless of age at menopause , postmenopausal women aged 50 years at the time of the examination had an 1.5 times increased probability of having dysglycemia compared with premenopausal women . additionally , age at menopause can be affected by various social and environmental factors ( 25,33 ) , which might be one possible explanation for the mixed results of the association of age at menopause and diabetes compared with existing studies . it has been established that a smoking habit is significantly associated with an early age at natural menopause ( 34 ) . factors of lower educational attainment ; being separated , widowed , or divorced ; and nonemployment have been associated with early natural menopause , whereas japanese ethnicity is associated with late age at natural menopause ( 33 ) . furthermore , bmi has been associated with age at menopause ( 35 ) . on the other hand , a recent study of women from five racial and ethnic groups indicated that there is no significant racial / ethnic difference in age at the final natural menstrual cycle after controlling for sociodemographic , lifestyle , and health factors ( 36 ) . further prospective investigations are needed to assess whether early menopause would increase the risk of developing diabetes across various ethnic groups while considering differences in demographic factors among study participants . recent reports indicated that early age at natural menopause is associated with an increased risk of ischemic stroke ( 37 ) and mortality ( 38 ) . nonetheless , both menopause and aging are nonmodifiable factors . regular physical activity may help to mitigate the tendency for weight gain and adverse changes in body composition and fat distribution that accompany aging and the menopausal transition ( 39 ) . high levels of habitual physical activity , such as walking , have been associated with a favorable cardiovascular risk profile in postmenopausal women ( 40 ) . further investigations are needed on whether different interventions for older postmenopausal women could control modifiable factors such as metabolically unhealthy obesity , dyslipidemia , hypertension , and lifestyle . the study participants were relatively lean , apparently healthy japanese government employees who underwent a routine health examination . thus , these individuals were more likely to pay attention to healthy lifestyle habits than those who did not have such an examination . the characteristics of the study participants , such as bmi and cardiometabolic factors , would influence the generalizability of the findings , although we analyzed these factors in multivariate models . additionally , limitations of the available data prevented a more in - depth analysis of factors that could influence an increased risk of developing diabetes ; therefore , we can not rule out the possibility that residual confounding influenced the results . because we did not include data on nutritional intake or other known risk factors for diabetes , such as sleep disturbances and depression , which are commonly observed in women at midlife , we did not have data on visceral fat or hormone replacement therapy , so that the ors might be over- or underestimated . nonetheless , the prevalence of women receiving hormone replacement therapy is considered to be low in japan . because the assessment of menopausal status is based on self - report , we can not deny the possibility of misclassification of menopausal status among the women studied . in conclusion , in this study of a large number of female and male japanese individuals , the postmenopausal state in women was significantly associated with the presence of type 2 diabetes and prediabetes , although the increased probability did not equal that in the men . the postmenopausal state was also associated with prediabetic hyperglycemia independently of age and demographic and metabolic factors among women without diabetes . menopause and older age might additively influence the elevated probability of dysglycemia in japanese women .

objectivefindings on the effect of menopause or age at menopause on the presence of hyperglycemia are controversial , and why women after menopause have a higher probability of having hyperglycemia than men in the same age range remains unknown.research design and methodswe reviewed data on 29,189 men , 6,308 premenopausal women , and 4,570 postmenopausal women in japan . odds ratios ( ors ) for diabetes or prediabetes indicated by american diabetes association criteria were calculated for men and for pre- and postmenopausal women.resultscompared with premenopausal women , women after natural menopause had an age - adjusted or of 1.40 ( 95% ci 1.031.89 ) for diabetes , and women after menopause by surgical or other causes had an age - adjusted or of 1.59 ( 1.072.37 ) . the age - adjusted or in men was 4.02 ( 3.155.14 ) . compared with premenopausal nondiabetic women , postmenopausal nondiabetic women had a significantly elevated or of 1.33 ( 1.201.48 ) for prediabetes ; nondiabetic men had an or of 1.93 ( 1.772.10 ) independently of age and demographic and metabolic factors . even among women aged < 50 years , postmenopausal status was significantly associated with an elevated or ( 1.50 [ 1.181.91 ] ) for dysglycemia ( either diabetes or prediabetes ) . postmenopausal women aged 50 years had a particularly elevated or for dysglycemia , regardless of age at menopause.conclusionsthe postmenopausal state was significantly associated with the presence of dysglycemia independently of normal aging , although the increased probability in postmenopausal women did not equal that in men . among women , menopause and older age might additively influence the elevated probability of dysglycemia .

RESEARCH DESIGN AND METHODS Diagnosis of type 2 diabetes and prediabetes Assessment of the menopausal state and other variables Clinical measurements Statistical analysis RESULTS CONCLUSIONS

among the 41,931 individuals , this cross - sectional study included 41,700 individuals for whom data on sex and menopausal status were available ( 6,458 premenopausal women , 5,701 postmenopausal women , and 29,541 men ) . among those 41,700 individuals , we excluded 1,027 women who did not report a cause for menopause ( natural , surgical , or other ) . after the exclusions , 40,673 individuals ( 6,458 premenopausal women , 3,630 women in natural menopause , 943 women in surgical menopause , 101 postmenopausal women by other causes , and 29,541 men ) were available for analysis . we excluded 81 women aged 65 years who had been in the premenopausal category because their persistent vaginal bleeding after the age 65 was not likely a result of menses but of pathologic processes ( 12 ) . subsequently , 40,067 individuals ( 6,308 premenopausal women , 3,552 women in natural menopause , 1,018 women in surgical menopause or another cause , and 29,189 men ) were included in the current analysis . with regard to women with missing data on age at menopause ( n = 154 ) , we excluded them only for the analysis of the relationship between age at menopause and dysglycemia . diagnosis of type 2 diabetes was made according to american diabetes association criteria ( 22 ) of a fasting plasma glucose ( fpg ) level 7.0 mmol / l ( 126 mg / dl ) , self - reported clinician - diagnosed diabetes or the use of hypoglycemic agents or insulin , or hba1c 6.5% ( 48 mmol / mol ) . if so , they were asked to indicate the reason for menopause ( natural , surgical , or other ) and the age at which menopause occurred ( 39 , 4044 , 4549 , or 50 years ) . parental history of diabetes , smoking habit ( never , former , or current ) , physical activity habit ( any physical activity for 2030 min or longer at least once weekly ) , and self - reported history of medical treatment for hypertension or diabetes were also assessed by the questionnaire for both men and women . logistic regression analysis was performed to calculate odds ratios ( ors ) and 95% cis . we initially investigated whether there was a difference in the association of dysglycemia between men and women and then calculated ors for dysglycemia for postmenopausal women ( regardless of cause ) and for men , with premenopausal women as the reference group . because few women reported an age at menopause of < 39 years , we categorized age at menopause into three groups ( < 45 , 4549 , and 50 years ) for the analysis . to investigate effect modifications , we performed logistic regression analysis with adjustment for age ( model 1 ) ; age and other demographic factors ( bmi , parental history of diabetes , physical activity habit , and smoking habit ) ( model 2 ) ; and age , demographic , and metabolic factors ( hypertension indicated by systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or medical treatment and hdl cholesterol and log - transformed triglyceride levels ) ( model 3 ) . in an additional analysis , we stratified women according age at the time of examination ( < 50 or 50 years ) because the mean age of menopause has been considered to be 50 years ( 9,11,14,21,24,25 ) . a combined effect of older age at the time of the examination and the postmenopausal condition on the presence of dysglycemia ( either prediabetes or type 2 diabetes ) was assessed , with premenopausal women aged < 50 years as the reference group . we then conducted a stratified analysis based on age at the time of examination ( < 50 or 50 years ) and calculated ors for dysglycemia across categories of age at menopause , with the premenopausal state as the reference group for women aged < 50 or 50 years . diagnosis of type 2 diabetes was made according to american diabetes association criteria ( 22 ) of a fasting plasma glucose ( fpg ) level 7.0 mmol / l ( 126 mg / dl ) , self - reported clinician - diagnosed diabetes or the use of hypoglycemic agents or insulin , or hba1c 6.5% ( 48 mmol / mol ) . if so , they were asked to indicate the reason for menopause ( natural , surgical , or other ) and the age at which menopause occurred ( 39 , 4044 , 4549 , or 50 years ) . parental history of diabetes , smoking habit ( never , former , or current ) , physical activity habit ( any physical activity for 2030 min or longer at least once weekly ) , and self - reported history of medical treatment for hypertension or diabetes were also assessed by the questionnaire for both men and women . logistic regression analysis was performed to calculate odds ratios ( ors ) and 95% cis . we initially investigated whether there was a difference in the association of dysglycemia between men and women and then calculated ors for dysglycemia for postmenopausal women ( regardless of cause ) and for men , with premenopausal women as the reference group . because few women reported an age at menopause of < 39 years , we categorized age at menopause into three groups ( < 45 , 4549 , and 50 years ) for the analysis . to investigate effect modifications , we performed logistic regression analysis with adjustment for age ( model 1 ) ; age and other demographic factors ( bmi , parental history of diabetes , physical activity habit , and smoking habit ) ( model 2 ) ; and age , demographic , and metabolic factors ( hypertension indicated by systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or medical treatment and hdl cholesterol and log - transformed triglyceride levels ) ( model 3 ) . in an additional analysis , we stratified women according age at the time of examination ( < 50 or 50 years ) because the mean age of menopause has been considered to be 50 years ( 9,11,14,21,24,25 ) . a combined effect of older age at the time of the examination and the postmenopausal condition on the presence of dysglycemia ( either prediabetes or type 2 diabetes ) was assessed , with premenopausal women aged < 50 years as the reference group . we then conducted a stratified analysis based on age at the time of examination ( < 50 or 50 years ) and calculated ors for dysglycemia across categories of age at menopause , with the premenopausal state as the reference group for women aged < 50 or 50 years . premenopausal women were younger ( 42.1 [ 6.6 ] years ) compared with postmenopausal women . we did not observe a marked difference in bmi between premenopausal and postmenopausal women . among the premenopausal women , only 69 ( 1.1% ) had type 2 diabetes , whereas the prevalence rate was high at 3.8% in women after natural menopause and 4.0% after surgical menopause or menopause from other causes . characteristics of total women , premenopausal women , postmenopausal women ( by cause ) , and men table 2 shows ors for type 2 diabetes and prediabetes among men and among women by menopausal status . men were 2.10 ( 95% ci 1.812.45 ) times more likely to have type 2 diabetes than the total number of women studied according to multivariate model 3 , which included age and demographic and metabolic factors . postmenopausal women had a significant association with type 2 diabetes ( 1.36 [ 1.011.82 ] ) compared with premenopausal women that was independent of age , bmi , smoking habit , physical activity habit , and parental history of diabetes ( model 2 ) , although the or was not as high as that in men ( 2.87 [ 2.233.69 ] ) . after adjustment for lipid measurements and hypertension ( model 3 ) , the or for the postmenopausal women was attenuated ( 1.17 [ 0.881.58 ] ) , and a significant association with the presence of type 2 diabetes remained only among the men ( 2.35 [ 1.823.03 ] ) . among the women , we did not find an obvious difference in the association of type 2 diabetes and menopausal status regardless of the cause of menopause . the association of prediabetes with menopause among individuals without type 2 diabetes showed that postmenopausal women had a significantly elevated or for prediabetes in model 3 ( 1.33 [ 1.201.48 ] ) compared with premenopausal women . additionally , the men had a significantly elevated or for prediabetes compared with postmenopausal women ( 1.93 [ 1.772.10 ] ) . regardless of age or demographic or metabolic factors , women with natural menopause or other causes of menopause had similarly elevated ors for prediabetes ( 1.36 [ 1.221.52 ] and 1.22 [ 1.041.43 ] , respectively ) . we observed that early age at menopause ( < 45 years ) was significantly associated with an elevated or for diabetes after adjustment for age ( 1.89 [ 1.212.96 ] ) or for age and demographic factors ( 1.73 [ 1.102.73 ] ) . we did not observe an association of early menopause with an increased probability of having prediabetes among individuals without type 2 diabetes . ors ( 95% ci ) for type 2 diabetes or prediabetes between women and men ( a ) , among pre- or postmenopausal women and men ( b and c ) , or among pre- or postmenopausal women according to age at menopause and men ( d ) figure 1 shows the combined effect of age at examination and menopausal status on the presence of dysglycemia ( either prediabetes or type 2 diabetes ) . although older age alone at the time of the examination ( 50 years ) was significantly associated with dysglycemia ( or 2.21 [ 95% ci 1.852.65 ] ) , postmenopausal status alone was also significantly associated with an elevated or for dysglycemia ( 1.50 [ 1.181.91 ] ) . the postmenopausal condition and older age additively influenced an elevated or because postmenopausal women aged 50 years had a markedly elevated or ( 3.69 [ 3.344.08 ] ) for dysglycemia . we stratified women by age at the time of the examination and investigated whether there was an association of age at menopause with the presence of dysglycemia ( table 3 ) . compared with premenopausal women , postmenopausal women who underwent menopause at < 45 or 4549 years had a 1.41 ( 0.982.02 ) and 1.59 ( 1.152.20 ) times increased or for dysglycemia , respectively , even among women aged < 50 years at the time of examination ( n = 5,991 ) . adjustment for demographic and metabolic factors ( multivariate model 2 ) attenuated the ors ( 1.18 [ 0.801.74 ] and 1.29 [ 0.911.82 ] , respectively ) . among women aged 50 years , the postmenopausal state was significantly associated with the presence of dysglycemia , regardless of age at which menopause occurred . in multivariate model 2 , postmenopausal women had a similarly elevated or for dysglycemia to premenopausal women , regardless of age at menopause ( < 45 years of age 1.58 [ 1.222.04 ] , 4549 years of age 1.62 [ 1.312.00 ] , 50 years of age 1.65 [ 1.371.99 ] ) . probability of having dysglycemia ( either prediabetes or type 2 diabetes ) through a combination of age at the time of examination and menopausal status . association of dysglycemia ( either prediabetes or type 2 diabetes ) and age at menopause among women aged < 50 or 50 years at the time of examination we found that older age and a postmenopausal state independently and additively influenced the high prevalence of dysglycemia in japanese women . even among women aged < 50 years at the time of examination , menopause was associated with the presence of dysglycemia . the study is unique because it compared the probability of dysglycemia among women across menopausal states with that among mainly middle - aged men who underwent health screening in japan . although the or for postmenopausal women was not high compared with that of the men , their ors for type 2 diabetes and prediabetes were significantly elevated independently of age compared with those in premenopausal women . whether menopausal status would influence the occurrence of diabetes independently of age and other confounding factors remains controversial because it is difficult to conduct studies to separate the effects of normal aging from the menopausal transition . a few studies showed a significant positive association of hyperglycemia with menopausal status after adjustment for age and other risk factors for diabetes ( 7,13,19 ) , but multivariate analyses in other studies showed no such associations ( 4,11,14,18,26 ) . in a cross - sectional multicenter study of italian women from outpatient menopausal clinics , those with natural menopause had a 1.38 times higher multivariate - adjusted or for diabetes than premenopausal women ( 13 ) . on the other hand , the researchers did not find a significant association in women with surgical menopause and diabetes ( 13 ) . a cross - sectional study of korean women suggested that in postmenopausal women , there is a significant association with the presence of hyperglycemia ( fasting glucose level 110 mg / dl or antidiabetes medications ) compared with premenopausal women that is independent of age and bmi ( 7 ) . in women at high risk for diabetes who participated in the diabetes prevention program , no association was found between natural menopause or bilateral oophorectomy and increased risk of developing diabetes after adjustment for age ( 12 ) . the present study shows a significant positive association between postmenopause ( regardless of cause ) and the presence of type 2 diabetes independently of normal aging compared with premenopause . diabetes and impaired fasting glycemia are reported to be more common in men than in women 3069 years of age , whereas the prevalence of isolated postload hyperglycemia , particularly impaired glucose tolerance , is reportedly higher in women than in men , especially in individuals > 70 years of age ( 27 ) . the lack of data on ogtt for the diagnosis of dysglycemia might lead to an underestimation of the associations between menopause and the prevalence of diabetes . the significant association of type 2 diabetes and postmenopausal status was particularly attenuated after adjustments for hypertension and blood lipid measurements , suggesting that when we consider the association of menopause with diabetes , we also should consider the influence of related metabolic factors . because the transition from the premenopausal to the postmenopausal state is associated with changes in body composition ( increased body fat mass , increased abdominal fat , and decreased lean body mass ) ( 3 ) and substantial metabolic changes , features of metabolic syndrome would occur in many women ( 17 ) . because data on body composition or visceral fat were not available for the current study , we could not assess whether differences in body composition across the menopausal state might have influenced the presence of dysglycemia , even if bmis across the menopausal state were relatively low . although we did not have data on reproductive hormone concentrations and can not explain the mechanism for the current observations , it was shown that natural menopause is characterized by increased relative androgenicity , which was reported to be associated with glucose metabolism ( 15 ) ; furthermore , reproductive hormone concentrations can vary by ethnicity and were shown to be confounded by ethnic disparities in body mass ( 32 ) . the current results show that among individuals without diabetes , prediabetic hyperglycemia is significantly associated with postmenopausal status independently of age and demographic and metabolic parameters . in a cross - sectional study of japanese women without diabetes , stepwise regression analysis showed natural menopause rather than age as a significant determinant of fpg concentrations ( 19 ) . on the other hand , among middle - aged women living in north taiwan , no significant difference in fpg , insulin levels , homeostasis model assessment of insulin resistance , and prevalence of hyperglycemia between premenopausal and postmenopausal women was shown ( 6 ) . the current results show that prediabetic hyperglycemia and the postmenopausal state are positively associated , suggesting that postmenopausal women might be at high risk for diabetes . in a prospective case - cohort study that included only postmenopausal women , earlier age at menopause the hazard ratio for diabetes was 32% higher in women who entered menopause before 40 years of age compared with those experiencing menopause at age 5054 ( 20 ) . a study in chinese postmenopausal women , however , showed no association between age at menopause and diabetes ( 21 ) . the results of the current cross - sectional investigation suggest that early age at menopause ( < 45 years ) might be more strongly associated with type 2 diabetes than menopause at 50 years . nonetheless , regardless of age at menopause , postmenopausal women aged 50 years at the time of the examination had an 1.5 times increased probability of having dysglycemia compared with premenopausal women . additionally , age at menopause can be affected by various social and environmental factors ( 25,33 ) , which might be one possible explanation for the mixed results of the association of age at menopause and diabetes compared with existing studies . it has been established that a smoking habit is significantly associated with an early age at natural menopause ( 34 ) . factors of lower educational attainment ; being separated , widowed , or divorced ; and nonemployment have been associated with early natural menopause , whereas japanese ethnicity is associated with late age at natural menopause ( 33 ) . furthermore , bmi has been associated with age at menopause ( 35 ) . on the other hand , a recent study of women from five racial and ethnic groups indicated that there is no significant racial / ethnic difference in age at the final natural menstrual cycle after controlling for sociodemographic , lifestyle , and health factors ( 36 ) . recent reports indicated that early age at natural menopause is associated with an increased risk of ischemic stroke ( 37 ) and mortality ( 38 ) . high levels of habitual physical activity , such as walking , have been associated with a favorable cardiovascular risk profile in postmenopausal women ( 40 ) . because we did not include data on nutritional intake or other known risk factors for diabetes , such as sleep disturbances and depression , which are commonly observed in women at midlife , we did not have data on visceral fat or hormone replacement therapy , so that the ors might be over- or underestimated . in conclusion , in this study of a large number of female and male japanese individuals , the postmenopausal state in women was significantly associated with the presence of type 2 diabetes and prediabetes , although the increased probability did not equal that in the men . the postmenopausal state was also associated with prediabetic hyperglycemia independently of age and demographic and metabolic factors among women without diabetes . menopause and older age might additively influence the elevated probability of dysglycemia in japanese women .

over the last decade or so , spiritual or existential well - being has been recognized as an important dimension of quality of life ( qol ) . taking this into account , world health organization ( who ) designed who 's quality of life spirituality , religiousness and personal beliefs ( whoqol - srpb ) scale to assess religious , spiritual , and personal beliefs as a domain of qol , which is distinct from psychological and social domains . the whoqol - srpb scale that has 32 questions , covering qol aspects related to spirituality , religiousness , and personal beliefs , which was developed from an extensive pilot test of 105 questions carried in 18 centers around the world . the whoqol - srpb scale acknowledges that some people follow a particular religion , while some others do not but believe in a higher spiritual entity . some , however , do not follow both , but do have strong personal beliefs ( such as scientific theory or a particular philosophical view ) that guide them in day - to - day activities . the whoqol - srpb makes allowance for these differing preferences as the questions are so framed that each individual can answer keeping one 's own particular belief system in mind . due to these , whoqol - srpb can be considered as a valuable instrument to assess spirituality and religiousness . the initial evaluation of the scale was not limited to ill population and this makes it an ideal measure to study the spiritual qol in diverse populations , including healthy subjects . the 32 items of srpb are supposed to be used in conjunction with the whoqol-100 . the 32 questions are divided into 8 facets , each comprising 4 items and the facets are named as spiritual connection , meaning and purpose in life , experiences of awe and wonder , wholeness and integration , spiritual strength , inner peace , hope and optimism , and faith . responses to each item is rated on a 5-point likert scale ( ranging from 0 [ not at all ] to 5 [ an extreme amount ] ) . each facet score is derived by averaging the score obtained from the responses to the 4 questions comprising that particular facet . as srpb is supposed to be used with whoqol-100 , the srpb domain consists of the 9 facets that include the spirituality domain of the original whoqol-100 scale and the remaining 8 srpb facets . the alpha value for various srpb facets are reported to be strong , ranging from meaning and purpose in life ( =0.77 ) to faith ( =0.95 ) . however , non - availability of this self - administered instrument in local language ( hindi in our context ) is an important limitation in using this scale , as a large proportion of population in india still is not very comfortable with english language . considering the fact that hindi is the dominant language spoken and understood by a large portion of the population in india and as whoqol-100 is available in hindi , it was considered that translation of the whoqol - srpb to hindi would be useful . accordingly , the aim of the present study was to translate the english version of the srpb facets of whoqol - srpb scale to hindi and evaluate its psychometric properties . the translation procedure was part of a larger study that attempted to study spirituality , religiousness , and personal beliefs of patients with schizophrenia . ethical review board of the institute approved the study and all participants were recruited after obtaining written informed consent . translation was carried out according to the methodology described by who . the 32 items were translated from english to hindi by 3 bilingual mental health professionals who had sufficient knowledge of both the languages ( spoken and written ) and had received formal education in both languages . the translators were knowledgeable about both the english - speaking and hindi - speaking cultures and idioms and had undertaken translation and development of other health - related instruments in the past . once the translated versions were available , an expert panel ( comprising three members other than the translators ) was convened . these members were experts in psychological and social fields of health , as well as had experience in instrument development and translation . the translated versions were reviewed by the members of the expert panel who also had access to the english version of whoqol - srpb instrument . the goal in this step was to identify the best translated version , make alternate suggestions , and by consensus resolve the difficulties in expressed meaning of the translated items . the members focused on overall quality of translation , meaning of words , and ease of understandability / comprehensibility of the language . the panel members questioned some words or expressions and made individual suggestions , which were discussed jointly . the changes that were agreed upon by all three members were incorporated and a draft of translated version was developed . the draft of translated version was then given to another 3 bilingual mental health professionals for back - translation . the back - translators had no knowledge or access to the english version of whoqol - srpb instrument . during the process of back - translation , emphasis was also on conceptual and cultural equivalence and not merely linguistic equivalence . the back - translated versions were compared with the english version of whoqol - srpb instrument by the expert panel and the hindi translated version was modified to remove the ambiguity in the meaning of words or phrases by consensus after reviewing all the 3 back - translations and a revised version was made . to further improve the quality of translation , the revised version was given to ten members of the community in which it was to be used . after these respondents completed the questionnaire , they were interviewed to enquire as to whether they were able to comprehend as to what each item was attempting to address . any words they did not understand or any word or expressions that they found unacceptable were also enquired into . the respondents were also asked to make suggestions regarding the use of words , phrases / idioms and framing of questions that in their opinion would improve the understandability of the instrument . the expert panel reviewed all the suggestions received and after thorough discussion those deemed appropriate were incorporated and final translated version was accepted . the final hindi version was assessed for cross language concordance and test - retest reliability . for this , participants were recruited by convenient sampling from those who were attending the psychiatry services of the institute . the participants included primary caregivers of patients , hospital staff , and patients with schizophrenia in remission . for studying the cross language equivalence , bilingual participants who were proficient in hindi and english were recruited , while for studying test - retest reliability , individuals well versed with hindi were recruited . for cross language equivalence , a crossover design was followed with half the subjects ( selected randomly ) being given either english or hindi versions first . the other language version ( english or hindi as applicable ) was given one week later to the same participants . another set of participants was given the hindi version twice , one week apart for assessing test - retest reliability . two assessments were done 1 week apart so as to negate any actual change in qol status between the two ratings and remove memory bias due to too close observations . frequency and descriptive analyses were carried out for the demographic variables . paired t - test and intra - class correlation were studied to assess the cross language concordance between item scores and between facet scores of english and hindi versions . similarly , paired t - test and intra - class correlation were performed to assess the test - retest reliability by comparing the scores ( at baseline and 1 week later ) obtained on final hindi version . cronbach 's alpha was used to examine the internal consistency , and the spearman - brown sphericity coefficient was used to determine the split - half reliability of the hindi srpb scale . the 32 items were translated from english to hindi by 3 bilingual mental health professionals who had sufficient knowledge of both the languages ( spoken and written ) and had received formal education in both languages . the translators were knowledgeable about both the english - speaking and hindi - speaking cultures and idioms and had undertaken translation and development of other health - related instruments in the past . once the translated versions were available , an expert panel ( comprising three members other than the translators ) was convened . these members were experts in psychological and social fields of health , as well as had experience in instrument development and translation . the translated versions were reviewed by the members of the expert panel who also had access to the english version of whoqol - srpb instrument . the goal in this step was to identify the best translated version , make alternate suggestions , and by consensus resolve the difficulties in expressed meaning of the translated items . the members focused on overall quality of translation , meaning of words , and ease of understandability / comprehensibility of the language . the panel members questioned some words or expressions and made individual suggestions , which were discussed jointly . the changes that were agreed upon by all three members were incorporated and a draft of translated version was developed . the draft of translated version was then given to another 3 bilingual mental health professionals for back - translation . the back - translators had no knowledge or access to the english version of whoqol - srpb instrument . during the process of back - translation , emphasis was also on conceptual and cultural equivalence and not merely linguistic equivalence . the back - translated versions were compared with the english version of whoqol - srpb instrument by the expert panel and the hindi translated version was modified to remove the ambiguity in the meaning of words or phrases by consensus after reviewing all the 3 back - translations and a revised version was made . to further improve the quality of translation , the revised version was given to ten members of the community in which it was to be used . after these respondents completed the questionnaire , they were interviewed to enquire as to whether they were able to comprehend as to what each item was attempting to address . any words they did not understand or any word or expressions that they found unacceptable were also enquired into . the respondents were also asked to make suggestions regarding the use of words , phrases / idioms and framing of questions that in their opinion would improve the understandability of the instrument . the expert panel reviewed all the suggestions received and after thorough discussion those deemed appropriate were incorporated and final translated version was accepted . the final hindi version was assessed for cross language concordance and test - retest reliability . for this , participants were recruited by convenient sampling from those who were attending the psychiatry services of the institute . the participants included primary caregivers of patients , hospital staff , and patients with schizophrenia in remission . for studying the cross language equivalence , bilingual participants who were proficient in hindi and english were recruited , while for studying test - retest reliability , individuals well versed with hindi were recruited . for cross language equivalence , a crossover design was followed with half the subjects ( selected randomly ) being given either english or hindi versions first . the other language version ( english or hindi as applicable ) was given one week later to the same participants . another set of participants was given the hindi version twice , one week apart for assessing test - retest reliability . two assessments were done 1 week apart so as to negate any actual change in qol status between the two ratings and remove memory bias due to too close observations . frequency and descriptive analyses were carried out for the demographic variables . paired t - test and intra - class correlation were studied to assess the cross language concordance between item scores and between facet scores of english and hindi versions . similarly , paired t - test and intra - class correlation were performed to assess the test - retest reliability by comparing the scores ( at baseline and 1 week later ) obtained on final hindi version . cronbach 's alpha was used to examine the internal consistency , and the spearman - brown sphericity coefficient was used to determine the split - half reliability of the hindi srpb scale . for the process of cross language equivalence , the hindi and english versions were given to 23 participants , and for the test - retest evaluation , the scale was given to 20 participants . the mean age of the participants was 42.09 years ( range : 20 - 64 years ) . a majority of them had at least 15 years of schooling ( n=18 , 82.8% ) . as depicted in tables 1 and 2 , there were significant correlations between the english and hindi versions of the whoqol - srpb at the level of each facet and item / question of the whoqol - srpb . the intra - class correlation value for the various facets ranged from 0.86 to 0.96 . the intra - class correlation value was significant for each item and varied from 0.58 to 0.97 , except for 2 items as shown in table 2 . cross language concordance between the hindi and english versions of whoqol - srpb facets cross language concordance between the hindi and english versions of whoqol - srpb and test - retest reliability of hindi version of whoqol - srpb for this , the sample consisted of 20 participants . the mean age of the participants was 38.05 years ( sd 15.16 ; range : 20 - 70 years ) and there were equal number of males and females ( i.e. 10 each ) . the intra - class correlation coefficient was significant ( p<0.001 ) for each facet and ranged from 0.89 to 0.95 [ table 3 ] . at the item level , there was significant correlation between both the assessments , with significance level < 0.001 for 30 of the 32 items , < 0.01 for one item , and < 0.05 for the remaining 1 item [ table 2 ] . test - retest reliability of hindi version of whoqol - srpb facets for determining the internal consistency and split half reliability data of 43 participants , responses on hindi version of the 23 participants in the cross - language equivalence and baseline responses of the 20 participants of the test re - test reliability were used . the cronbach 's alpha ( as a measure of internal consistency ) was 0.93 , and the spearman - brown sphericity coefficient ( for assessing split - half reliability ) was 0.91 for the hindi version of srpb . for the english version , the cronbach 's alpha ( as a measure of internal consistency ) was 0.85 , and the spearman - brown sphericity coefficient ( for assessing split - half reliability ) was 0.77 for the english version of srpb . the mean age of the participants was 42.09 years ( range : 20 - 64 years ) . a majority of them had at least 15 years of schooling ( n=18 , 82.8% ) . as depicted in tables 1 and 2 , there were significant correlations between the english and hindi versions of the whoqol - srpb at the level of each facet and item / question of the whoqol - srpb . the intra - class correlation value for the various facets ranged from 0.86 to 0.96 . the intra - class correlation value was significant for each item and varied from 0.58 to 0.97 , except for 2 items as shown in table 2 . cross language concordance between the hindi and english versions of whoqol - srpb facets cross language concordance between the hindi and english versions of whoqol - srpb and test - retest reliability of hindi version of whoqol - srpb for this , the sample consisted of 20 participants . the mean age of the participants was 38.05 years ( sd 15.16 ; range : 20 - 70 years ) and there were equal number of males and females ( i.e. 10 each ) . the intra - class correlation coefficient was significant ( p<0.001 ) for each facet and ranged from 0.89 to 0.95 [ table 3 ] . at the item level , there was significant correlation between both the assessments , with significance level < 0.001 for 30 of the 32 items , < 0.01 for one item , and < 0.05 for the remaining 1 item [ table 2 ] . the mean age of the participants was 42.09 years ( range : 20 - 64 years ) . a majority of them had at least 15 years of schooling ( n=18 , 82.8% ) . as depicted in tables 1 and 2 , there were significant correlations between the english and hindi versions of the whoqol - srpb at the level of each facet and item / question of the whoqol - srpb . the intra - class correlation value for the various facets ranged from 0.86 to 0.96 . the intra - class correlation value was significant for each item and varied from 0.58 to 0.97 , except for 2 items as shown in table 2 . cross language concordance between the hindi and english versions of whoqol - srpb facets cross language concordance between the hindi and english versions of whoqol - srpb and test - retest reliability of hindi version of whoqol - srpb the mean age of the participants was 38.05 years ( sd 15.16 ; range : 20 - 70 years ) and there were equal number of males and females ( i.e. 10 each ) . the intra - class correlation coefficient was significant ( p<0.001 ) for each facet and ranged from 0.89 to 0.95 [ table 3 ] . at the item level , there was significant correlation between both the assessments , with significance level < 0.001 for 30 of the 32 items , < 0.01 for one item , and < 0.05 for the remaining 1 item [ table 2 ] . for determining the internal consistency and split half reliability data of 43 participants , responses on hindi version of the 23 participants in the cross - language equivalence and baseline responses of the 20 participants of the test re - test reliability were used . the cronbach 's alpha ( as a measure of internal consistency ) was 0.93 , and the spearman - brown sphericity coefficient ( for assessing split - half reliability ) was 0.91 for the hindi version of srpb . for the english version , data of 23 participants who participated in the cross - language equivalence were used . the cronbach 's alpha ( as a measure of internal consistency ) was 0.85 , and the spearman - brown sphericity coefficient ( for assessing split - half reliability ) was 0.77 for the english version of srpb . findings of the present study show that the hindi version of srpb as translated for this study is a psychometrically valid instrument evidenced by the fact that it has good test re - retest reliability , cross - language equivalence , internal consistency , and split half reliability . the methodology of translation and back translation used in this study is a well - established method to achieve the goal of translating to a conceptually equivalent and acceptable instrument that is culture sensitive and acceptable to the local population . in translation research , it is rightly emphasized that while translating an instrument merely the word / phrase to word / phrase is not sufficient but the conceptual meaning of the text has to be understood and then translated . other issue considered important while translating and validating an instrument is the consideration for cultural differences , because certain concepts , which may be native to one culture may be foreign to another . furthermore , certain features of the language , such as idioms , are very difficult to translate and make little sense in a different cultural context . in this study , the translation process took these into account . also , lay persons belonging to the community in which the scale is purported to be used were involved in the translation process so as to understand the ease and understandability of the language used in the scale . in the present study , the test - retest reliability at the facet level was good for all the facets . at the item level too , the test - retest reliability of each item was found to be very good . results of the present study are comparable to the reliability analysis of the original scale ( whoqol srpb group , 2006 ) that showed an alpha correlation coefficient for the srpb facets to be strong , ranging from 0.77 to 0.95 . an overall strong icc for all the facets in the present study indicates that the quality of translation is acceptable for use in hindi - speaking population . in the present study , the cross language equivalence at the facet level was good . at the item / question level too , there was significant correlation for 30 of the 32 items . these findings suggest that the hindi srpb version assesses the same concepts as the english srpb instrument and it has good test - retest reliability where repeatability is concerned . while validating the hindi version of whoqol-100 noted that despite some cross linguistic equivalence between hindi and english versions , there were significant differences in one - third of facet and domain scores . this should be understood in the light of the fact that the concepts of spirituality and religiousness are integral to the indian culture and most people have imbibed and internalized spirituality without any active effort . thus , the construct is not new to the indian population and improves the chances of comprehensibility of the items as opposed to certain items assessing other domains of qol . such findings suggest that the quality of cross - cultural translation relies heavily on the acceptability of a construct in that culture . findings of the present study reflect that although the internal consistency and split - half reliability of both hindi and english version of srpb are good , the same parameters for the hindi version were better than the english version . , who while studying the psychometric properties of the hindi translation of the whoqol-100 found lower reliability of facets in english version . the authors raised concern regarding application of the scale in subjects who are proficient in a language but are from a different culture . to conclude , the present study shows that cross - language equivalence , internal consistency , split - half reliability , and test - retest reliability of the hindi version of whoqol - srpb are excellent . the internal consistency and split - half reliability of the english version of whoqol - srpb was also found to be good . however , we did not examine the test - retest reliability for the english version , which is a limitation of present study . the predictive and convergent validity of the scale was also not measured in the present study . in future , the hindi version of the srpb can be used as a self - rated measure along with hindi version who - qol-100 to measure the different aspects of qol of various patients groups , their caregivers , and people in the community . the findings of the present study suggest that hindi version of whoqol - srpb as translated in this study is a valid instrument .

background : world health organization 's quality of life spirituality , religiousness and personal beliefs scale ( whoqol srpb ) is a valuable instrument for assessing spirituality and religiousness . the absence of this self - administered instrument in hindi , which is a major language in india , is an important limitation in using this scale.aim:to translate the english version of the srpb facets of whoqol - srpb scale to hindi and evaluate its psychometric properties.materials and methods : the srpb facets were translated into hindi using the world health organisation 's translation methodology . the translated hindi version was evaluated for cross - language equivalence , test - retest reliability , internal consistency , and split half reliability.results:hindi version was found to have good cross - language equivalence and test - retest reliability at the level of facets . twenty - six of the 32 items and 30 of the 32 items had a significant correlation ( <0.001 ) in cross language concordance and test - retest reliability data , respectively . the cronbach 's alpha was 0.93 , and the spearman - brown sphericity value was 0.91 for the hindi version of srpb.conclusions:the present study shows that cross - language equivalence , internal consistency , split - half reliability , and test - retest reliability of the hindi version of srpb ( of whoqol - srpb ) are excellent . thus , the hindi version of whoqol - srpb as translated in this study is a valid instrument .

INTRODUCTION MATERIALS AND METHODS Translation process Psychometric evaluation Statistical analysis RESULTS None Cross-language concordance Test-retest reliability Internal consistency and split-half reliability DISCUSSION CONCLUSION

over the last decade or so , spiritual or existential well - being has been recognized as an important dimension of quality of life ( qol ) . taking this into account , world health organization ( who ) designed who 's quality of life spirituality , religiousness and personal beliefs ( whoqol - srpb ) scale to assess religious , spiritual , and personal beliefs as a domain of qol , which is distinct from psychological and social domains . the whoqol - srpb scale that has 32 questions , covering qol aspects related to spirituality , religiousness , and personal beliefs , which was developed from an extensive pilot test of 105 questions carried in 18 centers around the world . the whoqol - srpb scale acknowledges that some people follow a particular religion , while some others do not but believe in a higher spiritual entity . some , however , do not follow both , but do have strong personal beliefs ( such as scientific theory or a particular philosophical view ) that guide them in day - to - day activities . the whoqol - srpb makes allowance for these differing preferences as the questions are so framed that each individual can answer keeping one 's own particular belief system in mind . due to these , whoqol - srpb can be considered as a valuable instrument to assess spirituality and religiousness . the initial evaluation of the scale was not limited to ill population and this makes it an ideal measure to study the spiritual qol in diverse populations , including healthy subjects . the 32 items of srpb are supposed to be used in conjunction with the whoqol-100 . the 32 questions are divided into 8 facets , each comprising 4 items and the facets are named as spiritual connection , meaning and purpose in life , experiences of awe and wonder , wholeness and integration , spiritual strength , inner peace , hope and optimism , and faith . responses to each item is rated on a 5-point likert scale ( ranging from 0 [ not at all ] to 5 [ an extreme amount ] ) . as srpb is supposed to be used with whoqol-100 , the srpb domain consists of the 9 facets that include the spirituality domain of the original whoqol-100 scale and the remaining 8 srpb facets . the alpha value for various srpb facets are reported to be strong , ranging from meaning and purpose in life ( =0.77 ) to faith ( =0.95 ) . however , non - availability of this self - administered instrument in local language ( hindi in our context ) is an important limitation in using this scale , as a large proportion of population in india still is not very comfortable with english language . considering the fact that hindi is the dominant language spoken and understood by a large portion of the population in india and as whoqol-100 is available in hindi , it was considered that translation of the whoqol - srpb to hindi would be useful . accordingly , the aim of the present study was to translate the english version of the srpb facets of whoqol - srpb scale to hindi and evaluate its psychometric properties . the translation procedure was part of a larger study that attempted to study spirituality , religiousness , and personal beliefs of patients with schizophrenia . ethical review board of the institute approved the study and all participants were recruited after obtaining written informed consent . the 32 items were translated from english to hindi by 3 bilingual mental health professionals who had sufficient knowledge of both the languages ( spoken and written ) and had received formal education in both languages . once the translated versions were available , an expert panel ( comprising three members other than the translators ) was convened . the translated versions were reviewed by the members of the expert panel who also had access to the english version of whoqol - srpb instrument . the goal in this step was to identify the best translated version , make alternate suggestions , and by consensus resolve the difficulties in expressed meaning of the translated items . the members focused on overall quality of translation , meaning of words , and ease of understandability / comprehensibility of the language . the changes that were agreed upon by all three members were incorporated and a draft of translated version was developed . the draft of translated version was then given to another 3 bilingual mental health professionals for back - translation . the back - translators had no knowledge or access to the english version of whoqol - srpb instrument . during the process of back - translation , emphasis was also on conceptual and cultural equivalence and not merely linguistic equivalence . the back - translated versions were compared with the english version of whoqol - srpb instrument by the expert panel and the hindi translated version was modified to remove the ambiguity in the meaning of words or phrases by consensus after reviewing all the 3 back - translations and a revised version was made . to further improve the quality of translation , the revised version was given to ten members of the community in which it was to be used . the respondents were also asked to make suggestions regarding the use of words , phrases / idioms and framing of questions that in their opinion would improve the understandability of the instrument . the final hindi version was assessed for cross language concordance and test - retest reliability . for this , participants were recruited by convenient sampling from those who were attending the psychiatry services of the institute . the participants included primary caregivers of patients , hospital staff , and patients with schizophrenia in remission . for studying the cross language equivalence , bilingual participants who were proficient in hindi and english were recruited , while for studying test - retest reliability , individuals well versed with hindi were recruited . for cross language equivalence , a crossover design was followed with half the subjects ( selected randomly ) being given either english or hindi versions first . another set of participants was given the hindi version twice , one week apart for assessing test - retest reliability . frequency and descriptive analyses were carried out for the demographic variables . paired t - test and intra - class correlation were studied to assess the cross language concordance between item scores and between facet scores of english and hindi versions . similarly , paired t - test and intra - class correlation were performed to assess the test - retest reliability by comparing the scores ( at baseline and 1 week later ) obtained on final hindi version . cronbach 's alpha was used to examine the internal consistency , and the spearman - brown sphericity coefficient was used to determine the split - half reliability of the hindi srpb scale . the 32 items were translated from english to hindi by 3 bilingual mental health professionals who had sufficient knowledge of both the languages ( spoken and written ) and had received formal education in both languages . the translators were knowledgeable about both the english - speaking and hindi - speaking cultures and idioms and had undertaken translation and development of other health - related instruments in the past . once the translated versions were available , an expert panel ( comprising three members other than the translators ) was convened . the translated versions were reviewed by the members of the expert panel who also had access to the english version of whoqol - srpb instrument . the goal in this step was to identify the best translated version , make alternate suggestions , and by consensus resolve the difficulties in expressed meaning of the translated items . the members focused on overall quality of translation , meaning of words , and ease of understandability / comprehensibility of the language . the panel members questioned some words or expressions and made individual suggestions , which were discussed jointly . the changes that were agreed upon by all three members were incorporated and a draft of translated version was developed . the draft of translated version was then given to another 3 bilingual mental health professionals for back - translation . the back - translators had no knowledge or access to the english version of whoqol - srpb instrument . during the process of back - translation , emphasis was also on conceptual and cultural equivalence and not merely linguistic equivalence . the back - translated versions were compared with the english version of whoqol - srpb instrument by the expert panel and the hindi translated version was modified to remove the ambiguity in the meaning of words or phrases by consensus after reviewing all the 3 back - translations and a revised version was made . to further improve the quality of translation , the revised version was given to ten members of the community in which it was to be used . the expert panel reviewed all the suggestions received and after thorough discussion those deemed appropriate were incorporated and final translated version was accepted . the final hindi version was assessed for cross language concordance and test - retest reliability . for this , participants were recruited by convenient sampling from those who were attending the psychiatry services of the institute . the participants included primary caregivers of patients , hospital staff , and patients with schizophrenia in remission . for studying the cross language equivalence , bilingual participants who were proficient in hindi and english were recruited , while for studying test - retest reliability , individuals well versed with hindi were recruited . for cross language equivalence , a crossover design was followed with half the subjects ( selected randomly ) being given either english or hindi versions first . another set of participants was given the hindi version twice , one week apart for assessing test - retest reliability . frequency and descriptive analyses were carried out for the demographic variables . paired t - test and intra - class correlation were studied to assess the cross language concordance between item scores and between facet scores of english and hindi versions . similarly , paired t - test and intra - class correlation were performed to assess the test - retest reliability by comparing the scores ( at baseline and 1 week later ) obtained on final hindi version . cronbach 's alpha was used to examine the internal consistency , and the spearman - brown sphericity coefficient was used to determine the split - half reliability of the hindi srpb scale . for the process of cross language equivalence , the hindi and english versions were given to 23 participants , and for the test - retest evaluation , the scale was given to 20 participants . the mean age of the participants was 42.09 years ( range : 20 - 64 years ) . as depicted in tables 1 and 2 , there were significant correlations between the english and hindi versions of the whoqol - srpb at the level of each facet and item / question of the whoqol - srpb . the intra - class correlation value for the various facets ranged from 0.86 to 0.96 . cross language concordance between the hindi and english versions of whoqol - srpb facets cross language concordance between the hindi and english versions of whoqol - srpb and test - retest reliability of hindi version of whoqol - srpb for this , the sample consisted of 20 participants . the mean age of the participants was 38.05 years ( sd 15.16 ; range : 20 - 70 years ) and there were equal number of males and females ( i.e. the intra - class correlation coefficient was significant ( p<0.001 ) for each facet and ranged from 0.89 to 0.95 [ table 3 ] . at the item level , there was significant correlation between both the assessments , with significance level < 0.001 for 30 of the 32 items , < 0.01 for one item , and < 0.05 for the remaining 1 item [ table 2 ] . test - retest reliability of hindi version of whoqol - srpb facets for determining the internal consistency and split half reliability data of 43 participants , responses on hindi version of the 23 participants in the cross - language equivalence and baseline responses of the 20 participants of the test re - test reliability were used . the cronbach 's alpha ( as a measure of internal consistency ) was 0.93 , and the spearman - brown sphericity coefficient ( for assessing split - half reliability ) was 0.91 for the hindi version of srpb . for the english version , the cronbach 's alpha ( as a measure of internal consistency ) was 0.85 , and the spearman - brown sphericity coefficient ( for assessing split - half reliability ) was 0.77 for the english version of srpb . the mean age of the participants was 42.09 years ( range : 20 - 64 years ) . as depicted in tables 1 and 2 , there were significant correlations between the english and hindi versions of the whoqol - srpb at the level of each facet and item / question of the whoqol - srpb . the intra - class correlation value for the various facets ranged from 0.86 to 0.96 . the intra - class correlation value was significant for each item and varied from 0.58 to 0.97 , except for 2 items as shown in table 2 . cross language concordance between the hindi and english versions of whoqol - srpb facets cross language concordance between the hindi and english versions of whoqol - srpb and test - retest reliability of hindi version of whoqol - srpb for this , the sample consisted of 20 participants . the mean age of the participants was 38.05 years ( sd 15.16 ; range : 20 - 70 years ) and there were equal number of males and females ( i.e. the intra - class correlation coefficient was significant ( p<0.001 ) for each facet and ranged from 0.89 to 0.95 [ table 3 ] . at the item level , there was significant correlation between both the assessments , with significance level < 0.001 for 30 of the 32 items , < 0.01 for one item , and < 0.05 for the remaining 1 item [ table 2 ] . the mean age of the participants was 42.09 years ( range : 20 - 64 years ) . as depicted in tables 1 and 2 , there were significant correlations between the english and hindi versions of the whoqol - srpb at the level of each facet and item / question of the whoqol - srpb . the intra - class correlation value was significant for each item and varied from 0.58 to 0.97 , except for 2 items as shown in table 2 . cross language concordance between the hindi and english versions of whoqol - srpb facets cross language concordance between the hindi and english versions of whoqol - srpb and test - retest reliability of hindi version of whoqol - srpb the mean age of the participants was 38.05 years ( sd 15.16 ; range : 20 - 70 years ) and there were equal number of males and females ( i.e. the intra - class correlation coefficient was significant ( p<0.001 ) for each facet and ranged from 0.89 to 0.95 [ table 3 ] . at the item level , there was significant correlation between both the assessments , with significance level < 0.001 for 30 of the 32 items , < 0.01 for one item , and < 0.05 for the remaining 1 item [ table 2 ] . for determining the internal consistency and split half reliability data of 43 participants , responses on hindi version of the 23 participants in the cross - language equivalence and baseline responses of the 20 participants of the test re - test reliability were used . the cronbach 's alpha ( as a measure of internal consistency ) was 0.93 , and the spearman - brown sphericity coefficient ( for assessing split - half reliability ) was 0.91 for the hindi version of srpb . for the english version , data of 23 participants who participated in the cross - language equivalence were used . the cronbach 's alpha ( as a measure of internal consistency ) was 0.85 , and the spearman - brown sphericity coefficient ( for assessing split - half reliability ) was 0.77 for the english version of srpb . findings of the present study show that the hindi version of srpb as translated for this study is a psychometrically valid instrument evidenced by the fact that it has good test re - retest reliability , cross - language equivalence , internal consistency , and split half reliability . the methodology of translation and back translation used in this study is a well - established method to achieve the goal of translating to a conceptually equivalent and acceptable instrument that is culture sensitive and acceptable to the local population . in translation research , it is rightly emphasized that while translating an instrument merely the word / phrase to word / phrase is not sufficient but the conceptual meaning of the text has to be understood and then translated . other issue considered important while translating and validating an instrument is the consideration for cultural differences , because certain concepts , which may be native to one culture may be foreign to another . in this study , the translation process took these into account . also , lay persons belonging to the community in which the scale is purported to be used were involved in the translation process so as to understand the ease and understandability of the language used in the scale . in the present study , the test - retest reliability at the facet level was good for all the facets . at the item level too , the test - retest reliability of each item was found to be very good . results of the present study are comparable to the reliability analysis of the original scale ( whoqol srpb group , 2006 ) that showed an alpha correlation coefficient for the srpb facets to be strong , ranging from 0.77 to 0.95 . an overall strong icc for all the facets in the present study indicates that the quality of translation is acceptable for use in hindi - speaking population . in the present study , the cross language equivalence at the facet level was good . at the item / question level too , there was significant correlation for 30 of the 32 items . these findings suggest that the hindi srpb version assesses the same concepts as the english srpb instrument and it has good test - retest reliability where repeatability is concerned . while validating the hindi version of whoqol-100 noted that despite some cross linguistic equivalence between hindi and english versions , there were significant differences in one - third of facet and domain scores . this should be understood in the light of the fact that the concepts of spirituality and religiousness are integral to the indian culture and most people have imbibed and internalized spirituality without any active effort . thus , the construct is not new to the indian population and improves the chances of comprehensibility of the items as opposed to certain items assessing other domains of qol . such findings suggest that the quality of cross - cultural translation relies heavily on the acceptability of a construct in that culture . findings of the present study reflect that although the internal consistency and split - half reliability of both hindi and english version of srpb are good , the same parameters for the hindi version were better than the english version . , who while studying the psychometric properties of the hindi translation of the whoqol-100 found lower reliability of facets in english version . the authors raised concern regarding application of the scale in subjects who are proficient in a language but are from a different culture . to conclude , the present study shows that cross - language equivalence , internal consistency , split - half reliability , and test - retest reliability of the hindi version of whoqol - srpb are excellent . the internal consistency and split - half reliability of the english version of whoqol - srpb was also found to be good . however , we did not examine the test - retest reliability for the english version , which is a limitation of present study . the predictive and convergent validity of the scale was also not measured in the present study . in future , the hindi version of the srpb can be used as a self - rated measure along with hindi version who - qol-100 to measure the different aspects of qol of various patients groups , their caregivers , and people in the community . the findings of the present study suggest that hindi version of whoqol - srpb as translated in this study is a valid instrument .

physiotherapeutic procedures should restore locomotor fitness in patients after anterior cruciate ligament ( acl ) reconstruction . normal locomotion depends on , among other factors , ligamentocapsular structure stability , knee joint range of movement and an appropriate strength level of knee joint muscles , as well as on proprioception . the aim of acl reconstruction is to restore anatomical and biomechanical properties of this ligament , necessary to maintain anteroposterior and anteromedial knee joint stability . reported that selection of grafts and their placement in the bone tunnel affect the reconstruction result . the tension and fixation of the graft , tunnel mobility and graft healing speed are crucial . after reconstruction , the patients range of movement is temporarily limited and the strength of knee joint flexor muscles ( sartorius , gracilis , semitendinosus , semimembranosus , biceps femoris , gastrocnemius ) and extensor ( quadriceps ) muscles deteriorates . it is assumed that on completion of the entire physiotherapeutic program , the muscle strength values should be returned to preoperative levels , both in static and dynamic conditions . one of the goals of physiotherapy after acl reconstruction is to restore the antagonist strength ratio of knee joint muscle groups . when planning physiotherapeutic procedures , it is necessary to determine when and to what extent can muscle strength be safely restored . these programs , apart from being based on common assumptions , differ in the selection of exercises , and the magnitude and time of load application after acl reconstruction . the factors conditioning load selection to restore muscle strength after autologic ligament reconstruction include a minimal period following surgery , necessary for graft healing in the bone tunnel [ 1012 ] . the use of growth factors may enhance and improve graft healing , stiffness and load - bearing ability . personal traits of the patients , particularly mentality , gender , age and level of physical activity , are important . the aim of this paper is to present the physiotherapeutic program applied after acl reconstruction at the rehabilitation centre for the college of physiotherapy in wrocaw and the evaluation of its effectiveness , reflected by increased strength values of knee joint extensor and flexor muscles . the tailored pt study protocol , developed by the first author , was implemented for many years at the rehabilitation centre of the college of physiotherapy in wrocaw . a detailed and expanded version of this paper is part of the first author s doctoral thesis . thirty - seven males ( see table 1 ) participated in individual physiotherapeutic programs applied from the first week to up to 8 months after the primary endoscopic reconstruction of isolated acl tear by mitek s method of johnson & johnson gateway company using the autografts of the patients own semimembranosus and gracilis muscles . right- and left - sided lateralization was noted in 35 and 2 patients , respectively . the orthopedic surgeon qualified the patients for the first and for the subsequent stages of the physiotherapeutic procedure , and examined them after the physiotherapeutic program had finished . this examination included lachman s test , anteroposterior dislocation test and evaluation of the stability of other knee structures , as well as the presence of pain and swelling compared to the uninvolved knee . in the studied sample , during the entire study period of physiotherapeutic program implementation , the measurement of tibial translation , as related to the knee , did not exceed 5 mm . the patients gave their informed consent to participate in the study and the study was approved by the bioethics committee of the university of physical education in wrocaw . each patient underwent an individual therapeutic program with a physiotherapist , on average 4 times a week for 2 hours each day . additionally , the patients were instructed how to perform the exercises correctly at home and informed of possible hazards of ligament injuries . the outline of the physiotherapeutic program is presented in table 2 detailed physiotherapeutic procedure which comprises the more detailed program is presented elsewhere . measurements of range of movement and knee joint / thigh circumferences were performed twice : during the 1 ( i ) and 12 week ( ii ) after the reconstruction ( table 3 ) . the circumference was measured at the level of knee joint fissures and 10 cm above the patellar base in both legs . the measurements of extension and flexion range of knees were performed in a prone position . measurements of limb circumference was not taken prior to the surgical procedures as the patients were available only after surgery performed in another centre and then referred to undergo rehabilitation at the rehabilitation centre of the college of physiotherapy in wrocaw . the patients underwent a 12-minute warm up on a cycloergometer at the frequency of 60 rpm and 60 watts in 6 minutes . the warm - up was followed by a break and the measurements were started after 810 minutes . the measurements of muscle torque ( nm ) of knee joint extensor and flexor muscles of involved and uninvolved legs were performed under static and isokinetic conditions using a biodex 3 system ( biodex medical systems shirley , n.y . the lever arm leaning against the crus was placed at 40-cm from the knee joint axis . the measurements of maximal muscle torque of knee joint extensor and flexor muscles in static conditions were performed twice , during the 13 ( i ) and 21 ( ii ) week after acl reconstruction ( table 3 ) . the patients assumed a seated position in the measuring chair , with the hip joint at the angle of 80 and knee joint at 70 for extensor muscles and 30 for flexor muscles . the measurement was initiated with the start command and the result was recorded in the computer s memory . this was followed by a 1-minute break and then by the maximal muscle torque measurement of flexor muscles of the uninvolved knee . after finishing this procedure and a subsequent 1-minute break , the second measurement was performed , first for the extensor muscles , and , after a 1-minute break , for the flexor muscles of the uninvolved knee . the last measurement was followed by a 3-minute break to prepare the stand for the measurements of the operated leg . next , the measurement of maximal torque was taken for extensor and flexor muscles of the involved knee , according to the above procedure applied to the uninvolved knee . the measurement of peak torque for knee joint extensor and flexor muscles in isokinetic conditions was also performed twice , during the 16 ( i ) and 24 ( ii ) week after acl reconstruction ( table 3 ) , in a seated position with a hip joint angle of 80. the test consisted of 8 alternant movements : extension and flexion of the knee of the uninvolved leg at the angular velocity of 180/s . after a 3-minute break , the measurement of peak muscle torque was performed during a 5-fold alternant extension and flexion of the knee of the uninvolved leg at the angular velocity of 60/s . this was followed by a 3-minute break to prepare the stand for the measurements of the involved leg and to allow the patient to rest . next , the measurement of peak torque was taken under isokinetic conditions for the 2 groups of muscles of the operated knee , according to the above procedure performed for the uninvolved leg . the highest peak torques for the extensor and flexor muscles of the involved and uninvolved legs were selected for further analysis . the statistic analysis used student s t - test for dependent trials evaluating the differences between mean values in the studied group , and shapiro - wilk test evaluating the normality of features distribution . arithmetic transformations are presented with the accuracy to the second digit after the decimal point , with the numbers rounded according to generally accepted principles . thirty - seven males ( see table 1 ) participated in individual physiotherapeutic programs applied from the first week to up to 8 months after the primary endoscopic reconstruction of isolated acl tear by mitek s method of johnson & johnson gateway company using the autografts of the patients own semimembranosus and gracilis muscles . right- and left - sided lateralization was noted in 35 and 2 patients , respectively . the orthopedic surgeon qualified the patients for the first and for the subsequent stages of the physiotherapeutic procedure , and examined them after the physiotherapeutic program had finished . this examination included lachman s test , anteroposterior dislocation test and evaluation of the stability of other knee structures , as well as the presence of pain and swelling compared to the uninvolved knee . in the studied sample , during the entire study period of physiotherapeutic program implementation , the measurement of tibial translation , as related to the knee , did not exceed 5 mm . the patients gave their informed consent to participate in the study and the study was approved by the bioethics committee of the university of physical education in wrocaw . each patient underwent an individual therapeutic program with a physiotherapist , on average 4 times a week for 2 hours each day . additionally , the patients were instructed how to perform the exercises correctly at home and informed of possible hazards of ligament injuries . the outline of the physiotherapeutic program is presented in table 2 detailed physiotherapeutic procedure which comprises the more detailed program is presented elsewhere . measurements of range of movement and knee joint / thigh circumferences were performed twice : during the 1 ( i ) and 12 week ( ii ) after the reconstruction ( table 3 ) . the circumference was measured at the level of knee joint fissures and 10 cm above the patellar base in both legs . the measurements of extension and flexion range of knees were performed in a prone position . measurements of limb circumference was not taken prior to the surgical procedures as the patients were available only after surgery performed in another centre and then referred to undergo rehabilitation at the rehabilitation centre of the college of physiotherapy in wrocaw . the patients underwent a 12-minute warm up on a cycloergometer at the frequency of 60 rpm and 60 watts in 6 minutes . the warm - up was followed by a break and the measurements were started after 810 minutes . the measurements of muscle torque ( nm ) of knee joint extensor and flexor muscles of involved and uninvolved legs were performed under static and isokinetic conditions using a biodex 3 system ( biodex medical systems shirley , n.y . 11967 usa , model 333 - 250 , software biodex advantage ) . for both measurement conditions , the lever arm leaning against the crus was placed at 40-cm from the knee joint axis . the measurements of maximal muscle torque of knee joint extensor and flexor muscles in static conditions were performed twice , during the 13 ( i ) and 21 ( ii ) week after acl reconstruction ( table 3 ) . the patients assumed a seated position in the measuring chair , with the hip joint at the angle of 80 and knee joint at 70 for extensor muscles and 30 for flexor muscles . this was followed by a 1-minute break and then by the maximal muscle torque measurement of flexor muscles of the uninvolved knee . after finishing this procedure and a subsequent 1-minute break , the second measurement was performed , first for the extensor muscles , and , after a 1-minute break , for the flexor muscles of the uninvolved knee . the last measurement was followed by a 3-minute break to prepare the stand for the measurements of the operated leg . next , the measurement of maximal torque was taken for extensor and flexor muscles of the involved knee , according to the above procedure applied to the uninvolved knee . the measurement of peak torque for knee joint extensor and flexor muscles in isokinetic conditions was also performed twice , during the 16 ( i ) and 24 ( ii ) week after acl reconstruction ( table 3 ) , in a seated position with a hip joint angle of 80. the test consisted of 8 alternant movements : extension and flexion of the knee of the uninvolved leg at the angular velocity of 180/s . after a 3-minute break , the measurement of peak muscle torque was performed during a 5-fold alternant extension and flexion of the knee of the uninvolved leg at the angular velocity of 60/s . this was followed by a 3-minute break to prepare the stand for the measurements of the involved leg and to allow the patient to rest . next , the measurement of peak torque was taken under isokinetic conditions for the 2 groups of muscles of the operated knee , according to the above procedure performed for the uninvolved leg . the highest peak torques for the extensor and flexor muscles of the involved and uninvolved legs were selected for further analysis . measurements of range of movement and knee joint / thigh circumferences were performed twice : during the 1 ( i ) and 12 week ( ii ) after the reconstruction ( table 3 ) . the circumference was measured at the level of knee joint fissures and 10 cm above the patellar base in both legs . the measurements of extension and flexion range of knees were performed in a prone position . measurements of limb circumference was not taken prior to the surgical procedures as the patients were available only after surgery performed in another centre and then referred to undergo rehabilitation at the rehabilitation centre of the college of physiotherapy in wrocaw . the patients underwent a 12-minute warm up on a cycloergometer at the frequency of 60 rpm and 60 watts in 6 minutes . the warm - up was followed by a break and the measurements were started after 810 minutes . the measurements of muscle torque ( nm ) of knee joint extensor and flexor muscles of involved and uninvolved legs were performed under static and isokinetic conditions using a biodex 3 system ( biodex medical systems shirley , n.y . 11967 usa , model 333 - 250 , software biodex advantage ) . for both measurement conditions , the lever arm leaning against the crus was placed at 40-cm from the knee joint axis . the measurements of maximal muscle torque of knee joint extensor and flexor muscles in static conditions were performed twice , during the 13 ( i ) and 21 ( ii ) week after acl reconstruction ( table 3 ) . the patients assumed a seated position in the measuring chair , with the hip joint at the angle of 80 and knee joint at 70 for extensor muscles and 30 for flexor muscles . this was followed by a 1-minute break and then by the maximal muscle torque measurement of flexor muscles of the uninvolved knee . after finishing this procedure and a subsequent 1-minute break , the second measurement was performed , first for the extensor muscles , and , after a 1-minute break , for the flexor muscles of the uninvolved knee . the last measurement was followed by a 3-minute break to prepare the stand for the measurements of the operated leg . next , the measurement of maximal torque was taken for extensor and flexor muscles of the involved knee , according to the above procedure applied to the uninvolved knee . the measurement of peak torque for knee joint extensor and flexor muscles in isokinetic conditions was also performed twice , during the 16 ( i ) and 24 ( ii ) week after acl reconstruction ( table 3 ) , in a seated position with a hip joint angle of 80. the test consisted of 8 alternant movements : extension and flexion of the knee of the uninvolved leg at the angular velocity of 180/s . after a 3-minute break , the measurement of peak muscle torque was performed during a 5-fold alternant extension and flexion of the knee of the uninvolved leg at the angular velocity of 60/s . this was followed by a 3-minute break to prepare the stand for the measurements of the involved leg and to allow the patient to rest . next , the measurement of peak torque was taken under isokinetic conditions for the 2 groups of muscles of the operated knee , according to the above procedure performed for the uninvolved leg . the highest peak torques for the extensor and flexor muscles of the involved and uninvolved legs were selected for further analysis . the statistic analysis used student s t - test for dependent trials evaluating the differences between mean values in the studied group , and shapiro - wilk test evaluating the normality of features distribution . arithmetic transformations are presented with the accuracy to the second digit after the decimal point , with the numbers rounded according to generally accepted principles . the circumference results are given in cm and the flexion / extension results are given in degrees ( ns not significant ) . the first test revealed a significant difference in knee joint circumference between the involved and uninvolved legs ( table 4 ) . the average value of knee joint circumference of the involved extremities was 2.8 cm ( 7.4% ) higher compared to the uninvolved legs , due to postoperative swelling . this difference disappeared in session ii , which was conducted during the 12th week of physiotherapy ( table 4 ) . conversely , thigh circumference measured 10 cm above the knee revealed a significant decrease in value for the involved knee joints compared to the uninvolved legs on average a decrease of 2.3 cm ( 4.6% ) . this difference , resulting from a loss of muscle mass , was eliminated in session ii ( table 4 ) . from table 4 it can be seen that the postoperative range of flexion was only 56 , which corresponded to a 53% deficit of the entire natural range as related to uninvolved knees . the patients also had a limited range of knee extension , which amounted to 19 during the first test . the 12-week physiotherapeutic program allowed the patients to restore the full range of flexion and extension of the knee , comparable to that of the uninvolved legs . the values of thigh circumference of the involved and uninvolved legs were already equalized during the 12 week of physiotherapy . during the 13 week of physiotherapy after acl reconstruction , significantly lower values for extensor and flexor muscle torque under static conditions were noted for the operated knees as compared to the uninvolved ones ( table 5 ) . compared with the 13 week of physiotherapy , during the 21 week , a statistically significant increase in values was noted for the studied muscle torque for extensor and flexor muscles of the operated knees . the values for torque of the operated legs were comparable to those obtained for the uninvolved knees , amounting to 94.4% and 96.7% of the values for extensor and flexor muscles , respectively , of the uninvolved knees ( table 5 ) . during the 16 week postoperatively , the peak torques for both angular velocities of flexor muscles of the involved knees were significantly lower than the values obtained for these muscles of the uninvolved knees ( tables 6a and 6b ) . in session ii ( 24 week of physiotherapy ) , the difference in torque for flexors of the involved knees and uninvolved legs was reduced for both angular velocities ( tables 6a and 6b ) . the value of extensor muscle torque of the involved knees , at the angular velocity of 180/s , reached 151.98 nm in session ii , showing a statistically significant increase of 66.57 nm compared to the measurement taken in session i. a similar direction of change , albeit at a higher level ( session ii 204.71 nm ; session i 104.60 nm ) , was obtained for the extensor muscle torque at the angular velocity of 60/s . in session ii the values obtained for extensor muscle torque of the involved knees at both angular velocities were higher than the values obtained in session i for extensor muscles of the uninvolved knees . however , when the values obtained in session ii for extensor muscle torque of the involved knees were compared with the torque values obtained in session ii for these muscles of the uninvolved knees , they were found to be about 9% lower at both angular velocities . these results were statistically significant ( tables 6a and 6b ) . in order to control the restoration process of muscle strength distribution during physiotherapeutic procedures , the flexion / extension ratio was used : it is assumed that the flexion / extension ratio , based on the measurement performed under static conditions , is about 50% . this means that flexor muscle strength makes up about half of the strength of knee extensor muscles . table 7 shows that the uninvolved legs diverged from the accepted norm ; however , this was due to the measurement being made at other angular positions . selection of these angular positions for the measurement of the studied muscle groups was , in our study , justified by clinical and biomechanical conditions ( avoiding exposure to shear forces and conflicting location of the ligament graft toward bone canals ) . in session i , this distribution of strength was found to be disturbed in the operated knees ( table 7 ) , due to the impairment of mainly extensor muscles ( table 5 ) . however , during the 21 week of rehabilitation ( session ii ) , the ratio value approached that obtained for the uninvolved legs ( table 7 ) . the circumference results are given in cm and the flexion / extension results are given in degrees ( ns not significant ) . the first test revealed a significant difference in knee joint circumference between the involved and uninvolved legs ( table 4 ) . the average value of knee joint circumference of the involved extremities was 2.8 cm ( 7.4% ) higher compared to the uninvolved legs , due to postoperative swelling . this difference disappeared in session ii , which was conducted during the 12th week of physiotherapy ( table 4 ) . conversely , thigh circumference measured 10 cm above the knee revealed a significant decrease in value for the involved knee joints compared to the uninvolved legs on average a decrease of 2.3 cm ( 4.6% ) . this difference , resulting from a loss of muscle mass , was eliminated in session ii ( table 4 ) . from table 4 it can be seen that the postoperative range of flexion was only 56 , which corresponded to a 53% deficit of the entire natural range as related to uninvolved knees . the patients also had a limited range of knee extension , which amounted to 19 during the first test . the 12-week physiotherapeutic program allowed the patients to restore the full range of flexion and extension of the knee , comparable to that of the uninvolved legs . the values of thigh circumference of the involved and uninvolved legs were already equalized during the 12 week of physiotherapy . during the 13 week of physiotherapy after acl reconstruction , significantly lower values for extensor and flexor muscle torque under static conditions were noted for the operated knees as compared to the uninvolved ones ( table 5 ) . compared with the 13 week of physiotherapy , during the 21 week , a statistically significant increase in values was noted for the studied muscle torque for extensor and flexor muscles of the operated knees . the values for torque of the operated legs were comparable to those obtained for the uninvolved knees , amounting to 94.4% and 96.7% of the values for extensor and flexor muscles , respectively , of the uninvolved knees ( table 5 ) . during the 16 week postoperatively , the peak torques for both angular velocities of flexor muscles of the involved knees were significantly lower than the values obtained for these muscles of the uninvolved knees ( tables 6a and 6b ) . in session ii ( 24 week of physiotherapy ) , the difference in torque for flexors of the involved knees and uninvolved legs was reduced for both angular velocities ( tables 6a and 6b ) . the value of extensor muscle torque of the involved knees , at the angular velocity of 180/s , reached 151.98 nm in session ii , showing a statistically significant increase of 66.57 nm compared to the measurement taken in session i. a similar direction of change , albeit at a higher level ( session ii 204.71 nm ; session i 104.60 nm ) , was obtained for the extensor muscle torque at the angular velocity of 60/s . in session ii the values obtained for extensor muscle torque of the involved knees at both angular velocities were higher than the values obtained in session i for extensor muscles of the uninvolved knees . however , when the values obtained in session ii for extensor muscle torque of the involved knees were compared with the torque values obtained in session ii for these muscles of the uninvolved knees , they were found to be about 9% lower at both angular velocities . in order to control the restoration process of muscle strength distribution during physiotherapeutic procedures , the flexion / extension ratio was used : it is assumed that the flexion / extension ratio , based on the measurement performed under static conditions , is about 50% . this means that flexor muscle strength makes up about half of the strength of knee extensor muscles . table 7 shows that the uninvolved legs diverged from the accepted norm ; however , this was due to the measurement being made at other angular positions . selection of these angular positions for the measurement of the studied muscle groups was , in our study , justified by clinical and biomechanical conditions ( avoiding exposure to shear forces and conflicting location of the ligament graft toward bone canals ) . in session i , this distribution of strength was found to be disturbed in the operated knees ( table 7 ) , due to the impairment of mainly extensor muscles ( table 5 ) . however , during the 21 week of rehabilitation ( session ii ) , the ratio value approached that obtained for the uninvolved legs ( table 7 ) . the initial outcomes of the postoperative rehabilitation program , as compared to the positive results of the orthopaedic examination , were the improvement of the range of movement of the involved knee joints , and elimination of swelling and reconstruction of thigh muscle mass ( circumference ) . in the first study ( performed during the 1st week following reconstruction ) , the decrease in thigh circumference of the involved legs amounted to 4% , whereas in the second study ( performed 12 weeks after reconstruction ) the deficit was reduced to just over 1% , which was not statistically significant . the results from the first 2 studies also demonstrated that 12 weeks of physiotherapy restored the range of movement of the operated knee joints to almost that of the uninvolved legs . during the 13 week postoperatively , a significant deficiency was found in operated knee extensor and flexor muscle torque , measured using the static mode ( by about 50% and 20% , respectively ) . as mentioned above , a gain in muscle mass was noted , manifested by increased thigh circumference values obtained in the second study during the 12 week of rehabilitation , before the increase in torque was measured using the static mode . this phenomenon does not conform to the sequence of training - related strength gain , involving primary strengthening of neuromuscular processes and activation of more functional units , which is then followed by an increase in muscle mass . the thigh circumferences were found to be similar after 12 weeks of rehabilitation ; these measurements were taken 10 cm above the knee joint . thigh circumference is frequently measured at the area of its maximal thickness and , according to martin s technique , amounts to approximately 55 cm in males with body mass similar to that of our patients . on the one hand , information on thigh circumference taken 10 cm above the knee joint fissure on the other hand , this is the region of knee joint upper recesses , whose contours may be somewhat larger , which changes the true value of knee circumference with muscle mass . therefore , for future studies 2 measurement levels should be used , and ultrasonography of the recess region may also prove useful . given this point of reference , it should be assumed that the deficiency in muscle mass has not yet been fully compensated . an increase in body mass is usually triggered by alternation of isometric and dynamic training with a high load and an appropriate intensity . the content of body mass is manifested by a physiological cross - section , significantly connected with muscle strength . intensive training with a high load during the first 2 stages , and even the first half of the third stage of the physiotherapeutic programme , is neither indicated nor applied . during the first 2 stages of the physiotherapeutic program , the patients should avoid exercises in open kinematic chains of extensor muscles of the involved knee and external rotators of the foreleg , which may result in the impact of high values of shear forces on the acl graft . therefore , due to biological and biomechanical conditioning related to healing rate and ligament reconstruction , the measurements , and next , isokinetic exercises , which were the basis for strength training intensification , were started as late as the 16 week postoperatively . at the end of the third stage and during the fourth stage of the physiotherapeutic program , other dynamic exercises were gradually introduced with additional load . subsequently , the exercises were applied in several planes of movement for the whole body , including rotations of the involved knee , ( pnf ) proprioceptive neuromuscular facilitation dynamic elements and other concentric - eccentric exercises of different dynamics , load magnitude and degree of difficulty . the torque results obtained for the studied muscle groups of the operated legs , performed using a static and an isokinetic mode , as compared to the results obtained for the uninvolved legs , indicate an unequal progress in torque increase during the sixth month postoperatively . during the 21 week after acl reconstruction , the values for extensor and flexor likewise , during the 24 week after the reconstruction , similar results were noted for flexor muscle peak torque at both rotational velocities 180 and 60/s of the involved and uninvolved knee joints . the values of extensor muscle peak torque of the operated knees at both angular velocities showed a significant improvement during the 24 week postoperatively compared to the 16th week postoperatively , and the results were approximately 91% of the values obtained at the same time for the uninvolved extremities . in other words , the values of the studied feature were on average 9% lower for the involved legs as compared to the values obtained for the uninvolved ones . nevertheless , the results of the statistical analysis of the entire sample and evaluation of individual patient s results suggest that in some patients it is still necessary to correct extensor muscle strength generation in the operated knees under dynamic conditions . the lack of a standard control group in the study should now be explained . in our opinion , the inclusion of such a group in the study would positively affect the quality and transparency of the methods used in the study as well as the reliability of the obtained results . the specific points of reference for our study were the results and effects of rehabilitation procedures , obtained in similar studies by other authors , as well as the comparison of the results of precise examinations of uninvolved limbs in each patient . upon comparison of our study results with those obtained by other authors , we noted that urabe et al . did not obtain equalization of extensor muscle strength of the involved knees even after 12 months of rehabilitation , and at that time they reported a strength deficiency ranging from 8% to 10% in the involved legs . such differences in time ( delay ) of muscle strength regain might have been due to the physiotherapeutic program . at our centre , the program of physiotherapy was implemented individually for each patient , on average 4 times a week for 6 months . the above - mentioned authors gradually instructed the patients how to carry out their physiotherapeutic program by themselves , at home . wit and mirowski et al . obtained similar results for both legs between the 12 and 18 month after the reconstruction . noted a deficit in extensor muscle strength of the involved knees compared to the uninvolved ones 2.5 years after acl reconstruction ; under isokinetic conditions at the level of 15.3% for the angular velocity 60/s and 11.3% for the angular velocity 180/s . andrade et al . found a deficit of 34% in extensor muscle strength values of the involved knees compared to the uninvolved ones . a deficit at the level of 33% was still present in the 8 month after reconstruction when tested under isokinetic conditions at the angular velocity of 60/s . our results should also be considered regarding the progress in training - related increase in strength . therefore , the physiotherapeutic program also included application of the load on the muscles of the uninvolved legs . this resulted in the increase in muscle strength being obtained in both the affected and the unaffected limbs . depending on the testing mode , the increase in strength of the uninvolved legs was noted to be between 18% and 30% for extensor muscles and between 15% and 22% for flexor muscles the extent of muscle strength increase observed in the involved legs was greater , since the decrease in strength level was greater following the reconstruction ( the baseline level of strength was lower ) . the increase ranged from 78% to 114% for extensor muscles and from 45% to 58% for flexor muscles ( figure 2 ) . the strength ratio of flexor to extensor muscles is frequently used to control the progress in physiotherapy that attempts to gain the same distribution of muscle strength in the sagittal plane , affecting both the involved and the uninvolved knees , and may be applied in prevention and evaluation of the risk of sports - related injuries of the knee joint . assessed the effect of pre - operative torque deficit in quadriceps femoris muscle on the results obtained after 2 years following acl reconstruction . other authors studied the correlation between changes in knee laxity after acl surgery related to open kinetic chain knee extensor training load . additionally , in our study sample it should be stressed , however , that despite significant progress , as demonstrated by the results for the studied features 24 weeks following the surgical procedure , the level of strength of extensor muscles of the involved knees was not fully restored under isokinetic conditions . this finding leads us to conclude that the period of physiotherapy should be extended beyond 6 months for some patients . six months of physiotherapy following acl reconstruction in males favorably affected muscle strength values of the involved and uninvolved knees under static and isokinetic conditions . application of individual loads resulted in similar levels of muscle strength for extensors and flexors measured under static conditions , and in flexors muscle of the operated knees and the uninvolved knees measured under isokinetic conditions during the sixth month of physiotherapy . after 6 months , a 9% deficit of extensor muscle strength under isokinetic conditions was observed in the involved knees , as compared to the uninvolved knees .

summarybackgroundthe aim of this paper is to present the physiotherapeutic program employed at the rehabilitation centre for the college of physiotherapy in wrocaw , poland and its effectiveness by demonstrating the increase in strength of knee joint extensor and flexor muscles of patients after anterior cruciate ligament ( acl ) reconstruction.material/methodsthirty-seven males participated in the physiotherapeutic program from the first week up to 8 months postoperatively . each patient underwent an individual therapeutic program . endoscopic reconstruction of a completely ruptured acl was performed using mitek s method ( graft harvesting from flexor muscles ) . all patients previously underwent orthopedic and functional examinations , including measurements of the range of movement , knee and thigh circumference and strength of flexor and extensor muscles of the involved and uninvolved leg using a biodex 3 system in both static and isokinetic modes.resultsthe outcome of the physiotherapeutic procedure , which is detailed in the paper , revealed a favorable effect of physiotherapy 6 months after acl reconstruction . the observed 9% deficit in extensor muscle strength measured under isokinetic conditions of the involved knees compared with the uninvolved knees led us to conclude that the period of physiotherapy should be extended beyond 6 months for some patients.conclusions1 . six months of physiotherapy following acl reconstruction in males favorably affected muscle strength values of the involved and uninvolved knees under static and isokinetic conditions . 2 . the application of individual loads during the sixth month of physiotherapy resulted in similar values of extensor and flexor muscle strength measured under static conditions , and flexor muscle strength measured under isokinetic conditions in involved and uninvolved knees .

Background Material and Methods Patients Physiotherapeutic program Tests Circumferences and range of knee movement Torque of knee muscles Statistic analysis Results Knee/thigh circumferences and range of movement Muscle torque in static mode Muscle torque in isokinetic mode Flexion/extension ratio: the ratio of flexor to extensor torque under static conditions Discussion Conclusions

physiotherapeutic procedures should restore locomotor fitness in patients after anterior cruciate ligament ( acl ) reconstruction . normal locomotion depends on , among other factors , ligamentocapsular structure stability , knee joint range of movement and an appropriate strength level of knee joint muscles , as well as on proprioception . after reconstruction , the patients range of movement is temporarily limited and the strength of knee joint flexor muscles ( sartorius , gracilis , semitendinosus , semimembranosus , biceps femoris , gastrocnemius ) and extensor ( quadriceps ) muscles deteriorates . it is assumed that on completion of the entire physiotherapeutic program , the muscle strength values should be returned to preoperative levels , both in static and dynamic conditions . one of the goals of physiotherapy after acl reconstruction is to restore the antagonist strength ratio of knee joint muscle groups . the aim of this paper is to present the physiotherapeutic program applied after acl reconstruction at the rehabilitation centre for the college of physiotherapy in wrocaw and the evaluation of its effectiveness , reflected by increased strength values of knee joint extensor and flexor muscles . the tailored pt study protocol , developed by the first author , was implemented for many years at the rehabilitation centre of the college of physiotherapy in wrocaw . thirty - seven males ( see table 1 ) participated in individual physiotherapeutic programs applied from the first week to up to 8 months after the primary endoscopic reconstruction of isolated acl tear by mitek s method of johnson & johnson gateway company using the autografts of the patients own semimembranosus and gracilis muscles . the orthopedic surgeon qualified the patients for the first and for the subsequent stages of the physiotherapeutic procedure , and examined them after the physiotherapeutic program had finished . each patient underwent an individual therapeutic program with a physiotherapist , on average 4 times a week for 2 hours each day . measurements of limb circumference was not taken prior to the surgical procedures as the patients were available only after surgery performed in another centre and then referred to undergo rehabilitation at the rehabilitation centre of the college of physiotherapy in wrocaw . the measurements of muscle torque ( nm ) of knee joint extensor and flexor muscles of involved and uninvolved legs were performed under static and isokinetic conditions using a biodex 3 system ( biodex medical systems shirley , n.y . the measurements of maximal muscle torque of knee joint extensor and flexor muscles in static conditions were performed twice , during the 13 ( i ) and 21 ( ii ) week after acl reconstruction ( table 3 ) . the patients assumed a seated position in the measuring chair , with the hip joint at the angle of 80 and knee joint at 70 for extensor muscles and 30 for flexor muscles . this was followed by a 1-minute break and then by the maximal muscle torque measurement of flexor muscles of the uninvolved knee . after finishing this procedure and a subsequent 1-minute break , the second measurement was performed , first for the extensor muscles , and , after a 1-minute break , for the flexor muscles of the uninvolved knee . next , the measurement of maximal torque was taken for extensor and flexor muscles of the involved knee , according to the above procedure applied to the uninvolved knee . the measurement of peak torque for knee joint extensor and flexor muscles in isokinetic conditions was also performed twice , during the 16 ( i ) and 24 ( ii ) week after acl reconstruction ( table 3 ) , in a seated position with a hip joint angle of 80. the test consisted of 8 alternant movements : extension and flexion of the knee of the uninvolved leg at the angular velocity of 180/s . next , the measurement of peak torque was taken under isokinetic conditions for the 2 groups of muscles of the operated knee , according to the above procedure performed for the uninvolved leg . the highest peak torques for the extensor and flexor muscles of the involved and uninvolved legs were selected for further analysis . thirty - seven males ( see table 1 ) participated in individual physiotherapeutic programs applied from the first week to up to 8 months after the primary endoscopic reconstruction of isolated acl tear by mitek s method of johnson & johnson gateway company using the autografts of the patients own semimembranosus and gracilis muscles . the orthopedic surgeon qualified the patients for the first and for the subsequent stages of the physiotherapeutic procedure , and examined them after the physiotherapeutic program had finished . each patient underwent an individual therapeutic program with a physiotherapist , on average 4 times a week for 2 hours each day . measurements of range of movement and knee joint / thigh circumferences were performed twice : during the 1 ( i ) and 12 week ( ii ) after the reconstruction ( table 3 ) . measurements of limb circumference was not taken prior to the surgical procedures as the patients were available only after surgery performed in another centre and then referred to undergo rehabilitation at the rehabilitation centre of the college of physiotherapy in wrocaw . the measurements of muscle torque ( nm ) of knee joint extensor and flexor muscles of involved and uninvolved legs were performed under static and isokinetic conditions using a biodex 3 system ( biodex medical systems shirley , n.y . the measurements of maximal muscle torque of knee joint extensor and flexor muscles in static conditions were performed twice , during the 13 ( i ) and 21 ( ii ) week after acl reconstruction ( table 3 ) . the patients assumed a seated position in the measuring chair , with the hip joint at the angle of 80 and knee joint at 70 for extensor muscles and 30 for flexor muscles . after finishing this procedure and a subsequent 1-minute break , the second measurement was performed , first for the extensor muscles , and , after a 1-minute break , for the flexor muscles of the uninvolved knee . next , the measurement of maximal torque was taken for extensor and flexor muscles of the involved knee , according to the above procedure applied to the uninvolved knee . the measurement of peak torque for knee joint extensor and flexor muscles in isokinetic conditions was also performed twice , during the 16 ( i ) and 24 ( ii ) week after acl reconstruction ( table 3 ) , in a seated position with a hip joint angle of 80. the test consisted of 8 alternant movements : extension and flexion of the knee of the uninvolved leg at the angular velocity of 180/s . after a 3-minute break , the measurement of peak muscle torque was performed during a 5-fold alternant extension and flexion of the knee of the uninvolved leg at the angular velocity of 60/s . next , the measurement of peak torque was taken under isokinetic conditions for the 2 groups of muscles of the operated knee , according to the above procedure performed for the uninvolved leg . the highest peak torques for the extensor and flexor muscles of the involved and uninvolved legs were selected for further analysis . measurements of limb circumference was not taken prior to the surgical procedures as the patients were available only after surgery performed in another centre and then referred to undergo rehabilitation at the rehabilitation centre of the college of physiotherapy in wrocaw . the measurements of muscle torque ( nm ) of knee joint extensor and flexor muscles of involved and uninvolved legs were performed under static and isokinetic conditions using a biodex 3 system ( biodex medical systems shirley , n.y . the measurements of maximal muscle torque of knee joint extensor and flexor muscles in static conditions were performed twice , during the 13 ( i ) and 21 ( ii ) week after acl reconstruction ( table 3 ) . the patients assumed a seated position in the measuring chair , with the hip joint at the angle of 80 and knee joint at 70 for extensor muscles and 30 for flexor muscles . after finishing this procedure and a subsequent 1-minute break , the second measurement was performed , first for the extensor muscles , and , after a 1-minute break , for the flexor muscles of the uninvolved knee . next , the measurement of maximal torque was taken for extensor and flexor muscles of the involved knee , according to the above procedure applied to the uninvolved knee . the measurement of peak torque for knee joint extensor and flexor muscles in isokinetic conditions was also performed twice , during the 16 ( i ) and 24 ( ii ) week after acl reconstruction ( table 3 ) , in a seated position with a hip joint angle of 80. the test consisted of 8 alternant movements : extension and flexion of the knee of the uninvolved leg at the angular velocity of 180/s . after a 3-minute break , the measurement of peak muscle torque was performed during a 5-fold alternant extension and flexion of the knee of the uninvolved leg at the angular velocity of 60/s . next , the measurement of peak torque was taken under isokinetic conditions for the 2 groups of muscles of the operated knee , according to the above procedure performed for the uninvolved leg . the highest peak torques for the extensor and flexor muscles of the involved and uninvolved legs were selected for further analysis . the first test revealed a significant difference in knee joint circumference between the involved and uninvolved legs ( table 4 ) . the average value of knee joint circumference of the involved extremities was 2.8 cm ( 7.4% ) higher compared to the uninvolved legs , due to postoperative swelling . from table 4 it can be seen that the postoperative range of flexion was only 56 , which corresponded to a 53% deficit of the entire natural range as related to uninvolved knees . the values of thigh circumference of the involved and uninvolved legs were already equalized during the 12 week of physiotherapy . during the 13 week of physiotherapy after acl reconstruction , significantly lower values for extensor and flexor muscle torque under static conditions were noted for the operated knees as compared to the uninvolved ones ( table 5 ) . compared with the 13 week of physiotherapy , during the 21 week , a statistically significant increase in values was noted for the studied muscle torque for extensor and flexor muscles of the operated knees . the values for torque of the operated legs were comparable to those obtained for the uninvolved knees , amounting to 94.4% and 96.7% of the values for extensor and flexor muscles , respectively , of the uninvolved knees ( table 5 ) . during the 16 week postoperatively , the peak torques for both angular velocities of flexor muscles of the involved knees were significantly lower than the values obtained for these muscles of the uninvolved knees ( tables 6a and 6b ) . the value of extensor muscle torque of the involved knees , at the angular velocity of 180/s , reached 151.98 nm in session ii , showing a statistically significant increase of 66.57 nm compared to the measurement taken in session i. a similar direction of change , albeit at a higher level ( session ii 204.71 nm ; session i 104.60 nm ) , was obtained for the extensor muscle torque at the angular velocity of 60/s . in session ii the values obtained for extensor muscle torque of the involved knees at both angular velocities were higher than the values obtained in session i for extensor muscles of the uninvolved knees . however , when the values obtained in session ii for extensor muscle torque of the involved knees were compared with the torque values obtained in session ii for these muscles of the uninvolved knees , they were found to be about 9% lower at both angular velocities . this means that flexor muscle strength makes up about half of the strength of knee extensor muscles . the first test revealed a significant difference in knee joint circumference between the involved and uninvolved legs ( table 4 ) . the average value of knee joint circumference of the involved extremities was 2.8 cm ( 7.4% ) higher compared to the uninvolved legs , due to postoperative swelling . conversely , thigh circumference measured 10 cm above the knee revealed a significant decrease in value for the involved knee joints compared to the uninvolved legs on average a decrease of 2.3 cm ( 4.6% ) . from table 4 it can be seen that the postoperative range of flexion was only 56 , which corresponded to a 53% deficit of the entire natural range as related to uninvolved knees . the patients also had a limited range of knee extension , which amounted to 19 during the first test . the values of thigh circumference of the involved and uninvolved legs were already equalized during the 12 week of physiotherapy . during the 13 week of physiotherapy after acl reconstruction , significantly lower values for extensor and flexor muscle torque under static conditions were noted for the operated knees as compared to the uninvolved ones ( table 5 ) . compared with the 13 week of physiotherapy , during the 21 week , a statistically significant increase in values was noted for the studied muscle torque for extensor and flexor muscles of the operated knees . the values for torque of the operated legs were comparable to those obtained for the uninvolved knees , amounting to 94.4% and 96.7% of the values for extensor and flexor muscles , respectively , of the uninvolved knees ( table 5 ) . during the 16 week postoperatively , the peak torques for both angular velocities of flexor muscles of the involved knees were significantly lower than the values obtained for these muscles of the uninvolved knees ( tables 6a and 6b ) . in session ii ( 24 week of physiotherapy ) , the difference in torque for flexors of the involved knees and uninvolved legs was reduced for both angular velocities ( tables 6a and 6b ) . the value of extensor muscle torque of the involved knees , at the angular velocity of 180/s , reached 151.98 nm in session ii , showing a statistically significant increase of 66.57 nm compared to the measurement taken in session i. a similar direction of change , albeit at a higher level ( session ii 204.71 nm ; session i 104.60 nm ) , was obtained for the extensor muscle torque at the angular velocity of 60/s . in session ii the values obtained for extensor muscle torque of the involved knees at both angular velocities were higher than the values obtained in session i for extensor muscles of the uninvolved knees . however , when the values obtained in session ii for extensor muscle torque of the involved knees were compared with the torque values obtained in session ii for these muscles of the uninvolved knees , they were found to be about 9% lower at both angular velocities . in order to control the restoration process of muscle strength distribution during physiotherapeutic procedures , the flexion / extension ratio was used : it is assumed that the flexion / extension ratio , based on the measurement performed under static conditions , is about 50% . this means that flexor muscle strength makes up about half of the strength of knee extensor muscles . the initial outcomes of the postoperative rehabilitation program , as compared to the positive results of the orthopaedic examination , were the improvement of the range of movement of the involved knee joints , and elimination of swelling and reconstruction of thigh muscle mass ( circumference ) . in the first study ( performed during the 1st week following reconstruction ) , the decrease in thigh circumference of the involved legs amounted to 4% , whereas in the second study ( performed 12 weeks after reconstruction ) the deficit was reduced to just over 1% , which was not statistically significant . the results from the first 2 studies also demonstrated that 12 weeks of physiotherapy restored the range of movement of the operated knee joints to almost that of the uninvolved legs . as mentioned above , a gain in muscle mass was noted , manifested by increased thigh circumference values obtained in the second study during the 12 week of rehabilitation , before the increase in torque was measured using the static mode . during the first 2 stages of the physiotherapeutic program , the patients should avoid exercises in open kinematic chains of extensor muscles of the involved knee and external rotators of the foreleg , which may result in the impact of high values of shear forces on the acl graft . the torque results obtained for the studied muscle groups of the operated legs , performed using a static and an isokinetic mode , as compared to the results obtained for the uninvolved legs , indicate an unequal progress in torque increase during the sixth month postoperatively . during the 21 week after acl reconstruction , the values for extensor and flexor likewise , during the 24 week after the reconstruction , similar results were noted for flexor muscle peak torque at both rotational velocities 180 and 60/s of the involved and uninvolved knee joints . the values of extensor muscle peak torque of the operated knees at both angular velocities showed a significant improvement during the 24 week postoperatively compared to the 16th week postoperatively , and the results were approximately 91% of the values obtained at the same time for the uninvolved extremities . nevertheless , the results of the statistical analysis of the entire sample and evaluation of individual patient s results suggest that in some patients it is still necessary to correct extensor muscle strength generation in the operated knees under dynamic conditions . did not obtain equalization of extensor muscle strength of the involved knees even after 12 months of rehabilitation , and at that time they reported a strength deficiency ranging from 8% to 10% in the involved legs . noted a deficit in extensor muscle strength of the involved knees compared to the uninvolved ones 2.5 years after acl reconstruction ; under isokinetic conditions at the level of 15.3% for the angular velocity 60/s and 11.3% for the angular velocity 180/s . found a deficit of 34% in extensor muscle strength values of the involved knees compared to the uninvolved ones . therefore , the physiotherapeutic program also included application of the load on the muscles of the uninvolved legs . this resulted in the increase in muscle strength being obtained in both the affected and the unaffected limbs . depending on the testing mode , the increase in strength of the uninvolved legs was noted to be between 18% and 30% for extensor muscles and between 15% and 22% for flexor muscles the extent of muscle strength increase observed in the involved legs was greater , since the decrease in strength level was greater following the reconstruction ( the baseline level of strength was lower ) . the strength ratio of flexor to extensor muscles is frequently used to control the progress in physiotherapy that attempts to gain the same distribution of muscle strength in the sagittal plane , affecting both the involved and the uninvolved knees , and may be applied in prevention and evaluation of the risk of sports - related injuries of the knee joint . additionally , in our study sample it should be stressed , however , that despite significant progress , as demonstrated by the results for the studied features 24 weeks following the surgical procedure , the level of strength of extensor muscles of the involved knees was not fully restored under isokinetic conditions . this finding leads us to conclude that the period of physiotherapy should be extended beyond 6 months for some patients . six months of physiotherapy following acl reconstruction in males favorably affected muscle strength values of the involved and uninvolved knees under static and isokinetic conditions . application of individual loads resulted in similar levels of muscle strength for extensors and flexors measured under static conditions , and in flexors muscle of the operated knees and the uninvolved knees measured under isokinetic conditions during the sixth month of physiotherapy . after 6 months , a 9% deficit of extensor muscle strength under isokinetic conditions was observed in the involved knees , as compared to the uninvolved knees .

bisphosphonates ( bps ) are agents currently used to treat osteoporosis and decrease the risk of osteoporosis - related fractures . since the introduction and development of these drugs , increased bone mineral density ( bmd ) in the lumbar spine and hip ( total hip or neck ) has been the main focus for evaluation of their effectiveness along with decreasing the risk of fragility fracture . even though the effect of bp agents in preventing osteoporotic fracture is eminent , their strong association with atypical femoral fracture ( aff ) after a long - term administration has recently been reported . excessive suppression of bone remodeling after a long - term bp intake has been thought to deteriorate bone quality and to cause pre - fracture lesions . the lateral femoral cortex is affected , not the medial femoral cortex , presenting as periosteal callus and the dreaded black line on radiographs . the lateral femoral cortex in the subtrochanteric area is under a considerable tensile stress during the gait cycle , while the medial cortex in this area is under a compressive stress . cortical bone is weaker upon tensile stress than in compressive stress , which means the lateral cortex is more subject to structural damage and repair . on the other hand , differences in the pharmacokinetics of alendronate and risedronate can cause difference in bone mineral deposition , which are known to be affected by mineral affinity , farnesyl pyrophosphate synthetase inhibition , and the effect of bp binding to hydroxyapatite on the surface charge [ zeta potential ] . additionally , recent articles have reported the different risk of aff according to different bps . in this context , we speculated that the pharmacokinetic differences specific to each bp agent may cause different cortical responses at the subtrochanteric lateral cortex ( stlc ) . this might be one of the plausible explanations for the differences in aff risk among the bp agents . therefore , the purpose of this study was to retrospectively assess whether the response of the stlc is different according to two kinds of bp agents by tracing the changes of bmd using serial dual energy x - ray absorptiometry ( dxa ) examinations . this research protocol was in compliance with the health insurance portability and accountability act ( hipaa ) and was approved by the institutional review board ( irb ) . informed consent was waived by the irb for this retrospective study . from january 2004 through june 2013 , a total of 2,278 female individuals received dxa examination by a single technician at the kyungpook national university hospital . from these individuals , 149 subjects who had serial dxa examinations during two or more years after taking a bp ( alendronate or risedronate ) as nave users or simply undergoing a health check - up subjects who were already taking a bp at the time of the first dxa examination ( n=1,318 ) were excluded . among the remaining 960 subjects , subjects who underwent dxa examinations just once ( n=142 ) or had serial dxa examinations with less than a 2-year interval after bp use ( n=657 ) were also excluded . from the remaining 161 patients , subjects who acquired an endocrine disease that could possibly affect bmd ( n=2 ) , taking a steroid ( n=9 ) , or with acquired parkinson 's disease ( n=1 ) finally , 149 subjects whose first and follow - up dxa examinations were separated by a 2-to 4-year interval were included ( fig . 1 ) . among them , 38 did not take any anti - osteoporotic drugs ( control group ) , 48 were nave alendronate users ( the alendronate group ) , and 63 were nave risedronate users ( the risedronate group ) . the baseline characteristics of each group , including the mean age , time interval between dxa examinations , and body mass index ( bmi ) , were recorded based on the medical record . the medication duration of the patients in the medication groups was regarded as the time interval between dxa examinations since the included patients were nave patients and started the medication at the time of the first dxa examination . serial dxa examinations were performed in each patient by a technician using the same lunar prodigy scanner ( ge lunar , medison , wi , usa ) without changing the software or hardware of the dxa scanner . no events occurred that could have caused any changes in basic scanning skills during the study interval . posteroanterior ( pa ) spine and unilateral hip ( right hip in all patients ) dxa examinations were performed . the dxa examination was performed according to the vendor - recommended protocol for the scanning method and patient positioning . the entire scanning data source was backed up on separate storage devices at the institution from which the data were collected . the pre- and post - medication bmds for the spine and hip were measured in the alendronate and risedronate groups to validate whether the medication compliance was acceptable . the bmd of spine was measured at the l1-l4 spines ; whereas , the hip bmds were measured at the total hip , neck , trochanter , and ward 's area by placing region of interest ( roi ) provided by the vendor . bmd measurement was performed using ge - lunar encore software ( version 13.6 ) by the same technician who performed the dxa examination . technician precision was assessed with the commencement of this study by measuring the bmd twice from the 30 dxa images from our study according to the recommendation stated in the international society of clinical densitometry ( iscd ) guideline 2005 . the bmd of the subtrochanteric cortical bone was measured by the aforementioned technician who measured the bmd of the spine and hip . bmd was measured at the stlc and subtrochanteric medial cortex ( stmc ) for each patient at the level just distal to the lesser trochanter . as for the width , rois were drawn not violating the boundary of the cortices . the proximal to distal length of the roi was unified as 1 cm ( fig . 2 ) . all of the measured bmd data were recorded as g / cm . the bmd of the stmc was measured to assess whether that region was associated with changes in the stlc bmd . to assess the reliability of this measurement , 30 patients were randomly selected , and the technician measured the bmd again as described above with a time interval of 2 months , hal reflects the hip moment arm and we speculated that this value would reflect the load on the subtrochanteric cortex of the femur where the maximal strain is known to be loaded and could affect the change of bmd at the stlc . hal was measured along the extended femoral neck axis until the edge of the bone in each direction ( fig . the measurement was performed twice by an orthopedic surgeon on the image taken at the first dxa examination in each patient , and the mean values were recorded as cm . to test the difference in bmd changes between the first and the second dxa examinations , a standardization of the bmd change was needed because we believed that the same amount of bmd changes can not be considered the same when the baseline bmd was different . the percent changes were calculated for the stlc , stmc , spine , and total hip , and were assessed statistically as stated below considering two aspects : 1 ) to compare the percent changes of the total hip and spine bmd between the medication and control groups to ascertain the optimal compliance of the patients who took the medication , and 2 ) to test the difference in the lateral cortex percent changes according to the agents received . data were summarized as meansstandard deviations with ranges for continuous variables and as counts and percentages for categorical variables . the reproducibility of the bmd measurement of the subtrochanteric cortex was evaluated using the intraclass correlation coefficient ( icc ) . an icc value was evaluated as follows : 0 to 0.20 , poor agreement ; 0.21 to 0.40 , fair agreement ; 0.41 to 0.60 , moderate agreement ; 0.61 to 0.80 , substantial agreement ; 0.81 to 1.00 , nearly perfect agreement . a single measures icc value was obtained . to test whether there was a difference the percent change of bmd in the hip , spine , or stlc among the groups ( control , alendronate , or risedronate ) , an analysis of covariance ( ancova ) model was adopted to control five independent variables . the variables were age , bmi , bmd percent change of stmc , hal , and the time interval between dxa examinations . concurrently , we assessed whether any of the variables other than medication ( none , alendronate , or risedronate ) could affect the percent change of bmd . hal was included as one of the variables to be assessed to control geometry of the stlc that might affect the bmd change , which was mentioned above . bmd of stmc was included as a variable to further control unexpected effects on bmd at the subtrochanteric cortices other than geometry . using the ancova model , the least square means of the percent change for the hip bmd , spine bmd , and stlc bmd were obtained for each group , and multiple comparisons were performed to assess the group differences . all statistical analyses were performed using sas 9.2 software ( sas institute , cary , nc , usa ) . from january 2004 through june 2013 , a total of 2,278 female individuals received dxa examination by a single technician at the kyungpook national university hospital . from these individuals , 149 subjects who had serial dxa examinations during two or more years after taking a bp ( alendronate or risedronate ) as nave users or simply undergoing a health check - up subjects who were already taking a bp at the time of the first dxa examination ( n=1,318 ) were excluded . among the remaining 960 subjects , subjects who underwent dxa examinations just once ( n=142 ) or had serial dxa examinations with less than a 2-year interval after bp use ( n=657 ) were also excluded . from the remaining 161 patients , subjects who acquired an endocrine disease that could possibly affect bmd ( n=2 ) , taking a steroid ( n=9 ) , or with acquired parkinson 's disease ( n=1 ) finally , 149 subjects whose first and follow - up dxa examinations were separated by a 2-to 4-year interval were included ( fig . 1 ) . among them , 38 did not take any anti - osteoporotic drugs ( control group ) , 48 were nave alendronate users ( the alendronate group ) , and 63 were nave risedronate users ( the risedronate group ) . the baseline characteristics of each group , including the mean age , time interval between dxa examinations , and body mass index ( bmi ) , were recorded based on the medical record . the medication duration of the patients in the medication groups was regarded as the time interval between dxa examinations since the included patients were nave patients and started the medication at the time of the first dxa examination . serial dxa examinations were performed in each patient by a technician using the same lunar prodigy scanner ( ge lunar , medison , wi , usa ) without changing the software or hardware of the dxa scanner . no events occurred that could have caused any changes in basic scanning skills during the study interval . posteroanterior ( pa ) spine and unilateral hip ( right hip in all patients ) dxa examinations were performed . the dxa examination was performed according to the vendor - recommended protocol for the scanning method and patient positioning . the entire scanning data source was backed up on separate storage devices at the institution from which the data were collected . the pre- and post - medication bmds for the spine and hip were measured in the alendronate and risedronate groups to validate whether the medication compliance was acceptable . the bmd of spine was measured at the l1-l4 spines ; whereas , the hip bmds were measured at the total hip , neck , trochanter , and ward 's area by placing region of interest ( roi ) provided by the vendor . bmd measurement was performed using ge - lunar encore software ( version 13.6 ) by the same technician who performed the dxa examination . technician precision was assessed with the commencement of this study by measuring the bmd twice from the 30 dxa images from our study according to the recommendation stated in the international society of clinical densitometry ( iscd ) guideline 2005 . the bmd of the subtrochanteric cortical bone was measured by the aforementioned technician who measured the bmd of the spine and hip . bmd was measured at the stlc and subtrochanteric medial cortex ( stmc ) for each patient at the level just distal to the lesser trochanter . as for the width , rois were drawn not violating the boundary of the cortices . the proximal to distal length of the roi was unified as 1 cm ( fig . 2 ) . all of the measured bmd data were recorded as g / cm . the bmd of the stmc was measured to assess whether that region was associated with changes in the stlc bmd . to assess the reliability of this measurement , 30 patients were randomly selected , and the technician measured the bmd again as described above with a time interval of 2 months , not to be biased by the memory of the first measurement . hal reflects the hip moment arm and we speculated that this value would reflect the load on the subtrochanteric cortex of the femur where the maximal strain is known to be loaded and could affect the change of bmd at the stlc . hal was measured along the extended femoral neck axis until the edge of the bone in each direction ( fig . the measurement was performed twice by an orthopedic surgeon on the image taken at the first dxa examination in each patient , and the mean values were recorded as cm . to test the difference in bmd changes between the first and the second dxa examinations , a standardization of the bmd change was needed because we believed that the same amount of bmd changes can not be considered the same when the baseline bmd was different . the percent changes were calculated for the stlc , stmc , spine , and total hip , and were assessed statistically as stated below considering two aspects : 1 ) to compare the percent changes of the total hip and spine bmd between the medication and control groups to ascertain the optimal compliance of the patients who took the medication , and 2 ) to test the difference in the lateral cortex percent changes according to the agents received . data were summarized as meansstandard deviations with ranges for continuous variables and as counts and percentages for categorical variables . the reproducibility of the bmd measurement of the subtrochanteric cortex was evaluated using the intraclass correlation coefficient ( icc ) . an icc value was evaluated as follows : 0 to 0.20 , poor agreement ; 0.21 to 0.40 , fair agreement ; 0.41 to 0.60 , moderate agreement ; 0.61 to 0.80 , substantial agreement ; 0.81 to 1.00 , nearly perfect agreement . a single measures icc value was obtained . to test whether there was a difference the percent change of bmd in the hip , spine , or stlc among the groups ( control , alendronate , or risedronate ) , an analysis of covariance ( ancova ) model was adopted to control five independent variables . the variables were age , bmi , bmd percent change of stmc , hal , and the time interval between dxa examinations . concurrently , we assessed whether any of the variables other than medication ( none , alendronate , or risedronate ) could affect the percent change of bmd . hal was included as one of the variables to be assessed to control geometry of the stlc that might affect the bmd change , which was mentioned above . bmd of stmc was included as a variable to further control unexpected effects on bmd at the subtrochanteric cortices other than geometry . using the ancova model , the least square means of the percent change for the hip bmd , spine bmd , and stlc bmd were obtained for each group , and multiple comparisons were performed to assess the group differences . all statistical analyses were performed using sas 9.2 software ( sas institute , cary , nc , usa ) . the subject 's age , interval between the first and the second dxa examinations , bmi , and hal are summarized in table 1 . in the representative 30 cases , in which the cortical bmd at the subtrochanteric area was measured twice , the mean bmd values of the medial cortices at the first and second measurement were 2.110.28 g / cm ( range , 1.68 - 2.82 ) and 2.160.29 g / cm ( range , 1.59 - 2.78 ) , respectively . those of lateral cortices were 1.860.31 g / cm ( range , 1.252.61 ) and 1.800.36 g / cm ( range , 1.24 - 2.54 ) , respectively . overall icc values obtained from the two separate measurements of cortex bmd at the subtrochanteric area were 0.87 , which was in the range of near perfect agreement . in the ancova , the percentage change of the spine bmd was affected by medication ( p<0.001 ) and the time interval between dxa examinations ( p=0.026 ) . on this analysis , the increase in percent change of the spine bmd per month of dxa interval , which corresponds to the correlation estimatestandard error , was 0.110.05 . additionally , in the ancova of the percent change of the hip bmd , the percent change was also affected by medication ( p<0.001 ) and the time interval between dxa examinations ( p=0.029 ) . the increase in the percent change of the hip bmd per one month of dxa interval , which corresponds to the correlation estimatestandard error , was 0.070.03 . in the assessment of the bmd percent change difference of the spine among medication groups after the control of the dependent variables , the least square mean valuesstandard error of the bmd percent change of the spine in control , alendronate , and risedronate groups were -0.181.18 , 6.111.01 , and 4.900.90 , respectively . the risedronate ( adjusted p=0.003 ) and alendronate ( adjusted p < 0.001 ) groups showed a significantly higher percentage change than the control group in the multiple comparison analysis ( fig . 3a ) . in the assessment of the bmd percent change difference of the hip among medication groups after the control of dependent variables , the least square mean valuesstandard error of the bmd percent change of the hip in the control , alendronate , and risedronate groups were -1.830.75 , 1.130.64 , and 1.860.57 , respectively . the risedronate ( adjusted p<0.001 ) and alendronate ( adjusted p=0.01 ) groups showed a significantly higher percentage change than the control group in the multiple comparison analysis ( fig . the percent change of the stlc bmd ( p=0.009 ) and the stmc bmd ( p=0.004 ) were both affected by medication . on this analysis , the increase in the percent change of the stlc bmd with per 1% increase of stmc bmd , which corresponds to the correlation estimatestandard error between them , was 0.230.09 . the least square mean valuesstandard deviation of the bmd percent change in the control , alendronate , and risedronate groups were 1.461.50 , 2.231.26 , and 6.961.11 , respectively . the risedronate group showed a significantly higher percent change in bmd than the control ( adjusted p=0.012 ) or alendronate ( adjusted p=0.016 ) groups ( fig . in the representative 30 cases , in which the cortical bmd at the subtrochanteric area was measured twice , the mean bmd values of the medial cortices at the first and second measurement were 2.110.28 g / cm ( range , 1.68 - 2.82 ) and 2.160.29 g / cm ( range , 1.59 - 2.78 ) , respectively . those of lateral cortices were 1.860.31 g / cm ( range , 1.252.61 ) and 1.800.36 g / cm ( range , 1.24 - 2.54 ) , respectively . overall icc values obtained from the two separate measurements of cortex bmd at the subtrochanteric area were 0.87 , which was in the range of near perfect agreement . in the ancova , the percentage change of the spine bmd was affected by medication ( p<0.001 ) and the time interval between dxa examinations ( p=0.026 ) . on this analysis , the increase in percent change of the spine bmd per month of dxa interval , which corresponds to the correlation estimatestandard error , was 0.110.05 . additionally , in the ancova of the percent change of the hip bmd , the percent change was also affected by medication ( p<0.001 ) and the time interval between dxa examinations ( p=0.029 ) . the increase in the percent change of the hip bmd per one month of dxa interval , which corresponds to the correlation estimatestandard error , was 0.070.03 . in the assessment of the bmd percent change difference of the spine among medication groups after the control of the dependent variables , the least square mean valuesstandard error of the bmd percent change of the spine in control , alendronate , and risedronate groups were -0.181.18 , 6.111.01 , and 4.900.90 , respectively . the risedronate ( adjusted p=0.003 ) and alendronate ( adjusted p < 0.001 ) groups showed a significantly higher percentage change than the control group in the multiple comparison analysis ( fig . 3a ) . in the assessment of the bmd percent change difference of the hip among medication groups after the control of dependent variables , the least square mean valuesstandard error of the bmd percent change of the hip in the control , alendronate , and risedronate groups were -1.830.75 , 1.130.64 , and 1.860.57 , respectively . the risedronate ( adjusted p<0.001 ) and alendronate ( adjusted p=0.01 ) groups showed a significantly higher percentage change than the control group in the multiple comparison analysis ( fig . in the ancova , the percent change of the stlc bmd ( p=0.009 ) and the stmc bmd ( p=0.004 ) were both affected by medication . on this analysis , the increase in the percent change of the stlc bmd with per 1% increase of stmc bmd , which corresponds to the correlation estimatestandard error between them , was 0.230.09 . the least square mean valuesstandard deviation of the bmd percent change in the control , alendronate , and risedronate groups were 1.461.50 , 2.231.26 , and 6.961.11 , respectively . the risedronate group showed a significantly higher percent change in bmd than the control ( adjusted p=0.012 ) or alendronate ( adjusted p=0.016 ) groups ( fig . both drugs , alendronate and risedronate , increased the bmd of the spine and hip significantly after the years of administration on this study suggesting compliance of the patients enrolled was acceptable . after controlling the dependent variables of age , bmi , stmc percent change , hal , and the time interval between dxa examinations , we observed that the increase in the stlc bmd of the risedronate group was significantly higher than that of the alendronate group . our study can be considered disputable as bmd gain at the spine and hip has been known to be larger in alendronate user groups than in risedronate . however , although there is no debate about the superiority of alendronate to risedronate in bmd gain at the trabecular bone , there is still debate about this superiority at the cortical bone . additionally , on the basis that the patient has a normal mechanical axis of the lower limb , the tension strain on the stlc of the femoral shaft is highest in the subtrochanteric area during the gait cycle . the strain is approximately 2,000 , which could cause a microcrack in the stlc during daily activities . the cortical bone under tensile stress in the stlc is expected to be subject to high metabolic and remodeling demand . considering this aspect , although several investigators raised objection , thickening of the stlc was once thought to be an important factor in the development of aff in the long term bp users . we believe that the difference in pharmacokinetics between alendronate and risedronate can affect the increase in bmd differently at the stlc , where the metabolic and remodeling demand is high as mentioned above , and the difference supports our observation like follows . the kinetic binding affinity of bps for hydroxyapatite in ascending order are clodronate < risedronate < ibandronate < alendronate < pamidronate < zoledronate . the inhibition power against farnesyl pyrophosphate synthetase in ascending order are clodronate < pamidronate < alendronate < ibandronate < risedronate < zoledronate . zeta potential is regarded as an independent property that does not correlate directly with affinity , but rather influences binding capacity . zeta potential is highest in alendronate and lowest in risedronate among the bps mentioned above . another previous article also reported that mineral binding affinity significantly affects the distribution of bps around the newly forming cortical bone osteons in the rib and trabecular hemiosteons of the vertebrae . bps with lower binding affinities to hydroxyapatite , such as risedronate , has a greater capacity to reach these areas deep in the bone , where the most vulnerable osteocytes reside . even without affecting the osteocytes , more widespread distribution of drug throughout the cortex could provide a more uniform reduction in remodeling . based on our results and the aforementioned articles regarding kinematics of these two bp agents , we postulated that the wide mineral distribution in the stlc and strong inhibition of farnesyl pyrophosphate synthetase resulted in larger bmd increase of stlc in the risedronate group than the alendronate group . we believe that the difference of bmd increase on this study between risedronate and alendronate might give us a plausible explanation about the difference of the aff risk between these bp agents . long - term use of bp has been regarded as having possible correlation with aff and osteonecrosis of the jaw . most of the reported aff cases were alendronate users , which might have been associated with the fact that alendronate was introduced in the market in 1995 and has been used for longer period of time compared to risedronate which was introduced in 2005 . the first comprehensive case series of aff associated with bp was reported in 2007 from singapore . the case series reported that 9 patients took alendronate for several years among the 13 patients who sustained aff . as for the large investigation comparing aff risk between the different bp user groups , an investigator group analyzed the data from the u.s . food and drug administration ( fda ) adverse event reporting system ( faers ) and from the international safety efforts regarding bp and aff . the investigators showed that the proportional reporting ratio and odds ratio were approximately two times higher in alendronate users than in risedronate users . on the other hand , schilcher et al . analyzed 59 cases of aff and found no differences in risk of aff between alendronate and risedronate user . however , they recently reported that multivariable adjusted odd ratio was almost three times higher in alendronate users than in risedronate users . although further study is needed to determine whether difference in bmd increase is the only parameter that explains why the alendronate group showed larger aff risk than the risedronate group , we believe that our result demonstrated that bmd at the stlc is worth including as one of the parameters . an article also demonstrated that various parameters including bmd gain and mechanical property was superior in risedronate group than in alendronate group , partly supporting our observation although the investigation was based on short - term experimental data obtained from ovariectomized rat . stlc has been attracting attention as the location for monitoring of aff occurrence by using various imaging modalities . localized periosteal or endosteal thickening of the stlc on radiograph was previously defined as a minor feature on the previous report released by a task force team of the american society for bone and mineral research in 2010 . however , the changes in the stlc have been recently upgraded to a major feature in their second report . among five major features of aff , detection of subtle change at the stlc has been scrutinized whether it can be used as a predictor for aff occurrence . we believe that our study results suggest that not only image - based morphological changes , but also bmd measurement at the stlc is feasible and can be used as a monitoring tool for investigation related to the occurrence of aff . on the basis , we should address that the bmd measurement at the medial and lateral cortices of the subtrochanteric femur were reproducible . no software supplied by the vendors has been developed to automatically check the bmd at the stlc and stmc , so there are concerns about the errors in repeated measurements of bmd in sequentially captured dxas . nevertheless , the overall icc value for the measurement of the cortical bmd was in the range of near perfect agreement . first of all , the number of enrolled patients and follow - up years was not large enough , which was inevitable due to retrospective nature of the study . second , direct evaluation of the mechanical strength of and stress on the femur in the subjects was impossible , which expectedly affected the changes in the bmd on the stlc . however , indirect mechanical consideration through hal in our study revealed the independent influence of medication on the stlc bmd . third , an additional study to verify the association between the subtrochanteric cortex bmd and aff risk needs to be further performed before recommending routine use of this measurement in clinical practice . fourth , we acknowledge that bmd measurement at the stlc and stmc is not an established method once more . however , we believe that this concern has been solved by reproducibility test for the measurement in our study . fifth , there was wide range of observational period in each group , which might have been substantial bias for the results . in conclusion , we observed a greater and statistically significant increase in bmd percent change at the stlc in the risedronate user group compared with the alendronate user and control groups , even though the implication of these changes must be qualified in the future .

backgroundto retrospectively assess whether the response of subtrochanteric lateral cortex ( stlc ) is different according to the bisphosphonate agents in terms of bone mineral density ( bmd ) change.methodsa total of 149 subjects , who had 2- to 4-year interval follow - up of bmd using dual energy x - ray absorptiometry ( dxa ) , were included in this retrospective study divided into following 3 groups : control group ( no consumption of any anti - osteoporotic drugs , n=38 ) , alendronate group ( nave alendronate users , n=48 ) , risedronate group ( nave risedronate users , n=63 ) . bmd was measured at the stlc and subtrochanteric medial cortex ( stmc ) in each patient by drawing rectangular rois at the bone cortices . the percent change of bmd at the stlc were compared between the aforementioned 3 groups by using analysis of covariance model to control five independent variables of age , body mass index , percent change of stmc , hip axis length , time interval between dxa examinations.resultsthe least square mean valuesstandard deviation of the percent change of bmd in the control , alendronate , and risedronate groups were 1.461.50 , 2.231.26 , and 6.961.11 , respectively . the risedronate group showed significantly higher change of bmd percentage compared with the control ( adjusted p=0.012 ) or alendronate ( adjusted p=0.016 ) groups.conclusionsthe percent change of bmd at the stlc in the risedronate user group was greater than the alendronate and control groups . the implication of these changes needs to be further verified .

INTRODUCTION METHODS 1. Subject enrollment 2. DXA examination 3. BMD measurement of the spine, hip, and subtrochanteric cortex 4. Measurement of hip axis length (HAL) 5. Measurement of BMD change and its assessment 6. Statistical analysis RESULTS 1. Reproducibility of the BMD measurement at the subtrochanteric cortex 2. Differences among the medication groups in BMD percent change of the spine and total hip 3. Differences among the medication groups in the STLC BMD percent change DISCUSSION CONCLUSION

since the introduction and development of these drugs , increased bone mineral density ( bmd ) in the lumbar spine and hip ( total hip or neck ) has been the main focus for evaluation of their effectiveness along with decreasing the risk of fragility fracture . on the other hand , differences in the pharmacokinetics of alendronate and risedronate can cause difference in bone mineral deposition , which are known to be affected by mineral affinity , farnesyl pyrophosphate synthetase inhibition , and the effect of bp binding to hydroxyapatite on the surface charge [ zeta potential ] . in this context , we speculated that the pharmacokinetic differences specific to each bp agent may cause different cortical responses at the subtrochanteric lateral cortex ( stlc ) . therefore , the purpose of this study was to retrospectively assess whether the response of the stlc is different according to two kinds of bp agents by tracing the changes of bmd using serial dual energy x - ray absorptiometry ( dxa ) examinations . from these individuals , 149 subjects who had serial dxa examinations during two or more years after taking a bp ( alendronate or risedronate ) as nave users or simply undergoing a health check - up subjects who were already taking a bp at the time of the first dxa examination ( n=1,318 ) were excluded . from the remaining 161 patients , subjects who acquired an endocrine disease that could possibly affect bmd ( n=2 ) , taking a steroid ( n=9 ) , or with acquired parkinson 's disease ( n=1 ) finally , 149 subjects whose first and follow - up dxa examinations were separated by a 2-to 4-year interval were included ( fig . among them , 38 did not take any anti - osteoporotic drugs ( control group ) , 48 were nave alendronate users ( the alendronate group ) , and 63 were nave risedronate users ( the risedronate group ) . the baseline characteristics of each group , including the mean age , time interval between dxa examinations , and body mass index ( bmi ) , were recorded based on the medical record . the medication duration of the patients in the medication groups was regarded as the time interval between dxa examinations since the included patients were nave patients and started the medication at the time of the first dxa examination . serial dxa examinations were performed in each patient by a technician using the same lunar prodigy scanner ( ge lunar , medison , wi , usa ) without changing the software or hardware of the dxa scanner . the pre- and post - medication bmds for the spine and hip were measured in the alendronate and risedronate groups to validate whether the medication compliance was acceptable . the bmd of spine was measured at the l1-l4 spines ; whereas , the hip bmds were measured at the total hip , neck , trochanter , and ward 's area by placing region of interest ( roi ) provided by the vendor . technician precision was assessed with the commencement of this study by measuring the bmd twice from the 30 dxa images from our study according to the recommendation stated in the international society of clinical densitometry ( iscd ) guideline 2005 . bmd was measured at the stlc and subtrochanteric medial cortex ( stmc ) for each patient at the level just distal to the lesser trochanter . the bmd of the stmc was measured to assess whether that region was associated with changes in the stlc bmd . to assess the reliability of this measurement , 30 patients were randomly selected , and the technician measured the bmd again as described above with a time interval of 2 months , hal reflects the hip moment arm and we speculated that this value would reflect the load on the subtrochanteric cortex of the femur where the maximal strain is known to be loaded and could affect the change of bmd at the stlc . hal was measured along the extended femoral neck axis until the edge of the bone in each direction ( fig . the measurement was performed twice by an orthopedic surgeon on the image taken at the first dxa examination in each patient , and the mean values were recorded as cm . to test the difference in bmd changes between the first and the second dxa examinations , a standardization of the bmd change was needed because we believed that the same amount of bmd changes can not be considered the same when the baseline bmd was different . the percent changes were calculated for the stlc , stmc , spine , and total hip , and were assessed statistically as stated below considering two aspects : 1 ) to compare the percent changes of the total hip and spine bmd between the medication and control groups to ascertain the optimal compliance of the patients who took the medication , and 2 ) to test the difference in the lateral cortex percent changes according to the agents received . to test whether there was a difference the percent change of bmd in the hip , spine , or stlc among the groups ( control , alendronate , or risedronate ) , an analysis of covariance ( ancova ) model was adopted to control five independent variables . the variables were age , bmi , bmd percent change of stmc , hal , and the time interval between dxa examinations . concurrently , we assessed whether any of the variables other than medication ( none , alendronate , or risedronate ) could affect the percent change of bmd . hal was included as one of the variables to be assessed to control geometry of the stlc that might affect the bmd change , which was mentioned above . using the ancova model , the least square means of the percent change for the hip bmd , spine bmd , and stlc bmd were obtained for each group , and multiple comparisons were performed to assess the group differences . from these individuals , 149 subjects who had serial dxa examinations during two or more years after taking a bp ( alendronate or risedronate ) as nave users or simply undergoing a health check - up subjects who were already taking a bp at the time of the first dxa examination ( n=1,318 ) were excluded . from the remaining 161 patients , subjects who acquired an endocrine disease that could possibly affect bmd ( n=2 ) , taking a steroid ( n=9 ) , or with acquired parkinson 's disease ( n=1 ) finally , 149 subjects whose first and follow - up dxa examinations were separated by a 2-to 4-year interval were included ( fig . among them , 38 did not take any anti - osteoporotic drugs ( control group ) , 48 were nave alendronate users ( the alendronate group ) , and 63 were nave risedronate users ( the risedronate group ) . the baseline characteristics of each group , including the mean age , time interval between dxa examinations , and body mass index ( bmi ) , were recorded based on the medical record . the medication duration of the patients in the medication groups was regarded as the time interval between dxa examinations since the included patients were nave patients and started the medication at the time of the first dxa examination . serial dxa examinations were performed in each patient by a technician using the same lunar prodigy scanner ( ge lunar , medison , wi , usa ) without changing the software or hardware of the dxa scanner . the pre- and post - medication bmds for the spine and hip were measured in the alendronate and risedronate groups to validate whether the medication compliance was acceptable . the bmd of spine was measured at the l1-l4 spines ; whereas , the hip bmds were measured at the total hip , neck , trochanter , and ward 's area by placing region of interest ( roi ) provided by the vendor . technician precision was assessed with the commencement of this study by measuring the bmd twice from the 30 dxa images from our study according to the recommendation stated in the international society of clinical densitometry ( iscd ) guideline 2005 . bmd was measured at the stlc and subtrochanteric medial cortex ( stmc ) for each patient at the level just distal to the lesser trochanter . the bmd of the stmc was measured to assess whether that region was associated with changes in the stlc bmd . to assess the reliability of this measurement , 30 patients were randomly selected , and the technician measured the bmd again as described above with a time interval of 2 months , not to be biased by the memory of the first measurement . hal reflects the hip moment arm and we speculated that this value would reflect the load on the subtrochanteric cortex of the femur where the maximal strain is known to be loaded and could affect the change of bmd at the stlc . hal was measured along the extended femoral neck axis until the edge of the bone in each direction ( fig . to test the difference in bmd changes between the first and the second dxa examinations , a standardization of the bmd change was needed because we believed that the same amount of bmd changes can not be considered the same when the baseline bmd was different . the percent changes were calculated for the stlc , stmc , spine , and total hip , and were assessed statistically as stated below considering two aspects : 1 ) to compare the percent changes of the total hip and spine bmd between the medication and control groups to ascertain the optimal compliance of the patients who took the medication , and 2 ) to test the difference in the lateral cortex percent changes according to the agents received . to test whether there was a difference the percent change of bmd in the hip , spine , or stlc among the groups ( control , alendronate , or risedronate ) , an analysis of covariance ( ancova ) model was adopted to control five independent variables . the variables were age , bmi , bmd percent change of stmc , hal , and the time interval between dxa examinations . concurrently , we assessed whether any of the variables other than medication ( none , alendronate , or risedronate ) could affect the percent change of bmd . hal was included as one of the variables to be assessed to control geometry of the stlc that might affect the bmd change , which was mentioned above . using the ancova model , the least square means of the percent change for the hip bmd , spine bmd , and stlc bmd were obtained for each group , and multiple comparisons were performed to assess the group differences . the subject 's age , interval between the first and the second dxa examinations , bmi , and hal are summarized in table 1 . in the representative 30 cases , in which the cortical bmd at the subtrochanteric area was measured twice , the mean bmd values of the medial cortices at the first and second measurement were 2.110.28 g / cm ( range , 1.68 - 2.82 ) and 2.160.29 g / cm ( range , 1.59 - 2.78 ) , respectively . in the ancova , the percentage change of the spine bmd was affected by medication ( p<0.001 ) and the time interval between dxa examinations ( p=0.026 ) . on this analysis , the increase in percent change of the spine bmd per month of dxa interval , which corresponds to the correlation estimatestandard error , was 0.110.05 . additionally , in the ancova of the percent change of the hip bmd , the percent change was also affected by medication ( p<0.001 ) and the time interval between dxa examinations ( p=0.029 ) . the increase in the percent change of the hip bmd per one month of dxa interval , which corresponds to the correlation estimatestandard error , was 0.070.03 . in the assessment of the bmd percent change difference of the spine among medication groups after the control of the dependent variables , the least square mean valuesstandard error of the bmd percent change of the spine in control , alendronate , and risedronate groups were -0.181.18 , 6.111.01 , and 4.900.90 , respectively . the risedronate ( adjusted p=0.003 ) and alendronate ( adjusted p < 0.001 ) groups showed a significantly higher percentage change than the control group in the multiple comparison analysis ( fig . in the assessment of the bmd percent change difference of the hip among medication groups after the control of dependent variables , the least square mean valuesstandard error of the bmd percent change of the hip in the control , alendronate , and risedronate groups were -1.830.75 , 1.130.64 , and 1.860.57 , respectively . the risedronate ( adjusted p<0.001 ) and alendronate ( adjusted p=0.01 ) groups showed a significantly higher percentage change than the control group in the multiple comparison analysis ( fig . the percent change of the stlc bmd ( p=0.009 ) and the stmc bmd ( p=0.004 ) were both affected by medication . on this analysis , the increase in the percent change of the stlc bmd with per 1% increase of stmc bmd , which corresponds to the correlation estimatestandard error between them , was 0.230.09 . the least square mean valuesstandard deviation of the bmd percent change in the control , alendronate , and risedronate groups were 1.461.50 , 2.231.26 , and 6.961.11 , respectively . the risedronate group showed a significantly higher percent change in bmd than the control ( adjusted p=0.012 ) or alendronate ( adjusted p=0.016 ) groups ( fig . in the representative 30 cases , in which the cortical bmd at the subtrochanteric area was measured twice , the mean bmd values of the medial cortices at the first and second measurement were 2.110.28 g / cm ( range , 1.68 - 2.82 ) and 2.160.29 g / cm ( range , 1.59 - 2.78 ) , respectively . in the ancova , the percentage change of the spine bmd was affected by medication ( p<0.001 ) and the time interval between dxa examinations ( p=0.026 ) . on this analysis , the increase in percent change of the spine bmd per month of dxa interval , which corresponds to the correlation estimatestandard error , was 0.110.05 . additionally , in the ancova of the percent change of the hip bmd , the percent change was also affected by medication ( p<0.001 ) and the time interval between dxa examinations ( p=0.029 ) . the increase in the percent change of the hip bmd per one month of dxa interval , which corresponds to the correlation estimatestandard error , was 0.070.03 . in the assessment of the bmd percent change difference of the spine among medication groups after the control of the dependent variables , the least square mean valuesstandard error of the bmd percent change of the spine in control , alendronate , and risedronate groups were -0.181.18 , 6.111.01 , and 4.900.90 , respectively . the risedronate ( adjusted p=0.003 ) and alendronate ( adjusted p < 0.001 ) groups showed a significantly higher percentage change than the control group in the multiple comparison analysis ( fig . in the assessment of the bmd percent change difference of the hip among medication groups after the control of dependent variables , the least square mean valuesstandard error of the bmd percent change of the hip in the control , alendronate , and risedronate groups were -1.830.75 , 1.130.64 , and 1.860.57 , respectively . the risedronate ( adjusted p<0.001 ) and alendronate ( adjusted p=0.01 ) groups showed a significantly higher percentage change than the control group in the multiple comparison analysis ( fig . in the ancova , the percent change of the stlc bmd ( p=0.009 ) and the stmc bmd ( p=0.004 ) were both affected by medication . on this analysis , the increase in the percent change of the stlc bmd with per 1% increase of stmc bmd , which corresponds to the correlation estimatestandard error between them , was 0.230.09 . the least square mean valuesstandard deviation of the bmd percent change in the control , alendronate , and risedronate groups were 1.461.50 , 2.231.26 , and 6.961.11 , respectively . the risedronate group showed a significantly higher percent change in bmd than the control ( adjusted p=0.012 ) or alendronate ( adjusted p=0.016 ) groups ( fig . both drugs , alendronate and risedronate , increased the bmd of the spine and hip significantly after the years of administration on this study suggesting compliance of the patients enrolled was acceptable . after controlling the dependent variables of age , bmi , stmc percent change , hal , and the time interval between dxa examinations , we observed that the increase in the stlc bmd of the risedronate group was significantly higher than that of the alendronate group . considering this aspect , although several investigators raised objection , thickening of the stlc was once thought to be an important factor in the development of aff in the long term bp users . we believe that the difference in pharmacokinetics between alendronate and risedronate can affect the increase in bmd differently at the stlc , where the metabolic and remodeling demand is high as mentioned above , and the difference supports our observation like follows . based on our results and the aforementioned articles regarding kinematics of these two bp agents , we postulated that the wide mineral distribution in the stlc and strong inhibition of farnesyl pyrophosphate synthetase resulted in larger bmd increase of stlc in the risedronate group than the alendronate group . most of the reported aff cases were alendronate users , which might have been associated with the fact that alendronate was introduced in the market in 1995 and has been used for longer period of time compared to risedronate which was introduced in 2005 . although further study is needed to determine whether difference in bmd increase is the only parameter that explains why the alendronate group showed larger aff risk than the risedronate group , we believe that our result demonstrated that bmd at the stlc is worth including as one of the parameters . no software supplied by the vendors has been developed to automatically check the bmd at the stlc and stmc , so there are concerns about the errors in repeated measurements of bmd in sequentially captured dxas . third , an additional study to verify the association between the subtrochanteric cortex bmd and aff risk needs to be further performed before recommending routine use of this measurement in clinical practice . in conclusion , we observed a greater and statistically significant increase in bmd percent change at the stlc in the risedronate user group compared with the alendronate user and control groups , even though the implication of these changes must be qualified in the future .

the metabolic syndrome has become a major public health challenge with an estimated 22% of us adults having this condition . a consensus group for the international diabetes federation defines metabolic syndrome as central obesity , plus any two of the following : raised triglycerides , reduced high - density lipoprotein ( hdl ) cholesterol , raised fasting plasma glucose , and raised blood pressure . the consensus group also recommends additional criteria that should be part of further research into the metabolic syndrome including tomographic assessment of visceral adiposity and liver fat , biomarkers of adipose tissue ( leptin , adiponectin ) , and glucose tolerance testing . the cause of the syndrome remains obscure but the pathophysiology seems to be largely attributable to insulin resistance , excessive flux of fatty acids , and a chronic proinflammatory state . therapeutics include lifestyle changes ( e.g. , weight reduction and increased physical activity ) and pharmaceutical agents , but prevention would be preferred . a mounting body of evidence indicates that certain adverse exposures during the perinatal period contribute to the development of the metabolic syndrome . this early life stage may offer an attractive point in the disease process for prevention and intervention strategies . in the 1970s forsdahl , using official statistical data on norwegian counties , reported that poverty during adolescence , followed by prosperity , was positively correlated with risk of death from coronary heart disease . although no biological mechanisms were identified , forsdahl speculated that some form of permanent damage caused by the nutritional deficit may be involved . in 1986 , barker and colleagues began publishing reports on the association between an adverse intrauterine environment , as determined primarily by low birth weight , and an increased risk of coronary heart disease later in life . these studies involved examining men and women in middle and late life whose body measurements at birth were recorded in the archives and records offices of britain [ 5 , 6 ] . upon further investigation , it was found that the correlation between low birth weight and heart disease was also present between low birth weight and type 2 diabetes with the prevalence of impaired glucose tolerance and type 2 diabetes falling progressively with increasing birth weight . moreover , in a follow - up study , an association between birth weight and the presence of metabolic syndrome was discovered . in this study metabolic syndrome was defined as impaired glucose tolerance , hypertension , and elevated triglycerides . in both men and women , of the 64-year - old men whose birth weights were 2.95 kg ( 6.5 pounds ) or less , 22% had metabolic syndrome and their risk of developing the syndrome was more than 10 times greater than that of men whose birth weights were more than 4.31 kg ( 9.5 pounds ) [ 8 , 9 ] . the association between metabolic syndrome and low birth weight prompted the authors to suggest that the metabolic syndrome , also called syndrome x , be renamed the small - baby syndrome . collectively , these studies generated the forsdahl - barker hypothesis , recognizing forsdahl as the original source of the idea and barker as the developer of the concept . in the decades following their initial publications , the forsdahl - barker hypothesis has become more well known as the fetal origins hypothesis and has produced a new branch of scientific knowledge and inquiry known as the developmental origins of health and disease ( dohad ) . historically , investigations on the fetal origins hypothesis have focused on maternal undernutrition and specific nutrient deficiencies . today , the world faces the dual burden of malnutrition that encompasses both under- and overnutrition . it is estimated that maternal and child undernutrition is the underlying cause of 3.5 million deaths globally . while at the same time overweight and obesity are major public health challenges in both developed and developing regions of the world . in 2005 , 33% of the world 's adult population ( 1.3 billion people ) was overweight or obese , and it is estimated that by 2030 , up to 57.8% of the world 's adult population ( 3.3 billion people ) will be overweight or obese . adding to the complexity of the dual burden of malnutrition is the influence of socioeconomic status . by and large , in developed countries lower socioeconomic status is associated with both lower birth weights among the offspring of women and a higher prevalence of obesity among women . as a result , failure to control for social position could obscure a relationship between high birth weight and subsequent obesity and obesity - related disorders . incidentally , although the vast majority of the literature concerning the dohad is focused on the relationship between small size at birth and increased incidence of disease in adult life , it is now recognized that higher incidences of disease occur in both those born small and those born large , thus reflecting a u - shaped curve [ 15 , 16 ] . this paper provides a brief overview of the putative concepts and mechanisms behind the dohad and assembles thoughts on how alterations in maternal consumption of specific nutrients may irreversibly direct fetal programming of the metabolic syndrome ( figure 1 ) . developmental plasticity is the ability of an organism to change its phenotype in response to changes in the environment . if this change or adaptation is permanent , it is considered a programming change and is associated with persistent effects in structure and/or function [ 18 , 19 ] . gluckman and hanson use the example of the meadow vole ( microtus pennsylvanicus ) to illustrate programming . meadow moles born in autumn have a thicker hair coat than those born in the spring . this occurs as a response of the fetus to maternally derived signals for day length . a thick coat reflects an adaptive programming response to help ensure survival in the cold environment that was predicted while in utero . humans have similar numbers of sweat glands at birth , but they are essentially nonfunctional . in the first few years of life a proportion of the glands become functional depending on the temperature to which the child is exposed . the hotter the conditions , the greater number of functional sweat glands . after a few years programming changes can also be experimentally - induced as demonstrated by examination of adult ( 100-day - old ) female rats that were given a single subcutaneous injection of testosterone at 2 , 5 , 10 , or 20 days of age . those that were injected between birth and 10 days of age had lasting changes in organ weight , specifically larger adrenal and pituitary weights and smaller ovarian and uterine weights , as well as absent corpora lutea , failure to develop normal female sexual behavior , and permanent sterility [ 22 , 23 ] . theses aberrations in morphology and physiology were much reduced or absent in those rats injected at 10 or 20 days of age , indicating that the period of plasticity during which rats are androgen sensitive is between birth and 10 days of age . in most cases programming however , the problem of mismatch occurs when individuals developmentally adapted to one environment are exposed to another . revisiting the functional activity of the sweat glands to provide an example , the child who has experienced hot conditions will be better adapted to such conditions in later life because more functioning sweat glands provide the ability to cool down faster . while the child who has experienced cool conditions faces the problem of mismatch and will be unable to cool down as easily in hot conditions because of fewer functioning sweat glands . other examples of this mismatch phenomenon include people whose birth weights were towards the lower end of normal that subsequently grow up in affluent societies being at increased risk for hypertension , type 2 diabetes , and cardiovascular disease [ 16 , 25 ] . the problem of mismatch is thought to be involved in the current epidemic of type 2 diabetes and cardiovascular disease in the young adult and middle - aged populations of india . several countries , including india , are undergoing swift economic and nutritional transitions , exposing individuals to conditions that promote weight gain . the current surge in metabolic and cardiovascular disease in india may be being fueled by a combination of undernutrition in early life and overnutrition in later life . in a study of indian children , those born small and where relatively fat and tall by 8 years of age had the most adverse risk profiles for cardiovascular disease , including components of the metabolic syndrome . the authors commented on the possible importance of preventing childhood obesity in the prevention of disease in the low birth weight members of the indian population . it is important to note that indians as a race are more prone to the development of the metabolic syndrome , compared to caucasians , as a result of having a phenotype of high fat mass and low lean mass . the thrifty phenotype hypothesis , put forth by hales and barker , proposes that poor fetal and early postnatal nutrition imposes mechanisms of nutritional thrift upon the growing individual . in conditions of severe intrauterine deprivation the developing fetus may lose functional and structural units such as pancreatic cells , nephrons , and cardiomyocytes . such changes have been deemed an adaptive mechanism to ensure the survival of the fetus . the thrifty phenotype hypothesis is based on a study of 468 men in which the percentage of those with impaired glucose tolerance or type 2 diabetes fell progressively with increasing birth weight and weight at 1 year . from this research it was hypothesized that poor intrauterine nutrition results in the growth of vital organs , specifically the brain , at the expense of other organs ( i.e. , endocrine pancreas resulting in cell hypoplasia ) . such adaptations may increase the chance of fetal survival by means of brain sparing but result in difficulty in coping with nutritional abundance as an adult . fetal salvage hypothesis which offers a different explanation for the insulin resistance seen in those affected by intrauterine growth restriction . in this alternate explanation it is not hypoplasia of the pancreatic cells that leads to impaired glucose tolerance , but rather it is that the fetus develops peripheral insulin resistance . it is this peripheral insulin resistance that ensures that adequate amounts of glucose are delivered to essential organs such as the brain with subsequent reduced delivery to nonessential tissues such as skeletal muscle . support for the fetal salvage hypothesis comes from intrauterine growth restriction studies in rats . in these rodent models , glucose transport ( glucose uptake and glucose transporter mrna and protein levels ) is reduced in lung and skeletal muscles of growth - restricted fetuses , whereas glucose transport is unaffected in brain [ 32 , 33 ] . the kidney is another organ thought to be affected by nutritional thrift . in both human and animal studies small offspring , due to maternal undernutrition and other causes , have reduced numbers of nephrons which has been strongly linked to hypertension [ 16 , 34 , 35 ] . in the human , nephrogenesis is completed in utero , and no new nephrons are formed after birth ; therefore a nephron deficit present at birth persists throughout life [ 29 , 36 ] . in a study of human infants affected by intrauterine growth restriction , a similar study found a 20% reduction in nephron number in human neonates with low birth weights as compared to normal birth weight neonates . additionally , researchers found a positive relationship between weight at birth and the number of glomeruli as well as a negative correlation between weight at birth and glomerular volume . this reduction in nephrons may reflect an adaptation that has the immediate advantage of energy and resource conservation but no long - term advantages [ 16 , 34 ] . catch - up growth , also known as compensatory growth , is where children return to their genetic trajectory for size after a period of growth delay or arrest . it may occur at any stage of growth but is most commonly observed in the first 2 years of life . studies have found that catch - up growth often results in overcompensation , whereby the organism exceeds normal weight and often has excessive fat deposition . this rapid and excessive growth has been associated with the development of adult obesity , insulin resistance , metabolic syndrome , and type 2 diabetes [ 3840 ] . in the avon longitudinal study of pregnancy and childhood ( alspac ) birth cohort , children who showed catch - up growth , as calculated by specified gains in standard deviation scores , between 0 and 2 years of age , were heavier , taller , and fatter ( body mass index , percentage body fat , and waist circumference ) at 5 years of age than other children . a study that evaluated glucose tolerance and plasma insulin concentrations in more than 1400 young adults ( 26 to 32 years old ) who had grown up in the city of delhi , india , found an association between thinness in infancy and the presence of impaired glucose tolerance or diabetes in young adulthood . as a group , the young adults in the study who had impaired glucose tolerance or diabetes were overweight . they were not , however , overweight or obese in childhood . instead , they were characterized by their high rate of gain in body mass after the age of 2 years . cianfarani and colleagues speculate that the tremendous effort to recover lost growth shortly after birth involves insulin and insulin - like growth factors ( igf ) . infants affected by intrauterine growth restriction have low concentrations of insulin and igf-1 at birth , and normalization of these parameters occurs in the postnatal period [ 42 , 43 ] . it is thought that tissues chronically depleted of insulin and igf-1 throughout fetal life and then suddenly exposed to increased concentrations of the two hormones shortly after birth may counteract the actions of insulin by developing insulin resistance . thus , in this proposed scenario , insulin resistance is serving as a metabolic defense mechanism to protect the organism against hypoglycemia . in addition to insulin resistance and type 2 diabetes , accelerated weight gain in early life has been associated with elevated blood pressure [ 44 , 45 ] and coronary heart disease . the implication of catch - up growth is that rapidly enhancing early childhood growth by a nutrient enriched diet may cause harm overtime and that encouraging slower growth rates may actually be beneficial . in a study that provides evidence of the benefits of steady ( i.e. , not accelerated by dietary means ) growth , investigators measured fasting concentrations of 32 - 33 split proinsulin , a marker of insulin resistance , in adolescents born preterm who had participated in randomized intervention trials of neonatal nutrition . fasting 32 - 33 split proinsulin concentration was greater in those given a nutrient - enriched diet than those given the lower - nutrient diet and was associated with greater weight gain in the first 2 weeks of life . excessive reactive oxygen species can cause modulation of gene expression and/or direct damage to cell membranes and other molecules at critical developmental windows . many believe that oxidative stress is the primary link between adverse fetal growth and later elevated risks of the metabolic syndrome , type 2 diabetes , and other disorders . smoking , hypertension / preeclampsia , inflammation / infection , obesity , and malnutrition are common causes of preterm and/or low birth weight as well as known sources of oxidative stress . malnutrition can directly lead to a pro - oxidative state by means of creating protein and micronutrient deficiencies . proteins provide amino acids needed for antioxidant synthesis , such as glutathione and albumin , and many micronutrients themselves are antioxidants , such as vitamins a , c , and e [ 40 , 48 ] . pancreatic cells are particularly sensitive to reactive oxygen species because they are low in enzymatic antioxidant defense equipment . it has been demonstrated that oxidative stress can blunt insulin secretion . with the susceptibility of pancreatic cells to oxidative stress , it is believed that early and ongoing exposures to oxidative insults could result in the eventual manifestations of the metabolic syndrome and related disorders . elevated oxidative stress has been observed among human infants born small for gestational age ( sga ) as compared to those appropriate for gestational age ( aga ) and among preterm as compared to term infants . it is important to note that preterm ( birth at < 37 weeks of pregnancy ) infants are generally of low birth weight but are not necessarily growth restricted or small for gestational age . a study using cord blood to compare the status of oxidative stress between sga infants born to undernourished mothers and aga infants born to healthy mothers found elevated oxidative stress , as determined by increased quantities of malondialdehyde ( one of the major products of lipid peroxidation ) , reduced quantities of the antioxidant glutathione , and decreased activity of the antioxidants superoxide dismutase and catalase in the sga infants as compared to the aga controls . a human study determined vitamin a , c , and e levels in the umbilical cord blood of term and preterm infants and the blood of their mothers taken at the time of delivery . maternal vitamin a and e levels were higher than cord values , yet there was a positive correlation between maternal and cord levels of these two vitamins . a and e , cord vitamin c levels were higher than maternal levels , and no significant correlation between cord and maternal vitamin c levels was present . in comparing vitamin levels in term and preterm infants , term babies had significantly higher levels of vitamins a , c , and e. the authors concluded that for vitamins a and e , neonatal levels are dependent on maternal levels and that preterm babies have fewer lipid - soluble antioxidant vitamins in their serum compared to term infants thus may be more susceptible to oxidative stress . gestational diabetes , a common cause of macrosomia , is associated with oxidative stress which could have effects on the developing infant . a study evaluating oxidative and antioxidative status in pregnant diabetic ( gestational diabetes or type 1 diabetes ) women between 26 and 32 weeks gestation demonstrated increased oxidative stress in those who were diabetic . levels of malondialdehyde were significantly higher , and vitamin a and e levels were significantly lower in all diabetic women than controls . moreover , glutathione peroxidase and superoxide dismutase activities were significantly reduced in diabetic women ; glutathione peroxidase was reduced in both those with gestational and type 1 diabetes , and superoxide dismutase was reduced only in type 1 diabetics . an increase in circulating glucocorticoids may play a role in early programming of adult disease . experimental studies in animals and epidemiological studies in humans have suggested an altered set - point of the hypothalamic - pituitary - adrenal ( hpa ) axis as an important long - term change that occurs in association with reduced fetal growth . pregnant animal models have shown that exposure to a variety of stressors , including nutrient restriction , results in the birth of offspring with elevated basal or stress - induced glucocorticoid secretion [ 54 , 55 ] . it is thought that maternal exposure to stressors during pregnancy subsequently leads to excessive fetal exposure to glucocorticoids resulting in persistent alterations in hpa axis activity . in support of this hypothesis are studies conducted in rats in which fetoplacental exposure to maternally administered steroids throughout gestation reduced birth weight and produced hypertensive adult offspring . in one such study , dexamethasone administration to pregnant rats on days 1520 of gestation resulted in offspring with reduced birth weight , elevated blood pressure , increased basal plasma corticosterone , lower mrna expression of hippocampal neuronal glucocorticoid receptor , and decreased gene expression of hippocampal mineralocorticoid receptor . in a sheep study , dams were treated with dexamethasone over 2 days ; treatment group 1 was treated during 2229 days of pregnancy and treatment group 2 was treated during 5966 days of pregnancy ( term 145 days ) . offspring from dams that had received dexamethasone during 2229 days gestation , but not days 5966 of gestation , had elevated blood pressures . such studies suggest that excess glucocorticoid exposure at certain developmental stages or windows programs higher blood pressure . another study of pregnant sheep found that severe brief undernutrition in late gestation altered the function of the hpa axis of adult offspring . those exposed to gestational undernourishment for 10 days demonstrated altered steroid levels including an increased adrenocorticotropic hormone ( acth ) response as compared to offspring from dams fed ad libitum or offspring from dams undernourished for 20 days . an epidemiological study in humans involving three populations found that adults who had lower birth weights had elevated fasting plasma concentrations of cortisol and that cortisol concentrations positively correlated with current blood pressure . the authors concluded that increased activity of the hpa axis may link low birth weight with raised blood pressure in adult life . in a study of over 300 men , plasma cortisol concentrations fell progressively with increasing birth weight ; this trend was independent of age and body mass index . raised plasma cortisol concentrations were significantly associated with higher blood pressure as well as plasma glucose concentrations and insulin resistance , suggesting that programming of the hpa axis may be a mechanism underlying the association between low birth weight and the metabolic syndrome in adult life . the hypothalamus plays a critical role in the regulation of appetite and body composition by way of responding to cues from neuropeptides . a series of studies , primarily in rodents , have explored the possibility that maternal nutrition during pregnancy may alter the level of energy intake in the offspring through inducing changes in the hypothalamic circuitry and in the expression , localization , and action of specific neuropeptides . appetite stimulating neuropeptides include neuropeptide y ( npy ) , agouti - related peptide ( agrp ) , and ghrelin . conversely , appetite suppressing neuropeptides include cocaine and amphetamine - related transcript ( cart ) , melanocyte - stimulating hormone ( msh ) , serotonin , insulin , and leptin [ 61 , 62 ] . of note is that the overweight and obese tend to develop resistance to insulin and leptin ; therefore these two neuropeptides are usually elevated in such groups . the hypothalamus has multiple nerve centers , or nuclei , that are sensitive to a variety of physiologic changes . hypothalamic nuclei include the arcuate nucleus ( arc ) , paraventricular nucleus ( pvn ) , and ventromedial nucleus ( vmn ) which are involved in a number of biological activities with a fair degree of overlap . in general , the arc is a conduit of many diverse signals involved in various functions such as energy homeostasis , while the pvn is associated with blood pressure and stress responses and the vmn with satiety . each also plays a role in the central nervous system regulation of food intake and body weight . studies in rats have found that exposure to overnutrition in the fetal or neonatal period can result in permanent changes in body fat mass and in the hypothalamic neuronal circuitry regulating appetite in the adult brain . drner , plagemann , and colleagues found immunocytochemical and morphometric aberrations in the hypothalamus of offspring of diabetic dams or in those exposed to milk from diabetic dams . in one of their early studies , offspring of diabetic dams had permanent hypoplasia of the vmn , decreased insulin responsiveness to glucose , impaired glucose tolerance , and increased susceptibility to diabetes . a later study examined the effects of exposure to milk from diabetic dams . in this study , offspring of control dams cross - fostered to diabetic dams developed early postnatal growth delay and showed structural and functional hypothalamic exposure to milk from a diabetic dam resulted in an upregulation of the appetite stimulants npy and agrp and a downregulation of the appetite suppressant msh . although the researchers expected to find changes in the vmn as in their previous study , they found increased total number of neurons in the pvn which , as mentioned previously , is associated with regulation of blood pressure . incidentally , their research team performed a human , clinical study and reported an association between the neonatal ingestion of breast milk from diabetic mothers and increased blood pressure during childhood . leptin has received a considerable amount of attention in regard to early programming of appetite and body composition . in a rodent study to establish whether neonatal ( postnatal day 313 ) leptin treatment can alleviate postnatal obesity and the associated metabolic sequelae that occur in the offspring of undernourished dams , leptin treatment reversed the programmed phenotype . the benefits of leptin treatment in these rats included a slowing of neonatal weight gain and normalized caloric intake , body weight , fat mass , and fasting plasma glucose and insulin . studies in leptin deficient ( lep / lep ) mice have demonstrated permanently disrupted neural projection pathways from the arc . in the adult lep / lep mice , leptin treatment did not reverse these neuroanatomical defects ; however treatment of neonatal lep / lep mice with exogenous leptin rescued the development of arc projections . epigenetics refers to all modifications to genes other than changes in the dna sequence itself and includes dna methylation and histone modifications [ 18 , 72 ] . dna methylation is a post - replication modification that is predominantly found in the cytosines of the dinucleotide sequence cytosine phosphate guanine ( cpg ) . changes in methylation status ( hyper- or hypomethylation ) have been associated with various health conditions including malignancies . histones are proteins that permit the packaging of dna into nucleosomes , the fundamental units of chromatin . histones are subject to a large number of post - translational modifications which are likely to control the structure and/or function of the chromatin fiber . different modifications may have distinct consequences such as transcriptional activation or dna repair . every cell in the body has the same genetic information ; what makes cells , tissues , and organs different is that different sets of genes are turned on or expressed . an increasing body of evidence supports the role of environmentally - induced epigenetic changes in disease susceptibility . experimental studies in agouti mice suggest a role for maternal diet in inducing epigenetic changes in the offspring [ 75 , 76 ] . those carrying the dominant agouti alleles a or a develop the complex set of traits collectively referred to as the yellow obese syndrome . the yellow obese syndrome encompasses the pleiotropic effects of yellow fur , obesity , hyperinsulinemia , hyperglycemia , and increased susceptibility to neoplasia . female a / a mice fed a methyl - supplemented diet with extra folate , vitamin b-12 , choline , and betaine 2 weeks prior to mating with male a / a agouti mice and throughout pregnancy and lactation passed along the agouti gene to their offspring intact , yet demonstrated a shift in distribution towards having more offspring with the pseudoagouti phenotype . pseudoagouti mice are brown , lean , healthy , and longer lived than their yellow siblings . this shift in the distribution in coat color was mediated by cpg methylation at the at the a locus . another example of environmentally - induced epigenetic changes is reduced pancreatic and duodenal homeobox 1 ( pdx1 ) , also known as insulin promoter factor 1 , in a rat model of intrauterine growth restriction . pdx1 is a transcription factor necessary for development and function of the insulin producing pancreatic cell . rats faced with intrauterine growth restriction had permanently reduced expression of pdx1 in cells and developed type 2 diabetes in adulthood [ 78 , 79 ] . reduced pdx1 transcription was mediated through a cascade of epigenetic modifications characterized by loss of upstream stimulatory factor-1 binding at the proximal promoter of pdx1 , recruitment of the histone deacetylase 1 and the corepressor sin3a , and deacetylation of histones h3 and h4 which culminated in the eventual silencing of pdx1 . the dutch famine of 1944 has been used by various investigators as an equivalent to an experimental study to investigate the effects of perinatal undernutrition in humans . the ongoing hunger winter families study contributed empirical support for the hypothesis that early - life environmental conditions cause epigenetic changes in humans that persist throughout life . individuals who had been exposed to famine during periconception had , 6 decades later , less dna methylation of the insulin - like growth factor ii ( igf2 ) gene compared with their unexposed , same - sex siblings . the dutch famine cohort has also been used to study the transgenerational effects of famine exposure . women exposed to famine while in the womb later had offspring with birth weights lower than offspring of women not exposed to famine . this effect of in utero exposure to famine on birth weight in the subsequent generation persisted after control for potential confounding and intervening variables . in a study of 2 prospective cohorts , godfrey and colleagues used dna extracted from umbilical cord tissue obtained at birth in children who were later assessed for adiposity at 9 years of age to measure methylation status of cpgs in the promoters of candidate genes . five candidate genes were selected based on a number of criteria which included animal data [ 8385 ] and correlations with overall gene methylation status and adiposity . methylation of retinoid x receptor- ( rxra ) chr9 : 136355885 + ( in cohort 1 and 2 ) and endothelial nitric oxide synthase ( enos ) chr7 : 150315553 + ( in cohort 1 only ) at birth was correlated with greater adiposity in later childhood . although the data is correlative and does not prove causality between dna methylation at birth and adiposity in childhood , the observation suggests that epigenetics is involved in fetal programming of later obesity . a sizable number of experimental studies , predominantly in rats , have explored the effects of protein restriction on fetal growth and later health . as discussed in the section on the thrifty phenotype hypothesis , protein restriction during pregnancy has been found to produce small offspring that have reduced numbers of nephrons which potentially contributes to the development of hypertension in adulthood [ 16 , 34 , 35 ] . low protein exposure in fetal rats has also been shown to result in reduced pancreatic cell mass at birth and reduced insulin secretion in later life presumably due to dietary - induced reduction in proliferation rate and increased apoptosis of cells . weanling ( 26-day - old ) rats exposed to low protein during gestation and lactation were smaller and had reduced serum insulin and increased serum glucose and triglycerides . in addition , low protein - exposed offspring had increased hepatic triglycerides and hepatic expression of lipogenic enzymes favoring fat synthesis . the authors suggested that the increased expression of fat - synthesizing enzymes may account for the increased levels of serum and liver triglycerides seen in those exposed to low protein and that these increases may predispose the offspring to excessive accumulation of fat and ultimately obesity and insulin resistance . rat studies indicate that gestational exposure to low levels of protein due to maternal protein restriction establishes a preference for high fat foods . when given the choice of selecting high fat , high protein , or high carbohydrate foods , 12-week - old offspring of dams fed a low protein diet throughout gestation consumed significantly more of the high fat food and significantly less of the high carbohydrate food than their control counterparts . at 30 weeks of age , there was no difference in the pattern of food selection between the two groups . their results suggest that early exposure to undernutrition programs a preference for fatty foods , and thus maternal nutrition may influence pathways involved in control of appetite or the perception of palatability in the offspring . although the literature on protein restriction and disease risk is quite extensive , it is largely based on rodent models with few having assessed the specific role of protein restriction in human risk of disease . one study of men and women in scotland sought to determine how diet of the mother in pregnancy influences blood pressure in the 40-year - old adult offspring . the authors found that the relations between the diet of mothers and the blood pressure of their offspring were complex . when maternal intake of animal protein was less than 50 g daily , a higher carbohydrate intake was associated with higher blood pressures in the offspring . conversely , when daily protein intake was above 50 g , lower carbohydrate intake was associated with higher blood pressure . an additional finding was that increases in blood pressure were associated with decreased placental size . their conclusions were that blood pressure in adulthood may be influenced by maternal intakes of protein and carbohydrates during pregnancy and that this may be mediated through effects on placental growth . all dietary carbohydrates can be converted into glucose . in both animal and human studies , intrauterine exposure to high sugar diets and/or hyperglycemia has been found to increase the risk of the metabolic syndrome . in rats , a high fructose diet for 2 weeks resulted in increases in systolic blood pressure as well as plasma insulin and triglyceride concentrations as compared to rats fed a normal chow . in another rat study early - and long - term exposure to a high sucrose diet ( hsd ) was assessed to determine whether such exposure alleviates the detrimental effects of sucrose feeding in later life . dams were fed either standard or hsd ( 70% calories as sucrose ) starting 1 week before breeding and throughout gestation and lactation . after weaning , all male offspring were fed hsd until the age of 20 weeks , then detailed metabolic and morphometric profiles were ascertained . offspring of sucrose - fed dams displayed higher adiposity and increases in triglyceride liver content together with higher low - density lipoprotein ( ldl ) cholesterol concentrations . although the significance and mechanisms are not clear , a somewhat perplexing observation was that of substantial increases in the insulin sensitivity of skeletal muscle together with higher concentrations of adiponectin in the offspring of sucrose - fed dams compared with the offspring of standard diet - fed dams . triglycerides , free fatty acids , overall glucose tolerance , and the insulin sensitivity of adipose tissue were comparable in both groups . data derived from the camden study of adolescent pregnancy implicated high sugar consumption with a twofold increased risk for delivering small for gestational age infants . additionally , evaluation of the data based on ethnicity led to the detection of a substantial decrease in the duration of gestation among puerto rican adolescents with high sugar diets , whereas there was no effect of a high sugar diet on gestation duration among black and white adolescents . the authors commented that other studies involving comparisons among ethnicities have found hispanic women to be more likely to have abnormalities of carbohydrate metabolism during pregnancy as evidenced by abnormal glucose tolerance tests and increased incidence of gestational diabetes [ 9395 ] . pregnancies complicated by maternal diabetes , in any form , place the offspring at risk for developing obesity and glucose intolerance [ 29 , 96 ] . in a study of 18- to 27-year - old women born to diabetic ( gestational diabetes or type 2 diabetes ) mothers , the risk of overweight was doubled in the offspring of diabetic mothers as compared with offspring from a background population . moreover , the risk of the metabolic syndrome was increased 4-fold for offspring of mothers with gestational diabetes and 2.5-fold for offspring of mothers with type 1 diabetes . offspring risk of the metabolic syndrome increased significantly with increasing maternal fasting blood glucose as well as 2-hour blood glucose following an oral glucose load . their findings indicate that intrauterine exposure to hyperglycemia contributes to the pathogenesis of the metabolic syndrome , thus offspring of diabetic mothers are risk groups for this condition . our laboratory has performed multiple experimental studies aimed at determining the role of high fat diet in maternal - fetal health . in one such study , mice exposed to a high saturated fat diet during gestation and lactation developed adult obesity , hyperglycemia , insulin resistance , and hypertension , despite being fed a standard rodent diet post - weaning . given that oxidative stress is a potential mechanism connecting fetal exposures to the development of the metabolic syndrome , a group of pregnant mice were supplemented with quercetin , a powerful antioxidant , which mitigated the detrimental effects of high fat diet exposure . similarly , bouanane and colleagues used a rat model to determine the effects of a cafeteria diet , that had 42% of energy from fat , on oxidant / antioxidant status as well as variety of metabolic markers . dams fed the cafeteria diet developed increased body weight , hyperglycemia , hyperinsulinemia , hyperleptinemia , hyperlipidemia , and an imbalance between oxidants / antioxidants favoring oxidative stress . male and female offspring displayed similar effects , which persisted after birth , suggesting an unrelenting effect of maternal diet on metabolism and body habitus of surviving offspring . in the development of a dietary - induced gestational diabetes mouse model , our laboratory demonstrated that consumption of a high saturated fat diet prior to conception and throughout pregnancy can result in insulin resistance and placental vascular damage and that these abnormalities could be a result of oxidative stress . frias and colleagues , using a nonhuman primate model to determine the effect of chronic high fat diet on pregnancy outcome , found that consumption of such a diet , independent of maternal obesity , led to a significant reduction in uterine blood flow , a rise in placental inflammation , and an increase in fetal risk of nonalcoholic fatty liver disease , as evidenced by increased levels of liver triglycerides and increased hepatic oxidative stress [ 101 , 102 ] . offspring of high fat diet dams also exhibited elevated hepatic expression of gluconeogenic enzymes and transcription factors . moreover , reversing the maternal high fat diet to a low fat diet during a subsequent pregnancy improved fetal hepatic triglyceride levels and partially normalized gluconeogenic enzyme expression . the authors concluded that a developing fetus is highly vulnerable to excess lipids , independent of maternal diabetes and/or obesity and that such exposure may increase the risk of pediatric fatty liver disease . human observational studies have also found high fat intakes to be related to pregnancy and birth outcome as well as long - term health of the offspring . in a case - control study of 912 women admitted to obstetric hospitals for spontaneous abortion , the risk of miscarriage was directly associated with consumption of the main types of fats , butter and oil , while a significant inverse relationship was established between the risk of spontaneous abortion and the consumption of green vegetables , fruits , milk , cheese , eggs , and fish . results of a comprehensive dietary review of women with a history of gestational diabetes found that women with recurrence of gestational diabetes in a subsequent pregnancy had significantly higher fat intakes as a percentage of total energy than women who did not have recurrence . lipids and lipid disorders play a central role in the development of the metabolic syndrome and its associated diseases . fatty streaks in the aorta have been observed in children as young as 3 years old and autopsies of young soldiers killed in the korean and vietnam wars revealed advanced coronary artery lesions [ 108 , 109 ] . similarly , the pathobiological determinants of atherosclerosis in youth ( pday ) study investigated atherosclerosis in autopsied persons 15 to 34 years old that died from various traumas and observed advanced lesions of atherosclerosis in adolescents and young adults and that serum lipoprotein concentrations were a risk factor . although micronutrient deficiencies are known causes of several well - characterized diseases , such as scurvy and rickets , the role of micronutrients in the fetal origins of adult disease has yet to be explored in detail . the section on oxidative stress provided examples on how micronutrients , as antioxidants , may be associated with various short- and long - term effects on the offspring . in relating maternal micronutrient intake with fetal glucocorticoid exposure , a mouse model of maternal dietary restriction of copper , zinc , and vitamin e demonstrated reduced activity level of placental 11-hydroxysteroid dehydrogenase-2 , an enzyme that protects the fetus from overexposure to maternal glucocorticoids . as mentioned in the section on the hypothalamic - pituitary - adrenal axis , fetal exposure to glucocorticoids is associated with small size at birth and insulin resistance and hypertension in adult life . indeed , in this study offspring exposed to the micronutrient restricted diet had significantly reduced body weight and crown - to - rump length at birth as well as increased systolic blood pressure and insulin levels post - weaning as compared to those exposed to a control diet . as discussed in the section on epigenetics , experimental studies on the impact of methyl - supplemented diets containing added folate and vitamin b-12 revealed the potential role of these micronutrients in inducing major changes in offspring phenotype , including the ability to impact the development of chronic diseases . studies on maternal dietary zinc restriction in rats indicate that zinc deficiency during intrauterine and postnatal growth can induce elevations in blood pressure and renal lesions in adulthood [ 114 , 115 ] . concerning the renal alterations identified , early exposure to zinc deficiency resulted in a decrease in glomerular filtration rate which was associated with a reduction in the number and size of nephrons . these animals also had proteinuria , higher lipid peroxidation end products , and evidence of increased renal apoptosis and fibrosis [ 114 , 115 ] . few human studies have explored the role of early micronutrient deficiencies in the development of adult metabolic disorders . of the existing reports , results are often conflicting and the significance is ambiguous . in two randomized controlled trials , both in nepal , one found that daily multiple micronutrient supplementation in pregnant women resulted in slightly lower blood pressure in the offspring at 2.5 years of age , while the other found no effects on blood pressure with maternal multiple micronutrient supplementation among 6- to 8-year - old offspring . as part of the pune ( india ) maternal nutrition study , higher maternal erythrocyte folate concentrations at 28 weeks gestation were associated with higher adiposity and insulin resistance in children 6 years of age . conversely , a study in england found that maternal folate intake at 18 or 32 weeks gestation was not associated with any measures of body composition in children at 9 years of age . clearly more work is needed to disentangle the intricate interactions among micronutrients and to determine how these interactions may be involved in the initiation and progression of chronic disease . it is now widely accepted that certain chronic diseases of adulthood may have their origins in the womb . studies discussed in this paper provide evidence that a mother 's diet during pregnancy can exert major effects on the short- and long - term health of her children including programming of the metabolic syndrome . the challenges at present are to identify common mechanisms and pathways involved in disparate perinatal malnutrition paradigms , deciphering physiological and/or pathological roles of specific nutrients , and to determine which components of the maternal diet may be best modified to optimize maternal health , placental integrity , birth outcome , and lifelong health of the offspring . the implications of this avenue of research , particularly to obstetric and preventative medicine , dictate that effective interdisciplinary communication and knowledge transfer occur and that the information generated is disseminated to the general public . a recent review provides encouraging evidence that prenatal nutritional advice appears effective in reducing the risk of preterm birth and that balanced energy and protein supplementation seem to improve fetal growth and may reduce the risk of stillbirth and infants born small for gestational age . whilst the evidence in support of the dohad is compelling , there are many caveats and controversies in the field including species differences , body weight verses body composition , and twin studies . first , species differences ( e.g. , maternal age , parity , gestation length , fetal number , and variability in fetoplacental development ) should be considered when results derived from animal studies are extrapolated to humans . rodents are particularly important to reproductive and obstetric studies because of the strong similarities between human and murine placentas [ 121123 ] ; however , there are many differences between rodents and humans particularly relating to early development . rodents are altricial species , and their organs mature after birth , which explains why postnatal factors may be more pertinent in rodent species , whereas pre- and perinatal factors play a vital role in human development . human infants of hyperglycemic mothers develop macrosomia , whereas hyperglycemic rodent dams typically have normally sized or small pups . the more precocious human cells produce fetal insulin prior to birth , thus increasing glucose transport and growth prenatally , which does not occur in the rodent , whose endocrine pancreas becomes functional postnatally [ 62 , 124 , 125 ] . second , studies relating early exposures to later life risk of disease have been largely based on body weight and/or body mass index ; however , these measurements provide no information on body composition . more recent studies have included the assessment of body composition , and evidence suggests that lower birth weights reflect increased relative adiposity , while higher birth weights reflect higher lean mass . the increased use of body composition analysis is likely to offer a more complete understanding as to why small babies are prone to the development of the metabolic syndrome , an association that likely involves increased adiposity and decreased lean mass at birth . finally , studies in twins , especially monozygotic twins , have caused considerable contention in the field [ 15 , 29 ] . the fetal origins hypothesis indicates that such individuals should have increased morbidity and mortality from metabolic and cardiovascular disease , but this has not been proven to be necessarily the case [ 15 , 29 , 126 ] . the dohad is a relatively new field of research and in time such discrepancies may be resolved . with a more complete understanding of the role of maternal health and nutrition in the initiation and progression of the metabolic syndrome and other disorders comes the hope of prevention of chronic diseases at their earliest beginnings .

chronic diseases such as type 2 diabetes and cardiovascular disease are the leading cause of death and disability worldwide . although the metabolic syndrome has been defined in various ways , the ultimate importance of recognizing this combination of disorders is that it helps identify individuals at high risk for both type 2 diabetes and cardiovascular disease . evidence from observational and experimental studies links adverse exposures in early life , particularly relating to nutrition , to chronic disease susceptibility in adulthood . such studies provide the foundation and framework for the relatively new field of developmental origins of health and disease ( dohad ) . although great strides have been made in identifying the putative concepts and mechanisms relating specific exposures in early life to the risk of developing chronic diseases in adulthood , a complete picture remains obscure . to date , the main focus of the field has been on perinatal undernutrition and specific nutrient deficiencies ; however , the current global health crisis of overweight and obesity demands that perinatal overnutrition and specific nutrient excesses be examined . this paper assembles current thoughts on the concepts and mechanisms behind the dohad as they relate to maternal nutrition , and highlights specific contributions made by macro- and micronutrients .

1. Introduction 2. Concepts and Mechanisms 3. Macronutrient Contributions 4. Micronutrient Contributions 5. Conclusions

the metabolic syndrome has become a major public health challenge with an estimated 22% of us adults having this condition . a consensus group for the international diabetes federation defines metabolic syndrome as central obesity , plus any two of the following : raised triglycerides , reduced high - density lipoprotein ( hdl ) cholesterol , raised fasting plasma glucose , and raised blood pressure . the consensus group also recommends additional criteria that should be part of further research into the metabolic syndrome including tomographic assessment of visceral adiposity and liver fat , biomarkers of adipose tissue ( leptin , adiponectin ) , and glucose tolerance testing . the cause of the syndrome remains obscure but the pathophysiology seems to be largely attributable to insulin resistance , excessive flux of fatty acids , and a chronic proinflammatory state . a mounting body of evidence indicates that certain adverse exposures during the perinatal period contribute to the development of the metabolic syndrome . this early life stage may offer an attractive point in the disease process for prevention and intervention strategies . in the 1970s forsdahl , using official statistical data on norwegian counties , reported that poverty during adolescence , followed by prosperity , was positively correlated with risk of death from coronary heart disease . in 1986 , barker and colleagues began publishing reports on the association between an adverse intrauterine environment , as determined primarily by low birth weight , and an increased risk of coronary heart disease later in life . upon further investigation , it was found that the correlation between low birth weight and heart disease was also present between low birth weight and type 2 diabetes with the prevalence of impaired glucose tolerance and type 2 diabetes falling progressively with increasing birth weight . in this study metabolic syndrome was defined as impaired glucose tolerance , hypertension , and elevated triglycerides . in both men and women , of the 64-year - old men whose birth weights were 2.95 kg ( 6.5 pounds ) or less , 22% had metabolic syndrome and their risk of developing the syndrome was more than 10 times greater than that of men whose birth weights were more than 4.31 kg ( 9.5 pounds ) [ 8 , 9 ] . the association between metabolic syndrome and low birth weight prompted the authors to suggest that the metabolic syndrome , also called syndrome x , be renamed the small - baby syndrome . collectively , these studies generated the forsdahl - barker hypothesis , recognizing forsdahl as the original source of the idea and barker as the developer of the concept . in the decades following their initial publications , the forsdahl - barker hypothesis has become more well known as the fetal origins hypothesis and has produced a new branch of scientific knowledge and inquiry known as the developmental origins of health and disease ( dohad ) . historically , investigations on the fetal origins hypothesis have focused on maternal undernutrition and specific nutrient deficiencies . it is estimated that maternal and child undernutrition is the underlying cause of 3.5 million deaths globally . while at the same time overweight and obesity are major public health challenges in both developed and developing regions of the world . in 2005 , 33% of the world 's adult population ( 1.3 billion people ) was overweight or obese , and it is estimated that by 2030 , up to 57.8% of the world 's adult population ( 3.3 billion people ) will be overweight or obese . adding to the complexity of the dual burden of malnutrition is the influence of socioeconomic status . incidentally , although the vast majority of the literature concerning the dohad is focused on the relationship between small size at birth and increased incidence of disease in adult life , it is now recognized that higher incidences of disease occur in both those born small and those born large , thus reflecting a u - shaped curve [ 15 , 16 ] . this paper provides a brief overview of the putative concepts and mechanisms behind the dohad and assembles thoughts on how alterations in maternal consumption of specific nutrients may irreversibly direct fetal programming of the metabolic syndrome ( figure 1 ) . in the first few years of life a proportion of the glands become functional depending on the temperature to which the child is exposed . in most cases programming however , the problem of mismatch occurs when individuals developmentally adapted to one environment are exposed to another . revisiting the functional activity of the sweat glands to provide an example , the child who has experienced hot conditions will be better adapted to such conditions in later life because more functioning sweat glands provide the ability to cool down faster . other examples of this mismatch phenomenon include people whose birth weights were towards the lower end of normal that subsequently grow up in affluent societies being at increased risk for hypertension , type 2 diabetes , and cardiovascular disease [ 16 , 25 ] . the problem of mismatch is thought to be involved in the current epidemic of type 2 diabetes and cardiovascular disease in the young adult and middle - aged populations of india . the current surge in metabolic and cardiovascular disease in india may be being fueled by a combination of undernutrition in early life and overnutrition in later life . in a study of indian children , those born small and where relatively fat and tall by 8 years of age had the most adverse risk profiles for cardiovascular disease , including components of the metabolic syndrome . the authors commented on the possible importance of preventing childhood obesity in the prevention of disease in the low birth weight members of the indian population . it is important to note that indians as a race are more prone to the development of the metabolic syndrome , compared to caucasians , as a result of having a phenotype of high fat mass and low lean mass . in conditions of severe intrauterine deprivation the developing fetus may lose functional and structural units such as pancreatic cells , nephrons , and cardiomyocytes . such changes have been deemed an adaptive mechanism to ensure the survival of the fetus . the thrifty phenotype hypothesis is based on a study of 468 men in which the percentage of those with impaired glucose tolerance or type 2 diabetes fell progressively with increasing birth weight and weight at 1 year . in this alternate explanation it is not hypoplasia of the pancreatic cells that leads to impaired glucose tolerance , but rather it is that the fetus develops peripheral insulin resistance . in both human and animal studies small offspring , due to maternal undernutrition and other causes , have reduced numbers of nephrons which has been strongly linked to hypertension [ 16 , 34 , 35 ] . in the human , nephrogenesis is completed in utero , and no new nephrons are formed after birth ; therefore a nephron deficit present at birth persists throughout life [ 29 , 36 ] . this rapid and excessive growth has been associated with the development of adult obesity , insulin resistance , metabolic syndrome , and type 2 diabetes [ 3840 ] . as a group , the young adults in the study who had impaired glucose tolerance or diabetes were overweight . they were not , however , overweight or obese in childhood . in addition to insulin resistance and type 2 diabetes , accelerated weight gain in early life has been associated with elevated blood pressure [ 44 , 45 ] and coronary heart disease . many believe that oxidative stress is the primary link between adverse fetal growth and later elevated risks of the metabolic syndrome , type 2 diabetes , and other disorders . proteins provide amino acids needed for antioxidant synthesis , such as glutathione and albumin , and many micronutrients themselves are antioxidants , such as vitamins a , c , and e [ 40 , 48 ] . with the susceptibility of pancreatic cells to oxidative stress , it is believed that early and ongoing exposures to oxidative insults could result in the eventual manifestations of the metabolic syndrome and related disorders . elevated oxidative stress has been observed among human infants born small for gestational age ( sga ) as compared to those appropriate for gestational age ( aga ) and among preterm as compared to term infants . a study using cord blood to compare the status of oxidative stress between sga infants born to undernourished mothers and aga infants born to healthy mothers found elevated oxidative stress , as determined by increased quantities of malondialdehyde ( one of the major products of lipid peroxidation ) , reduced quantities of the antioxidant glutathione , and decreased activity of the antioxidants superoxide dismutase and catalase in the sga infants as compared to the aga controls . gestational diabetes , a common cause of macrosomia , is associated with oxidative stress which could have effects on the developing infant . experimental studies in animals and epidemiological studies in humans have suggested an altered set - point of the hypothalamic - pituitary - adrenal ( hpa ) axis as an important long - term change that occurs in association with reduced fetal growth . raised plasma cortisol concentrations were significantly associated with higher blood pressure as well as plasma glucose concentrations and insulin resistance , suggesting that programming of the hpa axis may be a mechanism underlying the association between low birth weight and the metabolic syndrome in adult life . a series of studies , primarily in rodents , have explored the possibility that maternal nutrition during pregnancy may alter the level of energy intake in the offspring through inducing changes in the hypothalamic circuitry and in the expression , localization , and action of specific neuropeptides . appetite stimulating neuropeptides include neuropeptide y ( npy ) , agouti - related peptide ( agrp ) , and ghrelin . conversely , appetite suppressing neuropeptides include cocaine and amphetamine - related transcript ( cart ) , melanocyte - stimulating hormone ( msh ) , serotonin , insulin , and leptin [ 61 , 62 ] . of note is that the overweight and obese tend to develop resistance to insulin and leptin ; therefore these two neuropeptides are usually elevated in such groups . hypothalamic nuclei include the arcuate nucleus ( arc ) , paraventricular nucleus ( pvn ) , and ventromedial nucleus ( vmn ) which are involved in a number of biological activities with a fair degree of overlap . in general , the arc is a conduit of many diverse signals involved in various functions such as energy homeostasis , while the pvn is associated with blood pressure and stress responses and the vmn with satiety . in one of their early studies , offspring of diabetic dams had permanent hypoplasia of the vmn , decreased insulin responsiveness to glucose , impaired glucose tolerance , and increased susceptibility to diabetes . in this study , offspring of control dams cross - fostered to diabetic dams developed early postnatal growth delay and showed structural and functional hypothalamic exposure to milk from a diabetic dam resulted in an upregulation of the appetite stimulants npy and agrp and a downregulation of the appetite suppressant msh . although the researchers expected to find changes in the vmn as in their previous study , they found increased total number of neurons in the pvn which , as mentioned previously , is associated with regulation of blood pressure . histones are subject to a large number of post - translational modifications which are likely to control the structure and/or function of the chromatin fiber . every cell in the body has the same genetic information ; what makes cells , tissues , and organs different is that different sets of genes are turned on or expressed . the yellow obese syndrome encompasses the pleiotropic effects of yellow fur , obesity , hyperinsulinemia , hyperglycemia , and increased susceptibility to neoplasia . rats faced with intrauterine growth restriction had permanently reduced expression of pdx1 in cells and developed type 2 diabetes in adulthood [ 78 , 79 ] . reduced pdx1 transcription was mediated through a cascade of epigenetic modifications characterized by loss of upstream stimulatory factor-1 binding at the proximal promoter of pdx1 , recruitment of the histone deacetylase 1 and the corepressor sin3a , and deacetylation of histones h3 and h4 which culminated in the eventual silencing of pdx1 . the dutch famine of 1944 has been used by various investigators as an equivalent to an experimental study to investigate the effects of perinatal undernutrition in humans . individuals who had been exposed to famine during periconception had , 6 decades later , less dna methylation of the insulin - like growth factor ii ( igf2 ) gene compared with their unexposed , same - sex siblings . although the data is correlative and does not prove causality between dna methylation at birth and adiposity in childhood , the observation suggests that epigenetics is involved in fetal programming of later obesity . a sizable number of experimental studies , predominantly in rats , have explored the effects of protein restriction on fetal growth and later health . as discussed in the section on the thrifty phenotype hypothesis , protein restriction during pregnancy has been found to produce small offspring that have reduced numbers of nephrons which potentially contributes to the development of hypertension in adulthood [ 16 , 34 , 35 ] . their results suggest that early exposure to undernutrition programs a preference for fatty foods , and thus maternal nutrition may influence pathways involved in control of appetite or the perception of palatability in the offspring . although the literature on protein restriction and disease risk is quite extensive , it is largely based on rodent models with few having assessed the specific role of protein restriction in human risk of disease . their conclusions were that blood pressure in adulthood may be influenced by maternal intakes of protein and carbohydrates during pregnancy and that this may be mediated through effects on placental growth . in both animal and human studies , intrauterine exposure to high sugar diets and/or hyperglycemia has been found to increase the risk of the metabolic syndrome . although the significance and mechanisms are not clear , a somewhat perplexing observation was that of substantial increases in the insulin sensitivity of skeletal muscle together with higher concentrations of adiponectin in the offspring of sucrose - fed dams compared with the offspring of standard diet - fed dams . additionally , evaluation of the data based on ethnicity led to the detection of a substantial decrease in the duration of gestation among puerto rican adolescents with high sugar diets , whereas there was no effect of a high sugar diet on gestation duration among black and white adolescents . in a study of 18- to 27-year - old women born to diabetic ( gestational diabetes or type 2 diabetes ) mothers , the risk of overweight was doubled in the offspring of diabetic mothers as compared with offspring from a background population . moreover , the risk of the metabolic syndrome was increased 4-fold for offspring of mothers with gestational diabetes and 2.5-fold for offspring of mothers with type 1 diabetes . offspring risk of the metabolic syndrome increased significantly with increasing maternal fasting blood glucose as well as 2-hour blood glucose following an oral glucose load . their findings indicate that intrauterine exposure to hyperglycemia contributes to the pathogenesis of the metabolic syndrome , thus offspring of diabetic mothers are risk groups for this condition . our laboratory has performed multiple experimental studies aimed at determining the role of high fat diet in maternal - fetal health . given that oxidative stress is a potential mechanism connecting fetal exposures to the development of the metabolic syndrome , a group of pregnant mice were supplemented with quercetin , a powerful antioxidant , which mitigated the detrimental effects of high fat diet exposure . frias and colleagues , using a nonhuman primate model to determine the effect of chronic high fat diet on pregnancy outcome , found that consumption of such a diet , independent of maternal obesity , led to a significant reduction in uterine blood flow , a rise in placental inflammation , and an increase in fetal risk of nonalcoholic fatty liver disease , as evidenced by increased levels of liver triglycerides and increased hepatic oxidative stress [ 101 , 102 ] . the authors concluded that a developing fetus is highly vulnerable to excess lipids , independent of maternal diabetes and/or obesity and that such exposure may increase the risk of pediatric fatty liver disease . in a case - control study of 912 women admitted to obstetric hospitals for spontaneous abortion , the risk of miscarriage was directly associated with consumption of the main types of fats , butter and oil , while a significant inverse relationship was established between the risk of spontaneous abortion and the consumption of green vegetables , fruits , milk , cheese , eggs , and fish . lipids and lipid disorders play a central role in the development of the metabolic syndrome and its associated diseases . fatty streaks in the aorta have been observed in children as young as 3 years old and autopsies of young soldiers killed in the korean and vietnam wars revealed advanced coronary artery lesions [ 108 , 109 ] . although micronutrient deficiencies are known causes of several well - characterized diseases , such as scurvy and rickets , the role of micronutrients in the fetal origins of adult disease has yet to be explored in detail . in relating maternal micronutrient intake with fetal glucocorticoid exposure , a mouse model of maternal dietary restriction of copper , zinc , and vitamin e demonstrated reduced activity level of placental 11-hydroxysteroid dehydrogenase-2 , an enzyme that protects the fetus from overexposure to maternal glucocorticoids . as discussed in the section on epigenetics , experimental studies on the impact of methyl - supplemented diets containing added folate and vitamin b-12 revealed the potential role of these micronutrients in inducing major changes in offspring phenotype , including the ability to impact the development of chronic diseases . studies on maternal dietary zinc restriction in rats indicate that zinc deficiency during intrauterine and postnatal growth can induce elevations in blood pressure and renal lesions in adulthood [ 114 , 115 ] . as part of the pune ( india ) maternal nutrition study , higher maternal erythrocyte folate concentrations at 28 weeks gestation were associated with higher adiposity and insulin resistance in children 6 years of age . studies discussed in this paper provide evidence that a mother 's diet during pregnancy can exert major effects on the short- and long - term health of her children including programming of the metabolic syndrome . the challenges at present are to identify common mechanisms and pathways involved in disparate perinatal malnutrition paradigms , deciphering physiological and/or pathological roles of specific nutrients , and to determine which components of the maternal diet may be best modified to optimize maternal health , placental integrity , birth outcome , and lifelong health of the offspring . the implications of this avenue of research , particularly to obstetric and preventative medicine , dictate that effective interdisciplinary communication and knowledge transfer occur and that the information generated is disseminated to the general public . a recent review provides encouraging evidence that prenatal nutritional advice appears effective in reducing the risk of preterm birth and that balanced energy and protein supplementation seem to improve fetal growth and may reduce the risk of stillbirth and infants born small for gestational age . whilst the evidence in support of the dohad is compelling , there are many caveats and controversies in the field including species differences , body weight verses body composition , and twin studies . rodents are particularly important to reproductive and obstetric studies because of the strong similarities between human and murine placentas [ 121123 ] ; however , there are many differences between rodents and humans particularly relating to early development . second , studies relating early exposures to later life risk of disease have been largely based on body weight and/or body mass index ; however , these measurements provide no information on body composition . more recent studies have included the assessment of body composition , and evidence suggests that lower birth weights reflect increased relative adiposity , while higher birth weights reflect higher lean mass . the increased use of body composition analysis is likely to offer a more complete understanding as to why small babies are prone to the development of the metabolic syndrome , an association that likely involves increased adiposity and decreased lean mass at birth . the fetal origins hypothesis indicates that such individuals should have increased morbidity and mortality from metabolic and cardiovascular disease , but this has not been proven to be necessarily the case [ 15 , 29 , 126 ] . the dohad is a relatively new field of research and in time such discrepancies may be resolved . with a more complete understanding of the role of maternal health and nutrition in the initiation and progression of the metabolic syndrome and other disorders comes the hope of prevention of chronic diseases at their earliest beginnings .

several minimally invasive spine technologies are undergoing vigorous development to obtain better practical results and social benefits in the clinical treatment of a variety of spinal disorders . therefore , a greater number of spinal surgeons and medical facilities are launching minimally invasive spine technologies . minimal invasiveness is a technology , a concept , a means , and a goal . minimal invasiveness is not merely a small incision ; the efforts to reduce wound length and injury to soft tissues , like skin , is also an important component of minimally invasive spine technologies . minimally invasive tlif uses a paramedian incision that spares the spinous process and interspinous ligament and retains the corresponding blood supply compared to the traditional open tlif . the direct exposure of the facet and transverse processes through the sacrospinalis muscle causes very little stripping damage to sacrospinalis muscle , and this exposure is helpful during postoperative recovery . in addition , minimally invasive tlif directly exposes the transverse process and facet joint via an intramuscular approach , which facilitates the placement of screws along the anatomical direction of the pedicle . a review of the relevant literatures from the past 10 years showed that minimally invasive tlif has primarily used bilateral paramedian incisions during treatment . this approach produces clearer clinical efficacy , but it also has some drawbacks , including the need for bilateral incisions and the effect of surgical scars on appearance . this study further investigated the clinical surgical feasibility of mast quadrant - assisted minimally invasive modified tlif with a small single posterior median incision based on previous clinical surgeries using mast quadrant - assisted minimally invasive modified tlif with a double paramedian incision . from march 2011 to march 2012 , 34 patients with single segmental lumbar degenerative disease ( 18 males and 16 females ) for whom conservative treatment was ineffective underwent minimally invasive modified tlif in huashan hospital , fudan university . the mast quadrant - assisted operation with a small single posterior median incision was performed in the single incision group . the clinical manifestations of patients in this group included symptoms of a unilateral or bilateral radiation to a lower extremity with or without obvious low back pain , with symptoms aggravated after walking . before surgery , patients were clearly confirmed to have lumbar degenerative diseases ( e.g. , lumbar intervertebral disc herniation , lumbar spinal canal stenosis , and degenerative lumbar spondylolisthesis ) using conventional lateral and dynamic lumbar x - ray examination , computed tomography scans of the lumbar spine with two - dimensional reconstruction , and lumbar spine magnetic resonance imaging ( mri ) examination . all patients had nonobvious symptom remission or recurrent symptoms after 6 months of regular conservative treatment . patients in this group were compared to the 37 patients with single - segment degenerative lumbar disease in the double incision group who received the mast quadrant - assisted minimally invasive modified tlif with a double paramedian incision between june 2010 and february 2011 in huashan hospital , fudan university . the average age and average weight of patients in the single incision group were 56.0 13.5 years and 68.3 8.2 kg , respectively , and the average age and average weight of patients in the double incision group were 54.8 12.7 years and 70.3 7.7 kg , respectively . the t - test results of the corresponding data between these two groups showed no significant differences ( p values were 0.696 and 0.270 , respectively ) [ table 1 ] . comparison of basic clinical data of patients between the two groups sd : standard deviation . modified transforaminal lumbar interbody fusion with a small single posterior median incision a c - arm fluoroscopy machine ( siemens , germany ) was used to locate surgical segments [ figure 1a ] . a posterior median longitudinal incision of approximately 4 cm was made , using the surgical space as the midline . skin and subcutaneous tissues were sequentially cut to expose the lumbodorsal fascia until it was free 2.02.5 cm from the symptomatic side or the side with serious symptoms . a longitudinal cut approximately 3.03.5 cm in length was made in the lumbodorsal fascia , and quadrant retractors were sequentially placed to establish a working channel [ figure 1b ] . the lower articular process and lower 1/22/3 of the lamina of the upper vertebra on the affected side , ligamentum flavum , and the thicker part of the upper articular process were cut with forceps under direct vision to fully decompress the nerve root and central canal . the affected segment of the intervertebral disc was exposed and excised , the intervertebral space was cleaned , and the intervertebral space was distracted to the appropriate height . the decompressed and excised autologous bone graft was trimmed to bone particles of an appropriate size and was implanted into the anterior disc space . a capstone intervertebral fusion device ( medtronic sofamor danek , usa ) filled with autologous bone particles was implanted under direct vision [ figure 1c and 1d ] . pedicle screws and rods ( medtronic sofamor danek , usa ) were further installed for fixation . a longitudinal cut was made in lumbodorsal fascia from the posterior median incision ( 2.02.5 cm ) to the contralateral free skin and subcutaneous tissues to establish a working channel for the placement of pedicle screws and rods for fixation on the contralateral side . during the bilateral pedicle screw placement process , the direction of quadrant retractor should be adjusted in order to obtain sufficient inner inclination for the screw placement . fenestration and decompression were performed based on the clinical symptoms and radiographic spinal stenosis conditions . after flushing and hemostasis , a negative pressure drainage system was placed on the side of the interbody fusion surgery . the bilateral lumbodorsal fascia was densely sutured , and the incision was closed [ figure 1e ] . the pedicle screws and intervertebral fusion device had a good location on x - ray immediately after surgery [ figure 1f and 1 g ] , and the length of the wound was approximately 4.0 cm postoperative 3 months follow - up [ figure 1h ] . surgical procedure of modified transforaminal lumbar interbody fusion with a small single posterior median incision . ( a ) preoperative localization of the surgical segment ; ( b ) 3.03.5 cm longitudinal incision was made in the lumbodorsal fascia , and expose the lamina on the symptomatic side ; ( c and d ) remove part of lamina , decompress the canal , and conduct the interbody fusion ; ( e ) the appearance and length of the wound after closing ; ( f and g ) the anteroposterior and lateral x - ray results immediately after surgery ; ( h ) the appearance and length of the wound at postoperative 3 months follow - up . modified transforaminal lumbar interbody fusion with a double paramedian incision surgical segments were located by fluoroscopy before surgery . a longitudinal paramedian incision approximately 3.5 cm in length was made using the surgical space as the midline . decompression was performed , and the interbody fusion operation was completed on the side with lower limb symptoms or serious symptoms . after flushing and hemostasis , a negative pressure drainage system was placed on the side of interbody fusion operation , and incisions were sutured layer by layer . operation indicators : statistical analysis of the operation time , intraoperative fluoroscopy time , intraoperative blood loss , incision length , and perioperative complications of the two groups.functional evaluation indicator : the oswestry disability index ( odi ) and visual analog scale ( vas ) scores of the two groups before surgery and 3 , 12 months postoperation.sacrospinalis muscle damage evaluation indicator : three months postoperation , patients in the two groups received electrophysiological examinations of sacrospinalis muscle at the surgical segment . the meb9400-k electromyography instrument ( nihon kohden , japan ) performed surface electromyography of sacrospinalis muscle at the surgical segment . the electromyography indicators of the sacrospinalis muscles , including the average discharge amplitude ( amp , v ) and average discharge frequency ( hz ) , were measured . in addition , a 3.0 t mri ( tr = 3000 , siemens , germany ) was used to performed continuous scanning of the sacrospinalis muscle at the surgical segment . a multifidus area of 1.5 cm 1.5 cm at the level of the midline fusion device was symmetrically selected to measure the t2 relaxation time.evaluation of fusion using radiological imaging : the brantigan - steffee fusion criteria evaluated the fusion conditions of patients in the two groups . operation indicators : statistical analysis of the operation time , intraoperative fluoroscopy time , intraoperative blood loss , incision length , and perioperative complications of the two groups . functional evaluation indicator : the oswestry disability index ( odi ) and visual analog scale ( vas ) scores of the two groups before surgery and 3 , 12 months postoperation . sacrospinalis muscle damage evaluation indicator : three months postoperation , patients in the two groups received electrophysiological examinations of sacrospinalis muscle at the surgical segment . the meb9400-k electromyography instrument ( nihon kohden , japan ) performed surface electromyography of sacrospinalis muscle at the surgical segment . the electromyography indicators of the sacrospinalis muscles , including the average discharge amplitude ( amp , v ) and average discharge frequency ( hz ) , were measured . in addition , a 3.0 t mri ( tr = 3000 , siemens , germany ) was used to performed continuous scanning of the sacrospinalis muscle at the surgical segment . a multifidus area of 1.5 cm 1.5 cm at the level of the midline fusion device was symmetrically selected to measure the t2 relaxation time . evaluation of fusion using radiological imaging : the brantigan - steffee fusion criteria evaluated the fusion conditions of patients in the two groups . the t - test was performed to evaluate changes in surgical indicators , sacrospinalis muscle injury evaluation indicator , vas scores , and odi scores before and after surgery of the two groups . from march 2011 to march 2012 , 34 patients with single segmental lumbar degenerative disease ( 18 males and 16 females ) for whom conservative treatment was ineffective underwent minimally invasive modified tlif in huashan hospital , fudan university . the mast quadrant - assisted operation with a small single posterior median incision was performed in the single incision group . the clinical manifestations of patients in this group included symptoms of a unilateral or bilateral radiation to a lower extremity with or without obvious low back pain , with symptoms aggravated after walking . before surgery , patients were clearly confirmed to have lumbar degenerative diseases ( e.g. , lumbar intervertebral disc herniation , lumbar spinal canal stenosis , and degenerative lumbar spondylolisthesis ) using conventional lateral and dynamic lumbar x - ray examination , computed tomography scans of the lumbar spine with two - dimensional reconstruction , and lumbar spine magnetic resonance imaging ( mri ) examination . all patients had nonobvious symptom remission or recurrent symptoms after 6 months of regular conservative treatment . patients in this group were compared to the 37 patients with single - segment degenerative lumbar disease in the double incision group who received the mast quadrant - assisted minimally invasive modified tlif with a double paramedian incision between june 2010 and february 2011 in huashan hospital , fudan university . the average age and average weight of patients in the single incision group were 56.0 13.5 years and 68.3 8.2 kg , respectively , and the average age and average weight of patients in the double incision group were 54.8 12.7 years and 70.3 7.7 kg , respectively . the t - test results of the corresponding data between these two groups showed no significant differences ( p values were 0.696 and 0.270 , respectively ) [ table 1 ] . comparison of basic clinical data of patients between the two groups sd : standard deviation . modified transforaminal lumbar interbody fusion with a small single posterior median incision a c - arm fluoroscopy machine ( siemens , germany ) was used to locate surgical segments [ figure 1a ] . a posterior median longitudinal incision of approximately 4 cm was made , using the surgical space as the midline . skin and subcutaneous tissues were sequentially cut to expose the lumbodorsal fascia until it was free 2.02.5 cm from the symptomatic side or the side with serious symptoms . a longitudinal cut approximately 3.03.5 cm in length was made in the lumbodorsal fascia , and quadrant retractors were sequentially placed to establish a working channel [ figure 1b ] . the lower articular process and lower 1/22/3 of the lamina of the upper vertebra on the affected side , ligamentum flavum , and the thicker part of the upper articular process were cut with forceps under direct vision to fully decompress the nerve root and central canal . the affected segment of the intervertebral disc was exposed and excised , the intervertebral space was cleaned , and the intervertebral space was distracted to the appropriate height . the decompressed and excised autologous bone graft was trimmed to bone particles of an appropriate size and was implanted into the anterior disc space . a capstone intervertebral fusion device ( medtronic sofamor danek , usa ) filled with autologous bone particles was implanted under direct vision [ figure 1c and 1d ] . pedicle screws and rods ( medtronic sofamor danek , usa ) were further installed for fixation . a longitudinal cut was made in lumbodorsal fascia from the posterior median incision ( 2.02.5 cm ) to the contralateral free skin and subcutaneous tissues to establish a working channel for the placement of pedicle screws and rods for fixation on the contralateral side . during the bilateral pedicle screw placement process , the direction of quadrant retractor should be adjusted in order to obtain sufficient inner inclination for the screw placement . fenestration and decompression were performed based on the clinical symptoms and radiographic spinal stenosis conditions . after flushing and hemostasis , a negative pressure drainage system was placed on the side of the interbody fusion surgery . the bilateral lumbodorsal fascia was densely sutured , and the incision was closed [ figure 1e ] . the pedicle screws and intervertebral fusion device had a good location on x - ray immediately after surgery [ figure 1f and 1 g ] , and the length of the wound was approximately 4.0 cm postoperative 3 months follow - up [ figure 1h ] . surgical procedure of modified transforaminal lumbar interbody fusion with a small single posterior median incision . ( a ) preoperative localization of the surgical segment ; ( b ) 3.03.5 cm longitudinal incision was made in the lumbodorsal fascia , and expose the lamina on the symptomatic side ; ( c and d ) remove part of lamina , decompress the canal , and conduct the interbody fusion ; ( e ) the appearance and length of the wound after closing ; ( f and g ) the anteroposterior and lateral x - ray results immediately after surgery ; ( h ) the appearance and length of the wound at postoperative 3 months follow - up . modified transforaminal lumbar interbody fusion with a double paramedian incision surgical segments were located by fluoroscopy before surgery . a 3.03.5 cm incision was made from the posterior midline to both sides . a longitudinal paramedian incision approximately 3.5 cm in length was made using the surgical space as the midline . decompression was performed , and the interbody fusion operation was completed on the side with lower limb symptoms or serious symptoms . bilateral placement of pedicle screws and rods was performed for fixation . after flushing and hemostasis , a negative pressure drainage system was placed on the side of interbody fusion operation , and incisions were sutured layer by layer . operation indicators : statistical analysis of the operation time , intraoperative fluoroscopy time , intraoperative blood loss , incision length , and perioperative complications of the two groups.functional evaluation indicator : the oswestry disability index ( odi ) and visual analog scale ( vas ) scores of the two groups before surgery and 3 , 12 months postoperation.sacrospinalis muscle damage evaluation indicator : three months postoperation , patients in the two groups received electrophysiological examinations of sacrospinalis muscle at the surgical segment . the meb9400-k electromyography instrument ( nihon kohden , japan ) performed surface electromyography of sacrospinalis muscle at the surgical segment . the electromyography indicators of the sacrospinalis muscles , including the average discharge amplitude ( amp , v ) and average discharge frequency ( hz ) , were measured . in addition , a 3.0 t mri ( tr = 3000 , siemens , germany ) was used to performed continuous scanning of the sacrospinalis muscle at the surgical segment . a multifidus area of 1.5 cm 1.5 cm at the level of the midline fusion device was symmetrically selected to measure the t2 relaxation time.evaluation of fusion using radiological imaging : the brantigan - steffee fusion criteria evaluated the fusion conditions of patients in the two groups . operation indicators : statistical analysis of the operation time , intraoperative fluoroscopy time , intraoperative blood loss , incision length , and perioperative complications of the two groups . functional evaluation indicator : the oswestry disability index ( odi ) and visual analog scale ( vas ) scores of the two groups before surgery and 3 , 12 months postoperation . sacrospinalis muscle damage evaluation indicator : three months postoperation , patients in the two groups received electrophysiological examinations of sacrospinalis muscle at the surgical segment . the meb9400-k electromyography instrument ( nihon kohden , japan ) performed surface electromyography of sacrospinalis muscle at the surgical segment . the electromyography indicators of the sacrospinalis muscles , including the average discharge amplitude ( amp , v ) and average discharge frequency ( hz ) , were measured . in addition , a 3.0 t mri ( tr = 3000 , siemens , germany ) was used to performed continuous scanning of the sacrospinalis muscle at the surgical segment . a multifidus area of 1.5 cm 1.5 cm at the level of the midline fusion device was symmetrically selected to measure the t2 relaxation time . evaluation of fusion using radiological imaging : the brantigan - steffee fusion criteria evaluated the fusion conditions of patients in the two groups . all data were analyzed using spss 17.0 ( spss inc . , chicago , il , usa ) . the t - test was performed to evaluate changes in surgical indicators , sacrospinalis muscle injury evaluation indicator , vas scores , and odi scores before and after surgery of the two groups . a total of 31 and 35 cases in the single incision and double incision groups , respectively , completed at least 12 months of systemic follow - up . the average operation times of the two groups were 149.2 28.2 min and 155.7 28.6 min , respectively . the average intraoperative fluoroscopy time of the two groups was 81.3 11.5 s and 86.3 11.7 s , respectively . the comparison of intraoperative blood loss and postoperative drainage between the two groups showed no significant differences , with p values of 0.909 and 0.343 , respectively . the total surgical incision lengths in the two groups were 4.4 0.7 cm and 7.5 0.4 cm , respectively , which were significantly different ( p = 0.000 ) . for the perioperative complications , one case in the single incision group had a postoperative superficial infection at the incision site , which recovered after enhanced dressing , and one case developed pulmonary infection , which recovered after antibiotic treatment . in the double incision group , two cases had pulmonary infections , and one case had fat liquefaction at the wound , which recovered after treatment [ table 2 ] . the odi and vas scores of patients in the two groups before surgery and at 3 , 12 months postoperation all significantly improved ( all p < 0.05 ) . the odi and vas scores at 3 and 12 months postoperation did not show significant differences between the two groups ( p 0.05 ) . the amp ( v ) , average discharge frequency of sacrospinalis muscle , and mr t2 relaxation time between groups were not significantly different ( p 0.05 ) [ table 3 ] . comparison of clinical function scores and sacrospinalis muscle damage between the two groups ( mean sd ) sd : standard deviation ; vas : visual analog scale ; odi : oswestry disability index ; mr : magnetic resonance . the radiological results of 12 months postoperation showed that there was no grade a and b of the brantigan - steffee fusion criteria in both groups ; two cases and three cases of grade c were in the single incision group and double incision group , respectively . the fusion rate of the two groups was 93.5% , 91.4% ( grade d and e cases ) , respectively ; there were no significant differences between the two groups ( p 0.05 ) . minimally invasive spine surgery ( miss ) is the combination of traditional spinal surgery with minimally invasive technology to ensure the effectiveness of spinal surgery . this technology tries to minimize the interference of surgery on the injury and physiological function of surrounding tissues to achieve smaller incisions , less tissue damage , lighter systemic reactions , faster recovery cycles , and better psychological effects . due to the disease characteristics and the treatment requirements of clinical diagnosis and treatment of spinal surgery , spinal surgery has as its goal the preservation of the following tissues and structures : nervous tissues , such as the spinal cord and nerve root , the inherent bony structure of the spine , the muscle fascia surrounding the spine , and the local skin surrounding the surgery site . therefore , the basic principle of miss can be summarized as follows : on the bases of the safe and effective decompression of nervous tissues , such as the spinal cord and nerve root , the destruction of inherent bony and ligamentous structures of the spine is minimized as much as possible , the damage of muscle and fascia tissues around the surgical area is reduced , and the length of the skin incision is reduced . in other words , from the earliest total laminectomy for decompression to unilateral lamina fenestration decompression , the destruction of bony and ligamentous structures of the lumbar spine is effectively decreased , which allows minimal invasiveness in the development of lumbar intervertebral disc herniation treatment . the use of the wiltse paraspinal approach to reduce sacrospinalis muscle damage is minimally invasive , and the transition from longitudinal incision to transverse incision in the anterior cervical spine to effectively minimize skin incision is also minimally invasive . however , it must be emphasized that the essence of minimal invasiveness is to require less surgical trauma to reach the equivalent or better efficacy than traditional open surgery . if miss only obtains a small incision in appearance using an advanced endoscope , percutaneous surgical technique , or other new high technologies but does not perform effective decompression for important tissues , such as spinal cord and nerve root , or it does not obtain better results than the traditional open surgery due to insufficient decompression or improper surgical operation , then this so - called minimal invasiveness is meaningless . a large number of the past studies have shown that minimally invasive tlif was better or equal to the traditional open tlif in clinical efficacy , and most comparative studies have shown that minimally invasive tlif did not significantly increase the operative - related complications . in addition , most of the clinical studies have confirmed that minimally invasive lumbar posterior surgery could significantly reduce the paraspinal muscles injuries compared with the traditional open approaches . on the bases of clinical performance of the mast quadrant - assisted minimally invasive modified tlif with a double paramedian incision , this study further investigated the clinical surgical feasibility of the minimally invasive modified tlif with a small single posterior median incision . this study found that the clinical efficacy of this minimally invasive modified tlif with a small single posterior median incision was the same as that of the previous double paramedian incision . this technology reduced the number of surgical incisions , which may have reduced the overall incidence of wound complications . although this modified clinical operation did not significantly increase clinical efficacy , it effectively shortened the length of surgical incisions and presented an excellent minimally invasive spinal operation concept because this technique allowed the safe and effective decompression of nerve structures such as nerve roots ( i.e. , the clinical efficacy and perioperative complications between the two groups were not significantly different ) and minimized the destruction and damage of inherent bony and ligamentous structures of the spine and muscle fascia tissues ( i.e. , the excised bony structure was the same , and the exposure and surgery were conducted through a paraspinal approach in which the muscle tissue injury was not increased ) . this surgical approach significantly shortened the skin incision length and minimized the potential effect of skin scarring on local appearance and the requirement of a second surgery . it should be noted that mast quadrant - assisted modified tlif with a small single posterior median incision may have some potential shortcomings , such as subcutaneous free might affect the blood supply of the skin , and increase in incision - related complications . however , the total surgical incision length of this technique was just 4.4 0.7 cm , without extensive subcutaneous free . in theory , the influence of the blood supply was relatively small . moreover , in this study , the patients in the single incision group did not have any wound - related complications . in addition , in order to obtain sufficient inner inclination for the screw placement , it is necessary to adjust the direction of quadrant retractors repeatedly . in conclusion , through this clinical comparison study , we considered the mast quadrant - assisted minimally invasive modified tlif with a small single posterior median incision to have excellent clinical operation feasibility compared to the minimally invasive tlif with a double paramedian incision . the minimally invasive modified tlif with a small single posterior median incision significantly shortened surgical incisions and exhibited similar clinical efficacy . this approach is an excellent concept for minimally invasive surgery , and patients are more willing to accept this technique .

background : the concept of minimally invasive techniques is to make every effort to reduce tissue damage . certainly , reducing skin incision is an important part of these techniques . this study aimed to investigate the clinical feasibility of mast quadrant - assisted modified transforaminal lumbar interbody fusion ( tlif ) with a small single posterior median incision.methods:during the period of march 2011 to march 2012 , 34 patients with single - segment degenerative lumbar disease underwent the minimally invasive modified tlif assisted by mast quadrant with a small single posterior median incision ( single incision group ) . the cases in this group were compared to 37 patients with single - segment degenerative lumbar disease in the double incision group . the perioperative conditions of patients in these two groups were statistically analyzed and compared . the oswestry disability index ( odi ) scores , visual analog scale ( vas ) scores , and sacrospinalis muscle damage evaluation indicators before operation and 3 , 12 months postoperation were compared.results:a total of 31 and 35 cases in the single incision and double incision groups , respectively , completed at least 12 months of systemic follow - up . the differences in perioperative conditions between the two groups were not statistically significant . the incision length of the single incision group was significantly shorter than that of the double incision group ( p < 0.01 ) . the odi and vas scores of patients in both groups improved significantly at 3 and 12 months postoperation . however , these two indicators at 3 and 12 months postoperation and the sacrospinalis muscle damage evaluation indicators at 3 months postoperation did not differ significantly between the two groups ( p 0.05).conclusions : mast quadrant - assisted modified tlif with a small single posterior median incision has excellent clinical feasibility compared to minimally invasive tlif with a double paramedian incision .

I M Patient data Surgical procedure Comparison of indicators Statistical analysis R D

several minimally invasive spine technologies are undergoing vigorous development to obtain better practical results and social benefits in the clinical treatment of a variety of spinal disorders . minimal invasiveness is not merely a small incision ; the efforts to reduce wound length and injury to soft tissues , like skin , is also an important component of minimally invasive spine technologies . minimally invasive tlif uses a paramedian incision that spares the spinous process and interspinous ligament and retains the corresponding blood supply compared to the traditional open tlif . the direct exposure of the facet and transverse processes through the sacrospinalis muscle causes very little stripping damage to sacrospinalis muscle , and this exposure is helpful during postoperative recovery . in addition , minimally invasive tlif directly exposes the transverse process and facet joint via an intramuscular approach , which facilitates the placement of screws along the anatomical direction of the pedicle . a review of the relevant literatures from the past 10 years showed that minimally invasive tlif has primarily used bilateral paramedian incisions during treatment . this study further investigated the clinical surgical feasibility of mast quadrant - assisted minimally invasive modified tlif with a small single posterior median incision based on previous clinical surgeries using mast quadrant - assisted minimally invasive modified tlif with a double paramedian incision . from march 2011 to march 2012 , 34 patients with single segmental lumbar degenerative disease ( 18 males and 16 females ) for whom conservative treatment was ineffective underwent minimally invasive modified tlif in huashan hospital , fudan university . the mast quadrant - assisted operation with a small single posterior median incision was performed in the single incision group . the clinical manifestations of patients in this group included symptoms of a unilateral or bilateral radiation to a lower extremity with or without obvious low back pain , with symptoms aggravated after walking . , lumbar intervertebral disc herniation , lumbar spinal canal stenosis , and degenerative lumbar spondylolisthesis ) using conventional lateral and dynamic lumbar x - ray examination , computed tomography scans of the lumbar spine with two - dimensional reconstruction , and lumbar spine magnetic resonance imaging ( mri ) examination . patients in this group were compared to the 37 patients with single - segment degenerative lumbar disease in the double incision group who received the mast quadrant - assisted minimally invasive modified tlif with a double paramedian incision between june 2010 and february 2011 in huashan hospital , fudan university . the average age and average weight of patients in the single incision group were 56.0 13.5 years and 68.3 8.2 kg , respectively , and the average age and average weight of patients in the double incision group were 54.8 12.7 years and 70.3 7.7 kg , respectively . the t - test results of the corresponding data between these two groups showed no significant differences ( p values were 0.696 and 0.270 , respectively ) [ table 1 ] . comparison of basic clinical data of patients between the two groups sd : standard deviation . modified transforaminal lumbar interbody fusion with a small single posterior median incision a c - arm fluoroscopy machine ( siemens , germany ) was used to locate surgical segments [ figure 1a ] . the lower articular process and lower 1/22/3 of the lamina of the upper vertebra on the affected side , ligamentum flavum , and the thicker part of the upper articular process were cut with forceps under direct vision to fully decompress the nerve root and central canal . the affected segment of the intervertebral disc was exposed and excised , the intervertebral space was cleaned , and the intervertebral space was distracted to the appropriate height . a longitudinal cut was made in lumbodorsal fascia from the posterior median incision ( 2.02.5 cm ) to the contralateral free skin and subcutaneous tissues to establish a working channel for the placement of pedicle screws and rods for fixation on the contralateral side . the pedicle screws and intervertebral fusion device had a good location on x - ray immediately after surgery [ figure 1f and 1 g ] , and the length of the wound was approximately 4.0 cm postoperative 3 months follow - up [ figure 1h ] . surgical procedure of modified transforaminal lumbar interbody fusion with a small single posterior median incision . ( a ) preoperative localization of the surgical segment ; ( b ) 3.03.5 cm longitudinal incision was made in the lumbodorsal fascia , and expose the lamina on the symptomatic side ; ( c and d ) remove part of lamina , decompress the canal , and conduct the interbody fusion ; ( e ) the appearance and length of the wound after closing ; ( f and g ) the anteroposterior and lateral x - ray results immediately after surgery ; ( h ) the appearance and length of the wound at postoperative 3 months follow - up . modified transforaminal lumbar interbody fusion with a double paramedian incision surgical segments were located by fluoroscopy before surgery . decompression was performed , and the interbody fusion operation was completed on the side with lower limb symptoms or serious symptoms . operation indicators : statistical analysis of the operation time , intraoperative fluoroscopy time , intraoperative blood loss , incision length , and perioperative complications of the two groups.functional evaluation indicator : the oswestry disability index ( odi ) and visual analog scale ( vas ) scores of the two groups before surgery and 3 , 12 months postoperation.sacrospinalis muscle damage evaluation indicator : three months postoperation , patients in the two groups received electrophysiological examinations of sacrospinalis muscle at the surgical segment . in addition , a 3.0 t mri ( tr = 3000 , siemens , germany ) was used to performed continuous scanning of the sacrospinalis muscle at the surgical segment . a multifidus area of 1.5 cm 1.5 cm at the level of the midline fusion device was symmetrically selected to measure the t2 relaxation time.evaluation of fusion using radiological imaging : the brantigan - steffee fusion criteria evaluated the fusion conditions of patients in the two groups . operation indicators : statistical analysis of the operation time , intraoperative fluoroscopy time , intraoperative blood loss , incision length , and perioperative complications of the two groups . functional evaluation indicator : the oswestry disability index ( odi ) and visual analog scale ( vas ) scores of the two groups before surgery and 3 , 12 months postoperation . sacrospinalis muscle damage evaluation indicator : three months postoperation , patients in the two groups received electrophysiological examinations of sacrospinalis muscle at the surgical segment . in addition , a 3.0 t mri ( tr = 3000 , siemens , germany ) was used to performed continuous scanning of the sacrospinalis muscle at the surgical segment . evaluation of fusion using radiological imaging : the brantigan - steffee fusion criteria evaluated the fusion conditions of patients in the two groups . the t - test was performed to evaluate changes in surgical indicators , sacrospinalis muscle injury evaluation indicator , vas scores , and odi scores before and after surgery of the two groups . from march 2011 to march 2012 , 34 patients with single segmental lumbar degenerative disease ( 18 males and 16 females ) for whom conservative treatment was ineffective underwent minimally invasive modified tlif in huashan hospital , fudan university . the mast quadrant - assisted operation with a small single posterior median incision was performed in the single incision group . the clinical manifestations of patients in this group included symptoms of a unilateral or bilateral radiation to a lower extremity with or without obvious low back pain , with symptoms aggravated after walking . , lumbar intervertebral disc herniation , lumbar spinal canal stenosis , and degenerative lumbar spondylolisthesis ) using conventional lateral and dynamic lumbar x - ray examination , computed tomography scans of the lumbar spine with two - dimensional reconstruction , and lumbar spine magnetic resonance imaging ( mri ) examination . patients in this group were compared to the 37 patients with single - segment degenerative lumbar disease in the double incision group who received the mast quadrant - assisted minimally invasive modified tlif with a double paramedian incision between june 2010 and february 2011 in huashan hospital , fudan university . the average age and average weight of patients in the single incision group were 56.0 13.5 years and 68.3 8.2 kg , respectively , and the average age and average weight of patients in the double incision group were 54.8 12.7 years and 70.3 7.7 kg , respectively . the t - test results of the corresponding data between these two groups showed no significant differences ( p values were 0.696 and 0.270 , respectively ) [ table 1 ] . comparison of basic clinical data of patients between the two groups sd : standard deviation . modified transforaminal lumbar interbody fusion with a small single posterior median incision a c - arm fluoroscopy machine ( siemens , germany ) was used to locate surgical segments [ figure 1a ] . the lower articular process and lower 1/22/3 of the lamina of the upper vertebra on the affected side , ligamentum flavum , and the thicker part of the upper articular process were cut with forceps under direct vision to fully decompress the nerve root and central canal . the affected segment of the intervertebral disc was exposed and excised , the intervertebral space was cleaned , and the intervertebral space was distracted to the appropriate height . a longitudinal cut was made in lumbodorsal fascia from the posterior median incision ( 2.02.5 cm ) to the contralateral free skin and subcutaneous tissues to establish a working channel for the placement of pedicle screws and rods for fixation on the contralateral side . the bilateral lumbodorsal fascia was densely sutured , and the incision was closed [ figure 1e ] . the pedicle screws and intervertebral fusion device had a good location on x - ray immediately after surgery [ figure 1f and 1 g ] , and the length of the wound was approximately 4.0 cm postoperative 3 months follow - up [ figure 1h ] . surgical procedure of modified transforaminal lumbar interbody fusion with a small single posterior median incision . ( a ) preoperative localization of the surgical segment ; ( b ) 3.03.5 cm longitudinal incision was made in the lumbodorsal fascia , and expose the lamina on the symptomatic side ; ( c and d ) remove part of lamina , decompress the canal , and conduct the interbody fusion ; ( e ) the appearance and length of the wound after closing ; ( f and g ) the anteroposterior and lateral x - ray results immediately after surgery ; ( h ) the appearance and length of the wound at postoperative 3 months follow - up . modified transforaminal lumbar interbody fusion with a double paramedian incision surgical segments were located by fluoroscopy before surgery . decompression was performed , and the interbody fusion operation was completed on the side with lower limb symptoms or serious symptoms . operation indicators : statistical analysis of the operation time , intraoperative fluoroscopy time , intraoperative blood loss , incision length , and perioperative complications of the two groups.functional evaluation indicator : the oswestry disability index ( odi ) and visual analog scale ( vas ) scores of the two groups before surgery and 3 , 12 months postoperation.sacrospinalis muscle damage evaluation indicator : three months postoperation , patients in the two groups received electrophysiological examinations of sacrospinalis muscle at the surgical segment . in addition , a 3.0 t mri ( tr = 3000 , siemens , germany ) was used to performed continuous scanning of the sacrospinalis muscle at the surgical segment . a multifidus area of 1.5 cm 1.5 cm at the level of the midline fusion device was symmetrically selected to measure the t2 relaxation time.evaluation of fusion using radiological imaging : the brantigan - steffee fusion criteria evaluated the fusion conditions of patients in the two groups . operation indicators : statistical analysis of the operation time , intraoperative fluoroscopy time , intraoperative blood loss , incision length , and perioperative complications of the two groups . functional evaluation indicator : the oswestry disability index ( odi ) and visual analog scale ( vas ) scores of the two groups before surgery and 3 , 12 months postoperation . sacrospinalis muscle damage evaluation indicator : three months postoperation , patients in the two groups received electrophysiological examinations of sacrospinalis muscle at the surgical segment . in addition , a 3.0 t mri ( tr = 3000 , siemens , germany ) was used to performed continuous scanning of the sacrospinalis muscle at the surgical segment . evaluation of fusion using radiological imaging : the brantigan - steffee fusion criteria evaluated the fusion conditions of patients in the two groups . the t - test was performed to evaluate changes in surgical indicators , sacrospinalis muscle injury evaluation indicator , vas scores , and odi scores before and after surgery of the two groups . a total of 31 and 35 cases in the single incision and double incision groups , respectively , completed at least 12 months of systemic follow - up . the average operation times of the two groups were 149.2 28.2 min and 155.7 28.6 min , respectively . the average intraoperative fluoroscopy time of the two groups was 81.3 11.5 s and 86.3 11.7 s , respectively . the comparison of intraoperative blood loss and postoperative drainage between the two groups showed no significant differences , with p values of 0.909 and 0.343 , respectively . the total surgical incision lengths in the two groups were 4.4 0.7 cm and 7.5 0.4 cm , respectively , which were significantly different ( p = 0.000 ) . for the perioperative complications , one case in the single incision group had a postoperative superficial infection at the incision site , which recovered after enhanced dressing , and one case developed pulmonary infection , which recovered after antibiotic treatment . in the double incision group , two cases had pulmonary infections , and one case had fat liquefaction at the wound , which recovered after treatment [ table 2 ] . the odi and vas scores of patients in the two groups before surgery and at 3 , 12 months postoperation all significantly improved ( all p < 0.05 ) . the odi and vas scores at 3 and 12 months postoperation did not show significant differences between the two groups ( p 0.05 ) . the amp ( v ) , average discharge frequency of sacrospinalis muscle , and mr t2 relaxation time between groups were not significantly different ( p 0.05 ) [ table 3 ] . comparison of clinical function scores and sacrospinalis muscle damage between the two groups ( mean sd ) sd : standard deviation ; vas : visual analog scale ; odi : oswestry disability index ; mr : magnetic resonance . the radiological results of 12 months postoperation showed that there was no grade a and b of the brantigan - steffee fusion criteria in both groups ; two cases and three cases of grade c were in the single incision group and double incision group , respectively . the fusion rate of the two groups was 93.5% , 91.4% ( grade d and e cases ) , respectively ; there were no significant differences between the two groups ( p 0.05 ) . due to the disease characteristics and the treatment requirements of clinical diagnosis and treatment of spinal surgery , spinal surgery has as its goal the preservation of the following tissues and structures : nervous tissues , such as the spinal cord and nerve root , the inherent bony structure of the spine , the muscle fascia surrounding the spine , and the local skin surrounding the surgery site . therefore , the basic principle of miss can be summarized as follows : on the bases of the safe and effective decompression of nervous tissues , such as the spinal cord and nerve root , the destruction of inherent bony and ligamentous structures of the spine is minimized as much as possible , the damage of muscle and fascia tissues around the surgical area is reduced , and the length of the skin incision is reduced . the use of the wiltse paraspinal approach to reduce sacrospinalis muscle damage is minimally invasive , and the transition from longitudinal incision to transverse incision in the anterior cervical spine to effectively minimize skin incision is also minimally invasive . a large number of the past studies have shown that minimally invasive tlif was better or equal to the traditional open tlif in clinical efficacy , and most comparative studies have shown that minimally invasive tlif did not significantly increase the operative - related complications . in addition , most of the clinical studies have confirmed that minimally invasive lumbar posterior surgery could significantly reduce the paraspinal muscles injuries compared with the traditional open approaches . on the bases of clinical performance of the mast quadrant - assisted minimally invasive modified tlif with a double paramedian incision , this study further investigated the clinical surgical feasibility of the minimally invasive modified tlif with a small single posterior median incision . this study found that the clinical efficacy of this minimally invasive modified tlif with a small single posterior median incision was the same as that of the previous double paramedian incision . although this modified clinical operation did not significantly increase clinical efficacy , it effectively shortened the length of surgical incisions and presented an excellent minimally invasive spinal operation concept because this technique allowed the safe and effective decompression of nerve structures such as nerve roots ( i.e. , the clinical efficacy and perioperative complications between the two groups were not significantly different ) and minimized the destruction and damage of inherent bony and ligamentous structures of the spine and muscle fascia tissues ( i.e. this surgical approach significantly shortened the skin incision length and minimized the potential effect of skin scarring on local appearance and the requirement of a second surgery . it should be noted that mast quadrant - assisted modified tlif with a small single posterior median incision may have some potential shortcomings , such as subcutaneous free might affect the blood supply of the skin , and increase in incision - related complications . however , the total surgical incision length of this technique was just 4.4 0.7 cm , without extensive subcutaneous free . moreover , in this study , the patients in the single incision group did not have any wound - related complications . in conclusion , through this clinical comparison study , we considered the mast quadrant - assisted minimally invasive modified tlif with a small single posterior median incision to have excellent clinical operation feasibility compared to the minimally invasive tlif with a double paramedian incision . the minimally invasive modified tlif with a small single posterior median incision significantly shortened surgical incisions and exhibited similar clinical efficacy . this approach is an excellent concept for minimally invasive surgery , and patients are more willing to accept this technique .

gastrointestinal ( gi ) problems in critically ill patients are common and associated with impaired outcome [ 14 ] . the hypothesis of the gut as a motor of multiple organ failure ( mof ) has repeatedly been proposed in the past [ 5 , 6 ] . despite this , the pathophysiological role of gi dysfunction in the clinical course of mof has not been sufficiently investigated . in a recent consensus statement , the working group on abdominal problems ( wgap ) of the european society of intensive care medicine proposed a terminology aiming to provide clinical definitions , although evidence - based criteria for these definitions were limited . the sequential organ failure assessment ( sofa ) score , widely used to assess organ dysfunction in critically ill patients , does not take gi dysfunction into account . a previous single - centre study demonstrated that the addition of a gif score based on the combination of feeding intolerance ( fi ) and intra - abdominal hypertension ( iah ) to the original sofa score improved the predictive power of the latter . the primary aim of this multicentre study was to develop a gi dysfunction score predicting 28-day mortality , among adult mechanically ventilated patients . a secondary aim was to study the possible additive value of gi dysfunction score to sofa score on outcome prediction . thus , the hypothesis tested was that symptoms of gi dysfunction could be used as predictors of outcome separately and/or as part of the sofa score . in this prospective , observational , multicentre study , 40 icus around the world participated . study units were asked to include consecutive adult patients ( 18 years and older ) with expected duration of mv of at least 6 h. patients who were spontaneously breathing on admission day were not included , even if they required mv later during their icu stay . patients in whom transvesical intra - abdominal pressure ( iap ) measurements were not possible for any reason , such as previous cystectomy , were excluded . the inclusion period ranged from two to four weeks in the different sites between october and december 2009 . informed consent was obtained from next of kin or waived ( due to the observational design ) according to local ethical rules . the study protocol was endorsed by the clinical trials working group of the world society of abdominal compartment syndrome ( wsacs trial number 013 , www.wsacs.org ) as well as by the wgap and eccrn of the esicm . power analysis based on earlier single - centre study indicated that 343 patients should be analyzed to detect a 5 % increase in the predictive capability between sofa and gif score ( based on the auc of the roc curve of the sofa score of 0.840 ( sd 0.25 ) ) . however , as the gif score for current study was not predefined , but had to be developed during the study , we aimed to enroll 500 patients . demographic and base - line clinical data ( clinical profile , previous surgery , presence / absence of sepsis , apache ii score and blood lactate concentration ) were collected on the day of icu admission . predefined gi symptoms , iap ( minimum , maximum and mean daily values ) , feeding details , sofa score with all its sub - scores , urine output , fluid balance , positive end - expiratory pressure , as well as serum albumin and c - reactive protein levels were documented on days 1 , 2 , 4 and 7 in the icu . caloric needs were calculated as 20 kcal / kg / day for day one and as 25 kcal / kg / day the following study days . the following definitions were used for uniform data collection : patient category : medical = no surgery within 4 weeks preceding icu admission ; elective surgical = surgery within 4 weeks preceding admission , scheduled > 24 h in advance ; emergency surgical = surgery within 4 weeks preceding admission , scheduled within 24 h of operation . gi symptoms were defined as follows : high gastric residual volumes ( grv ) = maximum grv above 500 ml at least once . absent bowel sounds ( bs ) = bs were not heard on careful auscultation . vomiting / regurgitation = visible vomiting or regurgitation in any amount . bowel distension = suspected or radiologically confirmed bowel dilatation in any bowel segment . gi bleeding = visible appearance of blood in vomits , nasogastric aspirate , or stool . feeding intolerance ( fi ) was considered present when less than 20 % of the calculated caloric needs were administered with enteral nutrition ( en ) and at the same time gi symptom(s ) were documented being a reason for withholding or reducing the en . intra - abdominal hypertension ( iah ) = mean iap of the day 12 mmhg and abdominal compartment syndrome ( acs ) = mean iap > 20 mmhg with new organ dysfunction or failure , with iap measured in the supine position with zero - point at mid - axillary line with a maximal instillation volume of 25 ml . statistical package for the social sciences ( ibm spss statistics 20.0 , somers , ny , usa ) software was used for statistical analysis . smirnov test with lilliefors correction was used to test normality of distribution . to compare groups , student s t - test ( normal distribution ) and mann whitney u test ( non - gaussian distribution ) univariate analyses of admission parameters were applied to identify the risk factors for 28-day mortality . parameters with p < 0.2 in univariate analysis were entered stepwise into a multiple logistic regression model to identify the best combination for prediction of 28-day mortality . meier curves and log - rank tests were used to compare survival of patients with and without gi symptoms . gi symptoms were entered separately into a regression model predicting mortality to evaluate the importance of individual gi symptoms . receiver operating characteristic ( roc ) curves were used to determine the likelihood ratios of different versions of possible gastrointestinal failure ( gif ) scores , the sofa score and the sofa with gif scores combined to predict the icu mortality . the optimal cut - off value was calculated from the roc curve analysis as the point with the greatest combined sensitivity and specificity . in this prospective , observational , multicentre study , 40 icus around the world participated . study units were asked to include consecutive adult patients ( 18 years and older ) with expected duration of mv of at least 6 h. patients who were spontaneously breathing on admission day were not included , even if they required mv later during their icu stay . patients in whom transvesical intra - abdominal pressure ( iap ) measurements were not possible for any reason , such as previous cystectomy , were excluded . the inclusion period ranged from two to four weeks in the different sites between october and december 2009 . informed consent was obtained from next of kin or waived ( due to the observational design ) according to local ethical rules . the study protocol was endorsed by the clinical trials working group of the world society of abdominal compartment syndrome ( wsacs trial number 013 , www.wsacs.org ) as well as by the wgap and eccrn of the esicm . power analysis based on earlier single - centre study indicated that 343 patients should be analyzed to detect a 5 % increase in the predictive capability between sofa and gif score ( based on the auc of the roc curve of the sofa score of 0.840 ( sd 0.25 ) ) . however , as the gif score for current study was not predefined , but had to be developed during the study , we aimed to enroll 500 patients . demographic and base - line clinical data ( clinical profile , previous surgery , presence / absence of sepsis , apache ii score and blood lactate concentration ) were collected on the day of icu admission . predefined gi symptoms , iap ( minimum , maximum and mean daily values ) , feeding details , sofa score with all its sub - scores , urine output , fluid balance , positive end - expiratory pressure , as well as serum albumin and c - reactive protein levels were documented on days 1 , 2 , 4 and 7 in the icu . caloric needs were calculated as 20 kcal / kg / day for day one and as 25 kcal / kg / day the following study days . the following definitions were used for uniform data collection : patient category : medical = no surgery within 4 weeks preceding icu admission ; elective surgical = surgery within 4 weeks preceding admission , scheduled > 24 h in advance ; emergency surgical = surgery within 4 weeks preceding admission , scheduled within 24 h of operation . gi symptoms were defined as follows : high gastric residual volumes ( grv ) = maximum grv above 500 ml at least once . absent bowel sounds ( bs ) = bs were not heard on careful auscultation . vomiting / regurgitation = visible vomiting or regurgitation in any amount . bowel distension = suspected or radiologically confirmed bowel dilatation in any bowel segment . gi bleeding = visible appearance of blood in vomits , nasogastric aspirate , or stool . feeding intolerance ( fi ) was considered present when less than 20 % of the calculated caloric needs were administered with enteral nutrition ( en ) and at the same time gi symptom(s ) were documented being a reason for withholding or reducing the en . intra - abdominal hypertension ( iah ) = mean iap of the day 12 mmhg and abdominal compartment syndrome ( acs ) = mean iap > 20 mmhg with new organ dysfunction or failure , with iap measured in the supine position with zero - point at mid - axillary line with a maximal instillation volume of 25 ml . statistical package for the social sciences ( ibm spss statistics 20.0 , somers , ny , usa ) software was used for statistical analysis . smirnov test with lilliefors correction was used to test normality of distribution . to compare groups , student s t - test ( normal distribution ) and mann whitney u test ( non - gaussian distribution ) univariate analyses of admission parameters were applied to identify the risk factors for 28-day mortality . parameters with p < 0.2 in univariate analysis were entered stepwise into a multiple logistic regression model to identify the best combination for prediction of 28-day mortality . meier curves and log - rank tests were used to compare survival of patients with and without gi symptoms . gi symptoms were entered separately into a regression model predicting mortality to evaluate the importance of individual gi symptoms . receiver operating characteristic ( roc ) curves were used to determine the likelihood ratios of different versions of possible gastrointestinal failure ( gif ) scores , the sofa score and the sofa with gif scores combined to predict the icu mortality . the optimal cut - off value was calculated from the roc curve analysis as the point with the greatest combined sensitivity and specificity . 377 patients from 40 icus were included . the study flow - chart is presented in fig . 1 . admission and day 1 characteristics with p < 0.2 for associations with mortality in univariate analyses are presented in table 1.fig . 1enrolment flow - charttable 1patient characteristics on admission and day 1 among survivors and nonsurvivorscharacteristicsall ( n = 377)survivors ( n = 260)nonsurvivors ( n = 117)p valueadmission age , years , median ( range)62 ( 1898)61 ( 1898)64 ( 2291)0.082 body mass index26 ( 2329)26 ( 2329)25(2229)0.086 medical profile , n ( % ) 142 ( 37.7)84 ( 32.3)58 ( 49.6)0.001 abdominal surgery , n ( % ) 118 ( 31.3)90 ( 34.6)28 ( 23.9)0.042day 1 apache ii score , points18 ( 1324)17 ( 1222)21 ( 1730)<0.001 sepsis , n ( % ) 137 ( 36.3)82 ( 31.5)55 ( 47.0)0.005 sofa score ( points)8 ( 510)7 ( 510)10 ( 714)<0.001 vasopressors , n ( % ) 263 ( 69.8)172 ( 66.2)91 ( 77.8)0.029 po2/fio2 ( mmhg)188 ( 108322)193 ( 115347)177 ( 97292)0.151 creatinine ( mol / l)99 ( 72164)91 ( 69139)126 ( 80188)<0.001 glasgow coma scale ( points)13 ( 615)14 ( 715)10 ( 415)<0.001 fluid balance ( l/24 h)+1.4 ( 0.42.9)+1.1 ( 0.22.7)+2.0 ( 1.03.5)0.001 urine output ( l/24 h)1.6 ( 0.92.6)1.8 ( 1.02.7)1.2 ( 0.42.3)<0.001 mean iap ( mmhg)9.8 ( 7.012.7)10.0 ( 7.312.8)9.0 ( 5.912.6)0.066 minimal app ( mmhg)62 ( 5271)62 ( 5372)60 ( 4670)0.033 number of gi symptoms0 ( 01)0 ( 01)1 ( 01)0.013 three or more gi symptoms , n ( % ) 18 ( 4.8)7 ( 2.7)11 ( 9.4)0.008data are median ( interquartile ranges ) if not stated otherwiseapache ii score acute physiology and chronic health evaluation ii ( 11 ) , sofa score sequential organ failure assessment ( 8) , po2/fio2 partial oxygen pressure in blood / content of oxygen in inspired air , iap intra - abdominal pressure , app abdominal perfusion pressure , gi gastrointestinal patient characteristics on admission and day 1 among survivors and nonsurvivors data are median ( interquartile ranges ) if not stated otherwise apache ii score acute physiology and chronic health evaluation ii ( 11 ) , sofa score sequential organ failure assessment ( 8) , po2/fio2 partial oxygen pressure in blood / content of oxygen in inspired air , iap intra - abdominal pressure , app abdominal perfusion pressure , gi gastrointestinal admission diagnosis was gastrointestinal in 27.3 % , ( including hepatopancreatic pathology in 6.6 % ) , pulmonary 19.1 % , cardiac 17.2 % , neurological 15.9 % and polytrauma in 8.7 % . most common reasons for admission were respiratory failure ( 22.5 % ) , shock ( 18.3 % ) , postoperative mv after major surgery ( 17.8 % ) and neurological deterioration ( 16.2 % ) . median duration of mv was 4.0 ( 213 ) , icu stay 7.0 ( 317 ) and hospital stay 19.0 ( 1028 ) days . mean apache ii score on admission was 19.0 ( sd 8.0 ) points , 278 patients ( 73.7 % ) were treated with vasoactive / inotropic agents . 142/377 patients ( 37.7 % ) had a medical profile , 78 ( 20.7 % ) were elective and 157 ( 41.6 % ) emergency surgery patients ; respective mortality rates were 40.8 , 17.9 and 28.7 % . one - third of the elective surgery patients underwent cardiovascular , one - third gi , and one - third other surgical procedures . daily and global incidences of gi symptoms and iah for all patients , and for survivors and non - survivors separately are presented in table 2 . the number of coincident gi symptoms was higher in non - survivors on each day . none of the patients had more than four gi symptoms simultaneously.table 2daily and global incidence of gastrointestinal ( gi ) symptoms , intra - abdominal hypertension and gastrointestinal failure among survivors and non - survivorsday 1day 2day 4day 7cumulativetotal number of patients377352264200377 survivors260244194147260 nonsurvivors1171067053117median ( iqr ) number of gi symptomscumulative maximum total1 ( 01)0 ( 01)0 ( 01)0 ( 01)1 ( 02 ) survivors0 ( 01)0 ( 01)0 ( 01)0 ( 01)1 ( 01 ) nonsurvivors1 ( 01)1 ( 01)1 ( 01)1 ( 01)1 ( 02 ) p value*<0.001<0.001<0.001<0.001<0.001absent bowel sounds total ( % ) 125 ( 33.2)82 ( 23.3)42 ( 15.9)29 ( 14.9)142 ( 37.7 ) survivors ( % ) 76 ( 29.2)45 ( 18.4)25 ( 12.9)15 ( 10.3)88 ( 33.8 ) nonsurvivors ( % ) 49 ( 41.9)37 ( 34.9)17 ( 24.3)14 ( 28.6)54 ( 46.2 ) p value*0.018<0.0010.0330.0090.022diarrhoea total ( % ) 26 ( 6.9)40 ( 11.2)46 ( 17.4)39 ( 19.5)81 ( 21.5 ) survivors ( % ) 16 ( 6.2)20 ( 8.2)31 ( 16.0)26 ( 17.7)53 ( 20.4 ) nonsurvivors ( % ) 10 ( 8.5)20 ( 18.9)15 ( 21.4)13 ( 24.5)28 ( 23.9 ) p value*0.3880.0060.2700.3140.498bowel distension total ( % ) 54 ( 14.3)53 ( 15.1)32 ( 12.9)19 ( 9.5)78 ( 20.7 ) survivors ( % ) 33 ( 12.7)34 ( 13.9)21 ( 11.5)10 ( 6.8)48 ( 18.5 ) nonsurvivors ( % ) 21 ( 17.9)19 ( 17.9)11 ( 16.7)9 ( 17.0)30 ( 25.6 ) p value*0.2020.3250.2750.0490.129vomiting / regurgitation total ( % ) 18 ( 4.8)11 ( 3.1)5 ( 1.9)7 ( 3.5)31 ( 15.5 ) survivors ( % ) 11 ( 4.2)7 ( 2.9)3 ( 1.5)5 ( 3.4)22 ( 8.5 ) nonsurvivors ( % ) 7 ( 6.0)4 ( 3.8)2 ( 2.9)2 ( 3.8)9 ( 7.7 ) p value*0.4450.7380.6111.0001.000high gastric residual volume total ( % ) 13 ( 3.4)8 ( 2.3)8 ( 3.0)8 ( 4.0)28 ( 7.4 ) survivors ( % ) 8 ( 3.1)5 ( 2.4)5 ( 2.6)5 ( 3.4)15 ( 5.8 ) nonsurvivors ( % ) 5 ( 4.3)3 ( 2.8)3 ( 4.3)3 ( 5.7)13 ( 11.1 ) p value*0.5560.7060.7120.6970.086gastrointestinal bleeding total ( % ) 16 ( 4.2)13 ( 3.7)5 ( 1.9)4 ( 2.0)24 ( 6.4 ) survivors ( % ) 7 ( 2.7)4 ( 1.6)2 ( 1.0)2 ( 1.4)11 ( 4.2 ) nonsurvivors ( % ) 9 ( 7.7)9 ( 8.5)3 ( 4.3)2 ( 3.8)13 ( 11.1 ) p value*0.0490.0030.1160.2700.020at least 1 gi symptom total168 ( 44.6)146 ( 41.7)109 ( 41.3)81 ( 40.5)227 ( 60.2 ) survivors105 ( 40.4)88 ( 33.8)71 ( 27.3)51 ( 19.6)148 ( 56.9 ) nonsurvivors63 ( 53.8)58 ( 49.6)38 ( 32.5)30 ( 25.6)79 ( 67.5 ) p value*0.0190.0010.0110.0090.0542 or more gi symptoms total62 ( 16.4)46 ( 13.1)21 ( 8.0)20 ( 10.0)76 ( 20.2 ) survivors39 ( 15.0)24 ( 9.2)13 ( 5.0)10 ( 3.8)44 ( 16.9 ) nonsurvivors23 ( 19.7)22 ( 18.8)8 ( 6.8)10 ( 8.5)32 ( 27.4 ) p value*0.2930.0090.2030.0170.0263 or more gi symptoms = gi failure total ( % ) 18 ( 4.8)11 ( 3.1)6 ( 2.3)3 ( 1.5)24 ( 6.4 ) survivors ( % ) 7 ( 2.7)2 ( 0.8)1 ( 0.5)1 ( 0.7)9 ( 3.5 ) nonsurvivors ( % ) 11 ( 9.4)9 ( 8.5)5 ( 7.1)2 ( 3.8)15 ( 12.8 ) p value*0.0080.0010.0060.1720.0014 or more gi symptoms total4 ( 1.1)11 ( 3.1)01 ( 0.5)16 ( 4.2 ) survivors2 ( 0.8)2 ( 0.8)01 ( 0.4)5 ( 1.9 ) nonsurvivors2 ( 1.7)9 ( 7.7)0011 ( 9.4 ) p value*0.5910.0010.002intra - abdominal hypertension total ( % ) 109 ( 28.9)96 ( 27.4)68 ( 25.8)40 ( 20.0)161 ( 42.7 ) survivors ( % ) 77 ( 29.6)71 ( 29.1)55 ( 28.4)32 ( 21.8)111(42.7 ) nonsurvivors ( % ) 32 ( 27.4)25 ( 23.6)13 ( 18.6)8 ( 15.1)50 ( 42.7 ) p value*0.7130.4210.1050.3051.000 * p values refer to comparisons between survivors and nonsurvivorsmaximal daily sum of gi symptoms daily and global incidence of gastrointestinal ( gi ) symptoms , intra - abdominal hypertension and gastrointestinal failure among survivors and non - survivors * p values refer to comparisons between survivors and nonsurvivors maximal daily sum of gi symptoms the incidence of absent bs was 37.7 % ( mortality rate 38.0 % ) , of overt gi bleeding 6.4 % ( mortality rate 54.2 % ) , of iah 42.7 % ( mortality rate 31.1 % ) and of acs 3.6 % ( mortality rate 38.5 % ) . prepyloric route for en was common , postpyloric route was used in 4.3 % on day 1 , increasing to 12.9 % on day 7 . multivariate regression analyses for 28 day mortality including the different gi symptoms , caloric intake < 80 % and iah are presented in table 3 . the occurrence of absent bs on day 1 , gi bleeding during the first two days and bowel distension on day 7 were independently associated with 28 day mortality , while vomiting , high grv , diarrhoea and the presence of iah were not predictive.table 3multivariate regression analyses with gi symptoms , failure of enteral nutrition , and intra - abdominal hypertension predicting 28 day survivalday 1p valueorlower ci 95 % upper ci 95 % absent bowel sounds0.0072.4571.2854.700 vomiting / regurgitation0.8770.9030.253.258 maximum grv > 500 ml0.8880.9100.2443.397 diarrhoea0.3871.7000.5115.659 bowel distension0.9160.9540.3982.289 gi bleeding0.0424.4041.05818.333 en < 80 % of caloric needs0.0320.3250.1160.906 iah0.3160.7080.3611.390day 2 absent bowel sounds0.4251.3680.6332.957 vomiting / regurgitation0.8871.1200.2345.352 maximum grv > 500 ml0.6731.3920.3006.469 diarrhoea0.4081.6400.5085.289 bowel distension0.7591.1620.4453.030 gi bleeding0.00819.0932.153169.336 en < 80 % of caloric needs0.0400.3550.1320.952 iah0.0620.4610.2041.041day 4 absent bowel sounds0.1921.7930.7464.310 vomiting / regurgitation0.3663.1470.26337.699 maximum grv > 500 ml0.3981.9950.4029.901 diarrhoea0.3611.6420.5674.758 bowel distension0.8291.1220.3963.173 gi bleeding0.1505.5950.53858.177 en < 80 % of caloric needs0.4401.4370.5733.605 iah0.1270.5120.2171.210day 7 absent bowel sounds0.1622.1570.7356.332 vomiting / regurgitation0.2300.1620.0083.158 maximum grv > 500 ml0.6361.4900.2857.790 diarrhea0.7931.1810.3424.083 bowel distension0.0367.0701.14043.859 gi bleeding0.2493.8220.39237.281 en < 80 % of caloric needs0.9510.9700.3642.582 iah0.1530.4280.1341.372the variables entered into the multivariate analysis were exclusively those listed abovesignificant findings are marked in boldgrv gastric residual volume , gi gastrointestinal , en enteral nutrition , iah intra - abdominal hypertension multivariate regression analyses with gi symptoms , failure of enteral nutrition , and intra - abdominal hypertension predicting 28 day survival the variables entered into the multivariate analysis were exclusively those listed above significant findings are marked in bold grv gastric residual volume , gi gastrointestinal , en enteral nutrition , iah intra - abdominal hypertension the reasons for withholding / stopping en were not documented in 58 % of the cases , and therefore in these cases the presence or absence of fi could not be assessed . en < 80 % of caloric needs on day 1 and 2 was associated with better survival . based on daily comparisons of survivors and nonsurvivors with different number of gi symptoms ( table 2 ) as well as kaplan meier curves with maximum number of gi symptoms , the cut - off point for gif was defined as three or more coincident gi symptoms listed above . gastrointestinal failure ( three or more coincident gi symptoms ) occurred in 24 patients ( 6.4 % ) and was associated with higher 28-day mortality ( 62.5 vs. 28.9 % ) ( fig . grey line shows less than three gi symptoms concomitantly during the first week in icu . black line shows at least three gi symptoms concomitantly during the first week in icu . p < 0.001 between the groups ( log - rank test ) kaplan meier survival plot for patients with gif versus without gif . grey line shows less than three gi symptoms concomitantly during the first week in icu . black line shows at least three gi symptoms concomitantly during the first week in icu . p < 0.001 between the groups ( log - rank test ) prediction of 28 day mortality in a statistical model including demographic data and admission day variables identified in univariate analyses , gi symptoms and sofa sub - scores on admission day is presented in table 4 . the occurrence of gif on day 1 was associated with a threefold increased mortality , being an independent predictor of mortality together with renal and neurological sofa sub - score . none of the gi symptoms alone nor iah or caloric intake < 80 % independently predicted mortality.table 4multivariate regression analysis with admission day variables predicting 28-day mortalityp valueodds ratio95 % ciage0.5421.0050.9901.019body mass index0.2070.9710.9291.016medical profile0.0831.5980.9402.716sepsis0.2231.4000.8152.406fluid balance day 10.8591.0001.0001.000three or more gi symptoms day 10.0353.1891.0829.396renal sofa sub - score<0.0011.4231.1691.733neurological sofa sub - score<0.0011.4441.2311.694haematologic sofa sub - score0.0731.2770.9771.668respiratory sofa sub - score0.3111.1130.9051.368hepatic sofa sub - score0.8040.9620.7101.305cardiovascular sofa sub - score0.8590.9820.8081.195nagelkerke r - square 0.253the table presents the final model of multivariate analysis after removal of clearly correlated variablessignificant findings are marked in boldgi gastrointestinal , sofa sequential organ failure assessment multivariate regression analysis with admission day variables predicting 28-day mortality nagelkerke r - square 0.253 the table presents the final model of multivariate analysis after removal of clearly correlated variables significant findings are marked in bold gi gastrointestinal , sofa sequential organ failure assessment regression analyses including daily sofa sub - scores and the number of gi symptoms revealed increasing number of gi symptoms as an independent predictor of mortality on day 2 and 7 with a tendency towards statistical significance on admission and day 4 ( table 5 ) . only the neurological sofa score predicted mortality on all study days , renal sofa score was predictive at three of the 4 days , haematologic sofa on one day , while none of the other sofa sub - scores predicted mortality.table 5regression analyses with daily sofa sub - scores and the number of gi symptoms as an additional sub - score predicting 28-day mortalitysofa sub - scores + number of gi symptoms , and survivalday 1p valueorlower ci 95 % upper ci 95 % sofa cardiovascular0.7571.0300.8541.242 sofa respiratory0.1331.1580.9561.403 sofa haematologic0.0751.2610.9771.628 sofa hepatic0.7740.9580.7181.280 sofa renal<0.0011.4411.1931.740 sofa neurological<0.0011.4691.2621.710 number of gi symptoms0.0891.2640.9651.656day 2 sofa cardiovascular0.7991.0250.8471.240 sofa respiratory0.2611.1190.9191.363 sofa haematologic0.2861.1510.8891.491 sofa hepatic0.7380.9400.6531.353 sofa renal0.0071.3091.0771.592 sofa neurological<0.0011.3311.1461.546 number of gi symptoms0.0021.6061.1842.179day 4 sofa cardiovascular0.9611.0060.8011.263 sofa respiratory0.4471.1050.8541.432 sofa haematologic0.3641.1560.8461.579 sofa hepatic0.7711.0610.7131.577 sofa renal0.0091.3811.0831.762 sofa neurological0.0011.3481.1221.620 number of gi symptoms0.0541.5050.9932.282day 7 sofa cardiovascular0.1331.2270.9401.603 sofa respiratory0.6561.0750.7821.478 sofa haematologic0.0451.5021.0082.237 sofa hepatic0.3710.8060.5031.292 sofa renal0.5881.0820.8141.438 sofa neurological0.0451.2381.0051.525 number of gi symptoms0.0101.8821.1643.042cumulative maximum sofa cardiovascular0.4541.0800.8831.320 sofa respiratory0.3901.1010.8841.371 sofa haematologic0.5611.0720.8471.357 sofa hepatic0.8881.0200.7771.338 sofa renal<0.0011.4751.2461.747 sofa neurological<0.0011.4521.2541.681 number of gi symptoms0.0821.2670.9711.655gi gastrointestinal , sofa sequential organ failure assessment regression analyses with daily sofa sub - scores and the number of gi symptoms as an additional sub - score predicting 28-day mortality gi gastrointestinal , sofa sequential organ failure assessment the best gif score with respect to mortality prediction included all six gi symptoms , but not iah , fi and/or caloric intake , giving points as follows : 0 = no gi symptoms ; 1 = 1 gi symptom ; 2 = 2 gi symptoms ; 3 = 3 gi symptoms and 4 4 gi symptoms . receiver operating characteristic curve analyses for sofa score alone , for the gif score based on the number of gi symptoms and their combination are presented in table 6 . roc curves including gif score were not significantly different from the roc curves of the sofa score alone.table 6roc analyses for sofa score alone and sofa combined with the score based on the number of gi symptomssofaaucse95 % ci day 10.7030.030.6430.762 day 20.6820.030.6160.748 day 40.6960.040.6200.772 day 70.6910.050.6020.780 cumulative maximum0.7320.030.6760.789number of gi symptoms day 10.5710.030.5080.635 day 20.6070.030.5410.673 day 40.5910.020.5120.670 day 70.6240.050.5330.714 cumulative maximum0.5810.010.5170.644sofa + number of gi symptoms day 10.7060.030.6470.766 day 20.6870.030.6220.752 day 40.6980.040.6230.772 day 70.7000.040.6140.785 cumulative maximum0.7340.030.6780.790gi gastrointestinal , sofa sequential organ failure assessment , cumulative maximum maximal daily score during the study roc analyses for sofa score alone and sofa combined with the score based on the number of gi symptoms gi gastrointestinal , sofa sequential organ failure assessment , cumulative maximum maximal daily score during the study the current prospective worldwide multicentre study including critically ill patients with an expected duration of mechanical ventilation of more than 6 h demonstrated that a large proportion of these patients had gi symptoms during the first week of admission . some specific symptoms , including absent bs , gi bleeding and bowel distension , as well as the total number of gi symptoms , were associated with 28 day mortality . however , the study failed to develop an additional dysfunction score that significantly improved mortality prediction of the sofa score . the total incidence , as well as the occurrence of the individual gi symptoms , was comparable to earlier observations [ 14 ] , despite the fact that the definitions for these symptoms differ somewhat between studies . the proportion of patients with two or more simultaneous gi symptoms was lower in the present study ( 20 % ) than in a previous single - centre study ( 36 % ) . an increasing number of gi symptoms was related to increased mortality in both studies . in the present study , absent bs , gi bleeding and bowel distension were the symptoms and signs associated with mortality , similar to earlier findings . another previously reported finding that a combination of iah and fi predicted outcome could not be confirmed in this study , as unfortunately there was a high rate of missing data for the reasons to withhold or reduce en . thus , although a final gif score is still not formulated , occurrence of gif is , independently of its exact formulation , associated with adverse outcome in all studies . a major limitation of assessment of gi symptoms is that some of the symptoms are subjective and poorly defined , the most questioned being absent bs . there is a consensus not using absent bs as a reason to withhold enteral nutrition . consistent association of absent bs ( despite the obvious limitations of this symptom ) with mortality is an important finding of our study . an explanation might be that absence of bs reflects severity of inflammation and hypoperfusion , but also deeper sedation and immobilisation often required for artificial organ support ( cardiac assist devices , ecmo , cvvh etc . ) . there is one previous observation that absent or abnormal bs are associated with higher mortality in univariate analysis . a high incidence of iah was observed in the study population ( 42.7 % compared to 2730 % in some previous studies [ 9 , 14 ] ) . the possibility to measure iap was an inclusion criterion , the reason being that previous studies have shown a relation between iah and mortality [ 9 , 15 ] . furthermore , the iap value is numerical and reproducible , and as such could be considered as a parameter for a sofa gi sub - score . the proportion of patients in whom transvesical pressure measurement is not possible ( mainly post - cystectomy patients ) is extremely small in a general icu population . in the present study , iah was not associated with increased mortality , confirming the findings of a recently published study . there are several possible reasons for failure to improve the predictive value of the sofa score by including a gi dysfunction score . first , there might be a type - ii statistical error , since we did not meet our enrolment goal . the goal was based on expected enrolment rates for a fixed study period , but on retrospect the actual enrolment control could have improved our study design . with inclusion of patients on mv for at least 6 h we aimed to minimize the inclusion of recovery room patients and concentrate on real icu patients . exclusion of spontaneously breathing patients was planned because of different pathophysiological patterns of iap during mv . unfortunately , selection bias must have occurred , as in some centres patient enrolment was unexpectedly low and at the same time severity of illness and associated mortality were higher than expected . in a previous study on gi dysfunction enrolling all mv patients staying in icu for 24 h and showing that gif score increased the predictive power of the sofa score mean apache ii score ( 14 vs. 19 ) and therefore also predicted mortality ( 19 vs. 32 % ) were lower than in the present study . compared to earlier studies in unselected icu patients , we also observed a rather limited performance of sofa score predicting mortality [ 17 , 18 ] . in particular , the cardiovascular subscore of sofa , usually the best - performing subscore [ 9 , 19 ] , had a low power in our study . the relative high proportion of patients receiving vasoactive drugs , resulting in high cardiovascular subscores , additionally confirms that the sickest patients were included . the inclusion of more severely ill patients and associated lower diversity of patients might explain that both sofa and gi score poorly predicted mortality . moreover , the fact that addition of gi dysfunction did not improve the predictive power of the sofa score may actually be an important finding of the study . it leads us to the hypothesis that in this general icu population of severely ill patients not primary gi failure due to abdominal pathology is the main problem , but rather secondary gi failure due to systemic inflammation and/or hypoperfusion . the majority of the patients did not reach their caloric needs via the enteral route , but in many cases the exact reasons were not documented . these missing data made it impossible to identify the impact of fi on outcome in this study . former studies have defined fi as < 80 % of caloric needs achieved after 4872 h in the icu or as withholding en for any gi reason . in both cases , this highly depends on the local feeding strategy and nutritional goals , which remain controversial for critically ill patients during the initial phase of critical illness [ 2123 ] . our observation of en < 80 % being associated with better survival is likely biased by not initiating enteral nutrition in patients with an expected oral intake within a couple of days . several biomarkers reflecting intestinal function have been suggested recently ( i - fabp , citrulline , d - lactate ) [ 25 , 26 ] . future studies should establish their place in clinical practice and establish their correlations with clinical gi signs and symptoms , as well as with prognosis . despite being the largest prospective multicentre international study to assess the gi dysfunction in mv patients first , most of the gi dysfunction definitions are subjective , an issue currently limiting the research in this area . second , missing data was a considerable problem in our study , mainly because the fi could not be identified in many cases . third , even though the inclusion of a wide variety of icus have made the results more generalizable , it might as well be considered as a limitation due to associated variations in treatment practice . fourth , the aimed number of patients was not reached in our study . a greater number of patients is needed to create a reliable score in future studies . fifth , the exclusion of patients with an expected short ventilation period makes our results apply to a population of more severely ill icu population . the current prospective worldwide multicentre study shows that a severely ill subgroup of mechanically ventilated icu patients frequently has gi symptoms and iah . absent bowel sounds , gi bleeding , and an increasing number of coincident gi symptoms were associated with 28-day mortality . based on the data of this study it was however not possible to develop a valid gi dysfunction score that improved the accuracy of the sofa score . this may either be due to data set limitations , definition problems , or may indicate that gi dysfunction is often secondary to and not the primary cause of other organ failure . inneke de laet : intensive care unit , ziekenhuis netwerk antwerpen , zna stuivenberg , antwerpen , belgium ; rob j. bosman : department of intensive care , onze lieve vrouwe gasthuis , amsterdam , the netherlands ; ioana grigoras , mihaela blaj : department of anesthesia and intensive care , university of medicine and pharmacy gr . iasi , romania ; willem stockman , piet lormans : department of anesthesia and critical care , heilig hart hospital roeselare - menen , roeselare , belgium ; carlos a. ordoez : intensive care unit and surgical department , fundacion valle del lili , universidad del valle , cali , colombia ; mikhail kirov : department of anesthesiology and intensive care medicine , northern state medical university , arkhangelsk , russia ; juan duchesne : surgical intensive care unit , section of trauma and critical care surgery , new york medical college westchester medical center , new orleans , usa ; nicola brienza : department of emergency and organ transplantation , university of bari , policlinico , bari , italy ; luis alejandro sanchez hurtado : department of intensive care , hospital especialidades antonio fraga mouret centro medico la raza imss , mexico city , mexico ; theodossis papavramidis : third department of surgery , ahepa university hospital , thessaloniki , greece ; kadri tamme : general intensive care unit , tartu university hospital , tartu , estonia ; guadalupe aguirre - avalos : department of intensive care , hospital civil de guadalajara fray antonio alcalde , guadalajara jalisco , mexico ; dariusz onichimowski : department of anaesthesiology and intensive care , the voivodal specialistic hospital , university of varmia and masuria in olsztyn , poland ; shaikh nissar : department of anesthesia and intensive care , hamad medical corporation ; doha - qatar ; andrey litvin : department of surgery , gomel regional clinical hospital , gomel , belarus ; karel balihar : department of intensive care , ist internal department , teaching hospital and medical faculty of charles university , pilsen , czech republic ; matti reinikainen : department of intensive care , north karelia central hospital , joensuu , finland ; ivana zykova : aro krajska nemocnice liberec , liberec , czech republic ; manhaz edalatnejad : internal medicine department , arak medical science university , arak , iran ; davide chiumello : dipartimento di anestesia , rianimazione ( intensiva e subintensiva ) e terapia del dolore , fondazione irccs ca granda - ospedale maggiore policlinico , milan , italy ; crystal wilson : department of critical care , foothills medical centre , calgary , alberta , canada ; javier izura : department of intensive care , hospital virgen del camino , navarra , spain ; caridad soler : department of intensive care , hermanos ameijeiras hospital , havana , cuba ; aleksandr koroljov : department of anaesthesiology and intensive care , east tallinn central hospital , tallinn , estonia ; milan kaska : academic department of surgery , charles university , medical faculty , hradec krlov , czech republic ; martin max : service des soins intensifs polyvalents , centre hospitalier de luxembourg , luxembourg ; mayada hussien : department of intensive care , theodor bilharz research institute , cairo , egypt ; pavel szturz : anesthesiology and resuscitation clinic , ostrava , czech republic ; ulrike holzinger : department of medicine iii - division of gastroenterology and hepatology , icu , medical university of vienna , vienna , austria ; raido paasma : department of anaesthesia and icu , prnu hospital , prnu , estonia ; ivan palibrk : department of anaesthesiology , clinical centre serbia , belgrade , serbia ; natasa kovac : department of anaesthesiology and intensive care , university hospital sestre milosrdnice , zagreb , croatia ; gaetan plantefeve : department of intensive care , victor dupouy hospital , argenteuil , france ; michael cheatham : department of surgical education , orlando regional medical center , orlando , florida , usa ; rao ivatury : department of surgery , division trauma , critical care and emergency general surgery , virginia commonwealth university medical center , richmond , virginia , usa ; ivan ramos palomino : intensive care unit , san gabriel , lima , peru ; pille parm : pulmonary intensive care unit , tartu university hospital , tartu , estonia ; piyush ranjan : department of general surgery , institute of post graduate medical education and research , kolkata , india ; gumersindo gonzlez daz : intensive care unit , hospital universitario morales meseguer , murcia , spain ; jan de waele , dieter debergh : department of critical care medicine , ghent university hospital , ghent , belgium inneke de laet : intensive care unit , ziekenhuis netwerk antwerpen , zna stuivenberg , antwerpen , belgium ; rob j. bosman : department of intensive care , onze lieve vrouwe gasthuis , amsterdam , the netherlands ; ioana grigoras , mihaela blaj : department of anesthesia and intensive care , university of medicine and pharmacy gr . iasi , romania ; willem stockman , piet lormans : department of anesthesia and critical care , heilig hart hospital roeselare - menen , roeselare , belgium ; carlos a. ordoez : intensive care unit and surgical department , fundacion valle del lili , universidad del valle , cali , colombia ; mikhail kirov : department of anesthesiology and intensive care medicine , northern state medical university , arkhangelsk , russia ; juan duchesne : surgical intensive care unit , section of trauma and critical care surgery , new york medical college westchester medical center , new orleans , usa ; nicola brienza : department of emergency and organ transplantation , university of bari , policlinico , bari , italy ; luis alejandro sanchez hurtado : department of intensive care , hospital especialidades antonio fraga mouret centro medico la raza imss , mexico city , mexico ; theodossis papavramidis : third department of surgery , ahepa university hospital , thessaloniki , greece ; kadri tamme : general intensive care unit , tartu university hospital , tartu , estonia ; guadalupe aguirre - avalos : department of intensive care , hospital civil de guadalajara fray antonio alcalde , guadalajara jalisco , mexico ; dariusz onichimowski : department of anaesthesiology and intensive care , the voivodal specialistic hospital , university of varmia and masuria in olsztyn , poland ; shaikh nissar : department of anesthesia and intensive care , hamad medical corporation ; doha - qatar ; andrey litvin : department of surgery , gomel regional clinical hospital , gomel , belarus ; karel balihar : department of intensive care , ist internal department , teaching hospital and medical faculty of charles university , pilsen , czech republic ; matti reinikainen : department of intensive care , north karelia central hospital , joensuu , finland ; ivana zykova : aro krajska nemocnice liberec , liberec , czech republic ; manhaz edalatnejad : internal medicine department , arak medical science university , arak , iran ; davide chiumello : dipartimento di anestesia , rianimazione ( intensiva e subintensiva ) e terapia del dolore , fondazione irccs ca granda - ospedale maggiore policlinico , milan , italy ; crystal wilson : department of critical care , foothills medical centre , calgary , alberta , canada ; javier izura : department of intensive care , hospital virgen del camino , navarra , spain ; caridad soler : department of intensive care , hermanos ameijeiras hospital , havana , cuba ; aleksandr koroljov : department of anaesthesiology and intensive care , east tallinn central hospital , tallinn , estonia ; milan kaska : academic department of surgery , charles university , medical faculty , hradec krlov , czech republic ; martin max : service des soins intensifs polyvalents , centre hospitalier de luxembourg , luxembourg ; mayada hussien : department of intensive care , theodor bilharz research institute , cairo , egypt ; pavel szturz : anesthesiology and resuscitation clinic , ostrava , czech republic ; ulrike holzinger : department of medicine iii - division of gastroenterology and hepatology , icu , medical university of vienna , vienna , austria ; raido paasma : department of anaesthesia and icu , prnu hospital , prnu , estonia ; ivan palibrk : department of anaesthesiology , clinical centre serbia , belgrade , serbia ; natasa kovac : department of anaesthesiology and intensive care , university hospital sestre milosrdnice , zagreb , croatia ; gaetan plantefeve : department of intensive care , victor dupouy hospital , argenteuil , france ; michael cheatham : department of surgical education , orlando regional medical center , orlando , florida , usa ; rao ivatury : department of surgery , division trauma , critical care and emergency general surgery , virginia commonwealth university medical center , richmond , virginia , usa ; ivan ramos palomino : intensive care unit , san gabriel , lima , peru ; pille parm : pulmonary intensive care unit , tartu university hospital , tartu , estonia ; piyush ranjan : department of general surgery , institute of post graduate medical education and research , kolkata , india ; gumersindo gonzlez daz : intensive care unit , hospital universitario morales meseguer , murcia , spain ; jan de waele , dieter debergh : department of critical care medicine , ghent university hospital , ghent , belgium

purposethe study aimed to develop a gastrointestinal ( gi ) dysfunction score predicting 28-day mortality for adult patients needing mechanical ventilation ( mv).methods377 adult patients from 40 icus with expected duration of mv for at least 6 h were prospectively studied . predefined gi symptoms , intra - abdominal pressures ( iap ) , feeding details , organ dysfunction and treatment were documented on days 1 , 2 , 4 and 7.resultsthe number of simultaneous gi symptoms was higher in nonsurvivors on each day . absent bowel sounds and gi bleeding were the symptoms most significantly associated with mortality . none of the gi symptoms alone was an independent predictor of mortality , but gastrointestinal failure ( gif)defined as three or more gi symptoms on day 1 in icu was independently associated with a threefold increased risk of mortality . during the first week in icu , gif occurred in 24 patients ( 6.4 % ) and was associated with higher 28-day mortality ( 62.5 vs. 28.9 % , p = 0.001 ) . adding the created subscore for gi dysfunction ( based on the number of gi symptoms ) to sofa score did not improve mortality prediction ( day 1 auroc 0.706 [ 95 % ci 0.6470.766 ] versus 0.703 [ 95 % ci 0.6430.762 ] in sofa score alone).conclusionsan increasing number of gi symptoms independently predicts 28 day mortality with moderate accuracy . however , it was not possible to develop a gi dysfunction score , improving the performance of the sofa score either due to data set limitations , definition problems , or possibly indicating that gi dysfunction is often secondary and not the primary cause of other organ failure .

Introduction Methods General Definitions Statistical analysis Results Discussion Conclusions Appendix Gastro-Intestinal Failure Trial Group

predefined gi symptoms , iap ( minimum , maximum and mean daily values ) , feeding details , sofa score with all its sub - scores , urine output , fluid balance , positive end - expiratory pressure , as well as serum albumin and c - reactive protein levels were documented on days 1 , 2 , 4 and 7 in the icu . predefined gi symptoms , iap ( minimum , maximum and mean daily values ) , feeding details , sofa score with all its sub - scores , urine output , fluid balance , positive end - expiratory pressure , as well as serum albumin and c - reactive protein levels were documented on days 1 , 2 , 4 and 7 in the icu . 1enrolment flow - charttable 1patient characteristics on admission and day 1 among survivors and nonsurvivorscharacteristicsall ( n = 377)survivors ( n = 260)nonsurvivors ( n = 117)p valueadmission age , years , median ( range)62 ( 1898)61 ( 1898)64 ( 2291)0.082 body mass index26 ( 2329)26 ( 2329)25(2229)0.086 medical profile , n ( % ) 142 ( 37.7)84 ( 32.3)58 ( 49.6)0.001 abdominal surgery , n ( % ) 118 ( 31.3)90 ( 34.6)28 ( 23.9)0.042day 1 apache ii score , points18 ( 1324)17 ( 1222)21 ( 1730)<0.001 sepsis , n ( % ) 137 ( 36.3)82 ( 31.5)55 ( 47.0)0.005 sofa score ( points)8 ( 510)7 ( 510)10 ( 714)<0.001 vasopressors , n ( % ) 263 ( 69.8)172 ( 66.2)91 ( 77.8)0.029 po2/fio2 ( mmhg)188 ( 108322)193 ( 115347)177 ( 97292)0.151 creatinine ( mol / l)99 ( 72164)91 ( 69139)126 ( 80188)<0.001 glasgow coma scale ( points)13 ( 615)14 ( 715)10 ( 415)<0.001 fluid balance ( l/24 h)+1.4 ( 0.42.9)+1.1 ( 0.22.7)+2.0 ( 1.03.5)0.001 urine output ( l/24 h)1.6 ( 0.92.6)1.8 ( 1.02.7)1.2 ( 0.42.3)<0.001 mean iap ( mmhg)9.8 ( 7.012.7)10.0 ( 7.312.8)9.0 ( 5.912.6)0.066 minimal app ( mmhg)62 ( 5271)62 ( 5372)60 ( 4670)0.033 number of gi symptoms0 ( 01)0 ( 01)1 ( 01)0.013 three or more gi symptoms , n ( % ) 18 ( 4.8)7 ( 2.7)11 ( 9.4)0.008data are median ( interquartile ranges ) if not stated otherwiseapache ii score acute physiology and chronic health evaluation ii ( 11 ) , sofa score sequential organ failure assessment ( 8) , po2/fio2 partial oxygen pressure in blood / content of oxygen in inspired air , iap intra - abdominal pressure , app abdominal perfusion pressure , gi gastrointestinal patient characteristics on admission and day 1 among survivors and nonsurvivors data are median ( interquartile ranges ) if not stated otherwise apache ii score acute physiology and chronic health evaluation ii ( 11 ) , sofa score sequential organ failure assessment ( 8) , po2/fio2 partial oxygen pressure in blood / content of oxygen in inspired air , iap intra - abdominal pressure , app abdominal perfusion pressure , gi gastrointestinal admission diagnosis was gastrointestinal in 27.3 % , ( including hepatopancreatic pathology in 6.6 % ) , pulmonary 19.1 % , cardiac 17.2 % , neurological 15.9 % and polytrauma in 8.7 % . none of the patients had more than four gi symptoms simultaneously.table 2daily and global incidence of gastrointestinal ( gi ) symptoms , intra - abdominal hypertension and gastrointestinal failure among survivors and non - survivorsday 1day 2day 4day 7cumulativetotal number of patients377352264200377 survivors260244194147260 nonsurvivors1171067053117median ( iqr ) number of gi symptomscumulative maximum total1 ( 01)0 ( 01)0 ( 01)0 ( 01)1 ( 02 ) survivors0 ( 01)0 ( 01)0 ( 01)0 ( 01)1 ( 01 ) nonsurvivors1 ( 01)1 ( 01)1 ( 01)1 ( 01)1 ( 02 ) p value*<0.001<0.001<0.001<0.001<0.001absent bowel sounds total ( % ) 125 ( 33.2)82 ( 23.3)42 ( 15.9)29 ( 14.9)142 ( 37.7 ) survivors ( % ) 76 ( 29.2)45 ( 18.4)25 ( 12.9)15 ( 10.3)88 ( 33.8 ) nonsurvivors ( % ) 49 ( 41.9)37 ( 34.9)17 ( 24.3)14 ( 28.6)54 ( 46.2 ) p value*0.018<0.0010.0330.0090.022diarrhoea total ( % ) 26 ( 6.9)40 ( 11.2)46 ( 17.4)39 ( 19.5)81 ( 21.5 ) survivors ( % ) 16 ( 6.2)20 ( 8.2)31 ( 16.0)26 ( 17.7)53 ( 20.4 ) nonsurvivors ( % ) 10 ( 8.5)20 ( 18.9)15 ( 21.4)13 ( 24.5)28 ( 23.9 ) p value*0.3880.0060.2700.3140.498bowel distension total ( % ) 54 ( 14.3)53 ( 15.1)32 ( 12.9)19 ( 9.5)78 ( 20.7 ) survivors ( % ) 33 ( 12.7)34 ( 13.9)21 ( 11.5)10 ( 6.8)48 ( 18.5 ) nonsurvivors ( % ) 21 ( 17.9)19 ( 17.9)11 ( 16.7)9 ( 17.0)30 ( 25.6 ) p value*0.2020.3250.2750.0490.129vomiting / regurgitation total ( % ) 18 ( 4.8)11 ( 3.1)5 ( 1.9)7 ( 3.5)31 ( 15.5 ) survivors ( % ) 11 ( 4.2)7 ( 2.9)3 ( 1.5)5 ( 3.4)22 ( 8.5 ) nonsurvivors ( % ) 7 ( 6.0)4 ( 3.8)2 ( 2.9)2 ( 3.8)9 ( 7.7 ) p value*0.4450.7380.6111.0001.000high gastric residual volume total ( % ) 13 ( 3.4)8 ( 2.3)8 ( 3.0)8 ( 4.0)28 ( 7.4 ) survivors ( % ) 8 ( 3.1)5 ( 2.4)5 ( 2.6)5 ( 3.4)15 ( 5.8 ) nonsurvivors ( % ) 5 ( 4.3)3 ( 2.8)3 ( 4.3)3 ( 5.7)13 ( 11.1 ) p value*0.5560.7060.7120.6970.086gastrointestinal bleeding total ( % ) 16 ( 4.2)13 ( 3.7)5 ( 1.9)4 ( 2.0)24 ( 6.4 ) survivors ( % ) 7 ( 2.7)4 ( 1.6)2 ( 1.0)2 ( 1.4)11 ( 4.2 ) nonsurvivors ( % ) 9 ( 7.7)9 ( 8.5)3 ( 4.3)2 ( 3.8)13 ( 11.1 ) p value*0.0490.0030.1160.2700.020at least 1 gi symptom total168 ( 44.6)146 ( 41.7)109 ( 41.3)81 ( 40.5)227 ( 60.2 ) survivors105 ( 40.4)88 ( 33.8)71 ( 27.3)51 ( 19.6)148 ( 56.9 ) nonsurvivors63 ( 53.8)58 ( 49.6)38 ( 32.5)30 ( 25.6)79 ( 67.5 ) p value*0.0190.0010.0110.0090.0542 or more gi symptoms total62 ( 16.4)46 ( 13.1)21 ( 8.0)20 ( 10.0)76 ( 20.2 ) survivors39 ( 15.0)24 ( 9.2)13 ( 5.0)10 ( 3.8)44 ( 16.9 ) nonsurvivors23 ( 19.7)22 ( 18.8)8 ( 6.8)10 ( 8.5)32 ( 27.4 ) p value*0.2930.0090.2030.0170.0263 or more gi symptoms = gi failure total ( % ) 18 ( 4.8)11 ( 3.1)6 ( 2.3)3 ( 1.5)24 ( 6.4 ) survivors ( % ) 7 ( 2.7)2 ( 0.8)1 ( 0.5)1 ( 0.7)9 ( 3.5 ) nonsurvivors ( % ) 11 ( 9.4)9 ( 8.5)5 ( 7.1)2 ( 3.8)15 ( 12.8 ) p value*0.0080.0010.0060.1720.0014 or more gi symptoms total4 ( 1.1)11 ( 3.1)01 ( 0.5)16 ( 4.2 ) survivors2 ( 0.8)2 ( 0.8)01 ( 0.4)5 ( 1.9 ) nonsurvivors2 ( 1.7)9 ( 7.7)0011 ( 9.4 ) p value*0.5910.0010.002intra - abdominal hypertension total ( % ) 109 ( 28.9)96 ( 27.4)68 ( 25.8)40 ( 20.0)161 ( 42.7 ) survivors ( % ) 77 ( 29.6)71 ( 29.1)55 ( 28.4)32 ( 21.8)111(42.7 ) nonsurvivors ( % ) 32 ( 27.4)25 ( 23.6)13 ( 18.6)8 ( 15.1)50 ( 42.7 ) p value*0.7130.4210.1050.3051.000 * p values refer to comparisons between survivors and nonsurvivorsmaximal daily sum of gi symptoms daily and global incidence of gastrointestinal ( gi ) symptoms , intra - abdominal hypertension and gastrointestinal failure among survivors and non - survivors * p values refer to comparisons between survivors and nonsurvivors maximal daily sum of gi symptoms the incidence of absent bs was 37.7 % ( mortality rate 38.0 % ) , of overt gi bleeding 6.4 % ( mortality rate 54.2 % ) , of iah 42.7 % ( mortality rate 31.1 % ) and of acs 3.6 % ( mortality rate 38.5 % ) . the occurrence of absent bs on day 1 , gi bleeding during the first two days and bowel distension on day 7 were independently associated with 28 day mortality , while vomiting , high grv , diarrhoea and the presence of iah were not predictive.table 3multivariate regression analyses with gi symptoms , failure of enteral nutrition , and intra - abdominal hypertension predicting 28 day survivalday 1p valueorlower ci 95 % upper ci 95 % absent bowel sounds0.0072.4571.2854.700 vomiting / regurgitation0.8770.9030.253.258 maximum grv > 500 ml0.8880.9100.2443.397 diarrhoea0.3871.7000.5115.659 bowel distension0.9160.9540.3982.289 gi bleeding0.0424.4041.05818.333 en < 80 % of caloric needs0.0320.3250.1160.906 iah0.3160.7080.3611.390day 2 absent bowel sounds0.4251.3680.6332.957 vomiting / regurgitation0.8871.1200.2345.352 maximum grv > 500 ml0.6731.3920.3006.469 diarrhoea0.4081.6400.5085.289 bowel distension0.7591.1620.4453.030 gi bleeding0.00819.0932.153169.336 en < 80 % of caloric needs0.0400.3550.1320.952 iah0.0620.4610.2041.041day 4 absent bowel sounds0.1921.7930.7464.310 vomiting / regurgitation0.3663.1470.26337.699 maximum grv > 500 ml0.3981.9950.4029.901 diarrhoea0.3611.6420.5674.758 bowel distension0.8291.1220.3963.173 gi bleeding0.1505.5950.53858.177 en < 80 % of caloric needs0.4401.4370.5733.605 iah0.1270.5120.2171.210day 7 absent bowel sounds0.1622.1570.7356.332 vomiting / regurgitation0.2300.1620.0083.158 maximum grv > 500 ml0.6361.4900.2857.790 diarrhea0.7931.1810.3424.083 bowel distension0.0367.0701.14043.859 gi bleeding0.2493.8220.39237.281 en < 80 % of caloric needs0.9510.9700.3642.582 iah0.1530.4280.1341.372the variables entered into the multivariate analysis were exclusively those listed abovesignificant findings are marked in boldgrv gastric residual volume , gi gastrointestinal , en enteral nutrition , iah intra - abdominal hypertension multivariate regression analyses with gi symptoms , failure of enteral nutrition , and intra - abdominal hypertension predicting 28 day survival the variables entered into the multivariate analysis were exclusively those listed above significant findings are marked in bold grv gastric residual volume , gi gastrointestinal , en enteral nutrition , iah intra - abdominal hypertension the reasons for withholding / stopping en were not documented in 58 % of the cases , and therefore in these cases the presence or absence of fi could not be assessed . gastrointestinal failure ( three or more coincident gi symptoms ) occurred in 24 patients ( 6.4 % ) and was associated with higher 28-day mortality ( 62.5 vs. 28.9 % ) ( fig . none of the gi symptoms alone nor iah or caloric intake < 80 % independently predicted mortality.table 4multivariate regression analysis with admission day variables predicting 28-day mortalityp valueodds ratio95 % ciage0.5421.0050.9901.019body mass index0.2070.9710.9291.016medical profile0.0831.5980.9402.716sepsis0.2231.4000.8152.406fluid balance day 10.8591.0001.0001.000three or more gi symptoms day 10.0353.1891.0829.396renal sofa sub - score<0.0011.4231.1691.733neurological sofa sub - score<0.0011.4441.2311.694haematologic sofa sub - score0.0731.2770.9771.668respiratory sofa sub - score0.3111.1130.9051.368hepatic sofa sub - score0.8040.9620.7101.305cardiovascular sofa sub - score0.8590.9820.8081.195nagelkerke r - square 0.253the table presents the final model of multivariate analysis after removal of clearly correlated variablessignificant findings are marked in boldgi gastrointestinal , sofa sequential organ failure assessment multivariate regression analysis with admission day variables predicting 28-day mortality nagelkerke r - square 0.253 the table presents the final model of multivariate analysis after removal of clearly correlated variables significant findings are marked in bold gi gastrointestinal , sofa sequential organ failure assessment regression analyses including daily sofa sub - scores and the number of gi symptoms revealed increasing number of gi symptoms as an independent predictor of mortality on day 2 and 7 with a tendency towards statistical significance on admission and day 4 ( table 5 ) . roc curves including gif score were not significantly different from the roc curves of the sofa score alone.table 6roc analyses for sofa score alone and sofa combined with the score based on the number of gi symptomssofaaucse95 % ci day 10.7030.030.6430.762 day 20.6820.030.6160.748 day 40.6960.040.6200.772 day 70.6910.050.6020.780 cumulative maximum0.7320.030.6760.789number of gi symptoms day 10.5710.030.5080.635 day 20.6070.030.5410.673 day 40.5910.020.5120.670 day 70.6240.050.5330.714 cumulative maximum0.5810.010.5170.644sofa + number of gi symptoms day 10.7060.030.6470.766 day 20.6870.030.6220.752 day 40.6980.040.6230.772 day 70.7000.040.6140.785 cumulative maximum0.7340.030.6780.790gi gastrointestinal , sofa sequential organ failure assessment , cumulative maximum maximal daily score during the study roc analyses for sofa score alone and sofa combined with the score based on the number of gi symptoms gi gastrointestinal , sofa sequential organ failure assessment , cumulative maximum maximal daily score during the study the current prospective worldwide multicentre study including critically ill patients with an expected duration of mechanical ventilation of more than 6 h demonstrated that a large proportion of these patients had gi symptoms during the first week of admission . this may either be due to data set limitations , definition problems , or may indicate that gi dysfunction is often secondary to and not the primary cause of other organ failure .

forest fire smoke can cool the planet in the daytime by scattering sunlight [ robock , 1988 , 1991 ; westphal and toon , 1991 ] . robock used the difference between forecasted and observed temperatures to suggest that forest fires in canada during 1981 and 1982 , siberia in 1987 , as well as in yellowstone national park in 1988 , cooled the surface under the smoke by 1.5 to 7c in the daytime , but did not have an observable impact on nighttime temperatures . using a numerical model , westphal and toon found a daytime cooling of 5c beneath a smoke plume over the northeastern u.s . , which originated from a fire in western canada in 1982 . the fire , located near yosemite national park , lasted from august to october [ peterson et al . , this exceptional event provides a good opportunity to further quantify radiative forcing by forest fires using modern global climate modeling approaches constrained by both remote and in situ data . the rim fire started on 17 august and spread rapidly until 31 august 2013 due to warm ambient temperatures , high nearsurface wind speeds , and low relative humidity [ peterson et al . , nasa 's studies of emissions and atmospheric composition , clouds and climate coupling by regional surveys field program ( seacrs ) [ toon et al . , multiple instruments on board the nasa dc8 aircraft provide a unique and rich data set on aerosol properties and chemical tracers in rim fire smoke . we use a climate model coupled with a sizeresolved aerosol model to simulate the rim fire smoke in order to examine if a relatively lowresolution model can correctly reproduce the physical and optical properties of rim fire smoke . in section 2 , we introduce the detailed modeling settings and emissions sources used ; in section 3 , we summarized observational data sets used in the study ; in section 4 , we evaluate the model performance on rim fire simulations ; in section 5 , we discuss the radiative impacts of rim fire smoke simulated by model ; in section 6 , we summarize the main findings of this study . physical and optical properties of rim fire smoke are simulated using the community earth system model , version 1 ( cesm1 ) coupled with a sectional aerosol microphysics model , the community aerosol and radiation model for atmospheres ( carma ) [ toon et al . , 1988 ; our version of cesm1/carma includes two groups of particles . the first group is composed of liquid droplets of sulfuric acid that have nucleated from the gas phase . the second group is an internal mixture of primary emitted organics , secondary organics , dust , sea salt , black carbon , and condensed sulfate . ammonia or nitrate is currently not included in carma . to compare with field observations , we extract the nearest model gridbox output ( 1.9 2.5 , 30 min for timestep ) along the flight track spatially and temporally . the core is composed of black carbon and dust , while the shell is composed of materials that are possibly in a liquid state including sulfate , organics , salt , and condensed water . at midvisible wavelengths , the refractive index of black carbon is assumed to be 1.75 0.443i and the index of the shell is assumed to be 1.43 + 0i according to hess et al . . aerosol optical properties ( scattering coefficient , absorption coefficient , single scattering albedo , and asymmetry parameter ) are passed to cesm1 's rapid radiative transfer model for global climate model radiation model [ iacono et al . , 2008 ] for online radiative calculation of forcing and heating rates . the optical properties vary spatially and temporally with dry particle size , relative humidity , black carbon amount , and dust amount [ yu et al . 2015a ] . to better resolve the rim fire smoke , we conducted runs with 1 horizontal resolution instead of the 2 resolution used in yu et al . [ 2015b ] . simulations were run for 5 years ( from 2007 to 2012 ) to spinup the aerosol and chemical tracers . the rim fire smoke was introduced in the sixth year of the model run ( i.e. , year 2013 ) . runs were nudged to offline meteorology ( temperature and winds ) using data from the modern era retrospectiveanalysis for research ( merra ) [ rienecker et al . , 2011 ] for the seacrs period . the nudging relaxes the model toward merra temperature and winds by 1% each time step ( i.e. , 30 min ) . the biomass burning emissions are determined using the daily quick fire emission dataset ( qfed ) [ darmenov and da silva , 2014 ] . emissions are tabulated in the qfed at 0.1 resolution , which we regrid to the model resolution of 1. qfed emissions for rim fires are evaluated and found not sufficient to resolve observed smoke amount we applied the correction factors generated by saide et al . for daily rim fire emissions ( 37.75 to 38.15n and 120.3 to 119.05w ) from 21 to 27 august . table 1 lists the adjusted daily biomass burning emission rate ( g / m / d ) for organic aerosol ( oa ) and black carbon for the rim fire . the ratio of the daily emissions of oa to black carbon ( bc ) ranges from about 26 to 36 . adjusted emission rate ( kg s m ) between 37.75 to 38.15n and 120.3 to 119.05w the injection height of rim fire smoke measured by differential absorption laser / high spectral resolution lidar was 35 km above the ground , which is roughly between 700 to 500 hpa [ peterson et al . we put the rim fire emissions at five pressure levels of cesm between 712 and 581 hpa , with a peak at 618 hpa . the emissions are vertically distributed in a gaussian distribution with a median injection height at 618 hpa and a width of 25 hpa . the peak location ( around 618 hpa ) is consistent with the location of the highest measured organic concentration along the rim fire smoke plumes . we also examined an alternative approaches to inject the smoke near surface or higher than the observed smoke peak , and we found modeled smoke matches observation the best when we inject the smoke near 618 hpa . the modeled particle size distribution is controlled by the size distribution at time of emission , particle microphysical process ( e.g. , coagulation , growth , evaporation , and deposition ) , and condensation of water . the initial particle size distribution for smoke emissions is based on a daily mean size distribution retrieved by aerosol robotic network ( aeronet ) at university of nevadareno on 26 august 2013 when rim fire smoke heavily impacted the site . the model outputs aerosol mass , number , compositions , size , and optical properties along the dc8 flight track ( shown in figure 1 ) when and where the measurements are taken . model 's spatial ( 0.9 1.25 ) and temporal ( 30 min ) resolution is lower than reported observational resolution ( 1 hz , about 200 m ) . simulated aerosol fields are interpolated using the nearest four model grid points and closest time step where and when the measurements are taken . concentration of oa in standard air ( unit : g / std m ) along the flight tracks of the dc8 from 26 to 27 august . starting points of flight of 26 and 27 august are denoted by the black text arrows . details of observational data sets on board of dc8 are documented in table 4 of toon et al . . table 2 lists aerosol properties used in this study and basic information of their instruments . aerosol properties and instruments used in this study las denotes tsi laser aerosol spectrometer . figure 1 shows the measured concentration of submicron oa along the flight tracks of the dc8 on 26 and 27 august as measured by the aerosol mass spectrometer ( ams ) [ dunlea et al . , we consider the smoke from california to montana with the highest concentration oa ( red dashed circle in figure 1 ) as the region of the smoke cloud , because it is most likely to have observable radiative effects due to its large aerosol concentration . figure 2 shows various aerosol properties in rim fire smoke observed by the ams , and the langley aerosol research group experiment ( large , 0.1 to 6.3 m in diameter ) , and as simulated by cesm / carma using the same aerosol size ranges . both model and observations suggest the effective radius ( around 0.14 m , measured by large laser aerosol spectrometer ) of smoke particles remains constant downwind , which is not shown in the figures in this paper . however , the lack of change in effective radius does suggest that no significant conversion of secondary organic aerosol or other gases to aerosols occurred as the smoke moved downwind . in addition observed ngstrm exponent ( ae ) of scattering ( 450 nm to 550 nm ) from large remains constantly along the smoke ( ranging from 1.9 to 2.2 , ae is derived from scattering coefficients measured by nephelometers ) . the smoke simulated in the model remains constant in altitude with limited variation , not much diurnal variations are shown in the model . the simulated oa mass concentration , particle number concentration , surface area concentration , and volume concentration in standard air averaged along the flight track within the dashed circle in figure 1 are within data variability ( 1 standard deviation ) . generally , the oa concentration from rim fire smoke peaks at around 600 hpa and decreases sharply by 23 orders of magnitude up to 400 hpa . the oa concentrations also decrease by 1 order of magnitude between 600 and 800 hpa . the mean of the simulated oa concentration , and the other particle concentrations , is lower than the mean observed between 550 to 600 hpa , although they are still within the variability . it is possible that the concentrations are low because the 1 model is not able to resolve subgrid smoke plumes near the source region . it is also possible that the initial injection profile assumed from 700 to 500 hpa with a peak at 600 hpa is not completely correct . the large spatial and temporal variabilities of smoke ( observed and modeled ) shown in figure 2 are partly because the aircraft is occasionally flying above or outside the smoke plume . ( a ) oa concentration , ( b ) particle number density , ( c)aerosol surface area density , and ( d ) aerosol volume density of standard air simulated by carma ( shown in solid red lines ) and observed in seacrs ( show in dashed blue lines ) . the grey shadings denote temporal and spatial variability of the model ( 1 standard deviation ) . data are averaged from california to montana along the flight track inside the dashed circle in figure 1 . figure 3 shows aerosol extinction along rim fire smoke observed by large ( in blue dashed line [ chen et al . , 2011 ] ) and noaa aerosol cavity ringdown extinction spectrometer ( in green dashed line [ langridge et al . , as shown in figure 3 the model underestimates the aerosol extinction coefficient in the smoke region between 550 and 650 hpa . scattering is measured with dual integrating nephelometers operating at less than 40% and 80% relative humidity so that the extinctions are adjusted to the ambient humidity [ ziemba et al . , absorption is measured by a particle soot absorption photometer . for the region below 650 hpa , the simulations are within the variability of the observations . extinction coefficients at midvisible wavelength simulated by carma ( red ) and observed by large ( blue ) , crds ( green ) . the grey shading denotes temporal and spatial variability of model ( 1 standard deviation ) . the comparisons in figure 2 and figure 3 suggest that cesm / carma generally captures the location and physical properties of rim fire smoke , although the simulations may underestimate concentrations . the underestimation may be a consequence of the 1 resolution being inadequate to fully capture the denser parts of the smoke plume . alternatively , the dailyaveraged input emissions ( without diurnal cycle ) may be an underestimate . figure 4 ( left ) illustrates the oa to bc mass ratio as a function of altitude simulated by carma ( in solid red line ) and calculated based on observational data sets ( in dashed blue line ) . both model and observation suggest that the ratio of oa to bc is quite large in the rim fire smoke . the data suggest that the ratio is about 4060 and the model about 3040 for pressures table 1 shows that the emission of primary oa from the fire is assumed to be 2636 times that of black carbon . . showed that no net secondary organic aerosol formation was observed in the rim fire plume , consistent with observations for most other wildfire plumes studied from aircraft [ cubison et al . the comparison , thus , suggests that the initial injected oatobc ratio may be too low . carma simulations are shown in red , while observations are shown in blue dashed lines . the error bars denote variability ( standard deviation ) of observations ; the grey shading denotes data variability of model . ( right ) single scattering albedo ( ssa ) at midvisible wavelength simulated by carma ( red ) and observed by large ( blue ) . the green lines denote calculated ssa using crds for dry extinction coefficient and pas for dry absorption coefficient . figure 4 ( right ) compares the simulated ssa in the rim fire smoke ( 0.91 ) with two sets of observations : one is humidified particle ssa measured by large [ ziemba et al . , 2013 ] , the other one is dry particle ssa measured by a combination of crds ( measure dry extinction coefficient ) and noaa aerosol photoacoustic absorption spectrometer ( pas , measure dry absorption coefficient ) . both measured ssa values are about 0.95 in the smoke region between 550 hpa and 700 hpa , which is larger than the modeled value ( 0.91 ) . as shown previously , we chose a relatively low value of the imaginary refractive index for bc , and we did not consider any absorption by brown carbon , which was present in this fire [ forrister et al . , both of these assumptions could bias the single scattering albedo high , rather than low as indicated by the observations . the single scattering albedo is likely too low in our simulations because the ratio of organic carbon to black carbon is about 25% too low , but larger particles could also reduce the ssa . the observed oatobc ratio declines above the rim fire smoke for pressures less than 550 hpa . the simulated ratio also declines to about 10 , and as a consequence the simulated single scattering albedo ( ssa ) at midvisible declines for pressures less than 550 hpa . using combined measurements of crds and pas , the observed ssa declines as low as 0.5 at 430 hpa ( not shown in figure 4 ) however , measured absorption coefficients above 500 hpa are close to the detection limit of pas ( 2 10 km ) . the lower ssa values at pressures below 550 hpa are partly due to the lower oa to bc ratio as shown in figure 4 ( left ) . another reason behind the lower ssa between 300 and 500 hpa is the presence of dust . figure 5 shows the modeled dust mass fraction for the size range between 0.2 and 3 m in diameter ( in dashed red lines ) ; modeled mass fractions for the size range between 0.1 and 17 m in diameter ( in dashed black lines ) ; in situ palms data ( particle analysis by laser mass spectrometry , detection limit : 0.22 m in diameter [ murphy et al . , 2006 ] ) are shown in blue lines . both model and observation suggest that dust mass fraction ( in the size range of 0.22 m ) is 1 to 5% in the upper troposphere ( 200 mb to 400 mb ) , while the model also suggests the total dust mass fraction could be as high as 820% . a simulation omitting dust emissions globally suggest the absence of dust ( dashed black lines in figure 4 , right ) leads to a ssa increase by up to 0.05 from 400 hpa to 500 hpa . dust mass fraction : the red dashed line denotes simulated in carma for aerosol in the size range of 0.2 to 2 m in diameter ; the black dashed line denotes simulated in carma for aerosol in the size range of 0.1 to 17 m in diameter ; the blue line denotes observations from palms for the size range of 0.2 to 2 m in diameter . figure 6a shows the modis midvisible aerosol optical depth ( aod , deep blue algorithm [ sayer et al . , 2013 ] ) on 27 august , and figure 6b shows the simulated midvisible aod by carma on 27 august with aeronet retrieved midvisible aod shown in filled circles . near the source region , both modis and aeronet see a value about 1 at midvisible , while the model predicts a value of 0.6 . the underestimation is likely because coarse model spatial resolution ( i.e. , one degree ) is not sufficient to resolve subgrid fire sources . downwind of the rim fire , modeled aod ( 0.30.6 ) , is close to observations . ( a ) modis deep blue midvisible aod of 27 august ; the grey area denotes no retrieval by modis . ( c ) net solar flux ( w m at midvisible ) at surface simulated in carma for the to rim fire smoke simulation minus the control , 20z22z of 27 august . ( d ) surface dimming efficiency for rim fire smoke for 20z22z of 27 august : surface dimming per aod of smoke ( w m per unit of midvisible aod ) . observation of midvisible aod ( level 2 ) by aeronet sites ( university of nevadareno : 39n , 119w ; rimrock : 46n , 116w ; missoula : 46n , 114w ; university of lethbridge : 49n , 112w ) close to the smoke , are shown in filled cycles . aeronet observations are mostly taken between 20 and 22z of 27 august . due to limited observation on 27 august , the aod data of university of nevadareno is taken at 23z of 27 august . to quantify the radiative impacts of rim fire smoke we conducted a control run with the same settings ( meteorology and initial conditions ) as in the base run but without black carbon and organic aerosols emitted in rim fire plumes . figure 6c shows simulated clearsky net radiative flux at the surface ( fsns , w m ) averaged from 20z to 22z of 27 august ( i.e. , 13:0015:00 local time of california ) from the run with rim fire smoke . the simulation suggests that rim fire smoke may prevent 46% of sunlight energy from reaching the surface . figure 6d illustrates the dimming efficiency ( defined as fsns difference per unit midvisible aod , w m per unit of aod ) calculated from the rim fire run and the control run ( 20z22z of 27 august ) . in the simulations the smoke is dimming the surface beneath it by 120140 w m per unit of midvisible aod this is consistent with the solar forcing efficiency of approximately 140 w m per unit midvisible aod measured by the broadband radiometers ( bbr ) and the spectrometer for skyscanning , sun tracking atmospheric research ( 4star ) on the dc8 as it flew gradient legs into and out of the smoke plume perpendicular to the smoke plume axis ( a. bucholtz , et al . , radiative forcing efficiencies and heating rates of forest fire smoke from the 2013 rim during seac4rs , submitted to journal of geophysical research , 2015 ) . the measured forcing efficiency of the smoke was derived from the slope of the net solar irradiance measured by the bbr versus the aod gradient measured by 4star . given high ssa observed ( 0.95 ) and modeled ( 0.91 ) , the surface dimming from rim fire smoke is mostly due to scattering rather than absorption of soot and brown carbon in the smoke . figure 7 shows simulated solar heating rate ( k / d ) difference between runs with and without smoke . up to 1.7 k / d solar heating rate is shown between 600 and 650 hpa near the source region with denser smoke , and near local noon . in the far end of the smoke , the heating rate becomes noisy due to the less dense smoke and the large solar zenith angles as sampling occurred late in the afternoon . given the fact the model with 1 resolution underestimates aod near source region by a factor of 23 as shown in figure 6c , the peak solar heating rate might be several times higher than 1.7 k / d near the source region . absorption of brown carbon [ jacobson , 2014 ] is not modeled in this study , but the single scatter albedo of the simulated smoke is too low . simulated solar heating rate ( k / d ) difference between runs with and without rim fire smoke along the dc8 flight track from 21z on 26 august to 22z on 27 august . the rim fire of 2013 , which consumed the third largest area in california history , produced a dense smoke plume . we simulate this plume for 26 and 27 august , when the smoke extended from the active fires in the sierra nevada mountains near yosemite national park , to southern canada and the great lakes . on these days the nasa dc8 made a large number of observations of the smoke plume properties as part of the seacrs field program . our goal is to determine if a climate model , with relatively coarse resolution , can correctly reproduce the smoke properties , and the radiative impact of the smoke . in table 3 , we list some assumptions and limitations of the model in simulating smoke 's physical and optical properties . the major limitations come from the uncertainties of rim fire emissions and the model 's coarse resolution . uncertainties on injection height , initial size distribution , smoke 's density , and smoke 's aging process can affect the smoke plume mass budget , size distributions , and lifetime . in addition , the smoke optical properties assumed in the model are also directly related to the dimming forcing calculations . assumptions in simulating radiative impact of rim fire smoke observations suggest the initial smoke aerosol concentrations peak between 550 and 650 hpa . using 1 spatial resolution , carma is able to reproduce smoke oa mass concentration , particle number concentration , particle surface area concentration , particle volume concentration , and extinction coefficient within observed data variability , although the simulated mean values for all the parameters or just extinction tend to be biased low with respect to mean observed values . the simulated single scattering albedo ( 0.9 ) is too low compared with observations ( 0.95 ) . surprisingly the simulated single scattering albedo ( ssa ) at mid visible wavelength is lower in the background air above the smoke plumes than in them , due to higher simulated and observed black carbon mass fraction in the aerosols above the main smoke layer and possibly due to the presence of dust . both simulations and palms observations suggest that the dust mass fraction in the upper troposphere is a few percent for particles smaller than 2 m in diameter , while carma simulations also suggests the dust mass fraction in upper troposphere is 820% of total aerosol mass . underestimates of the mean values of extinction coefficients and ssa are likely related to a combination of model resolution being too low , inaccurate emissions estimates , and/or injecting the emissions at a pressure that is slightly too high . the simulations suggest that scattering and absorption ( mostly scattering ) by the rim fire smoke reduced solar insolation at the surface at 20z22z on 27 august ( around local noon time ) by 2050 w m , which is roughly 46% of total solar radiation at the surface . the simulations also suggest that forest fire smoke may reduce surface solar flux with an efficiency of 120150 w m per unit aod . the peak of the simulated solar heating rate is 1.7 k / d , but the model may underestimate the heating rate by a factor of 23 especially near the source region because it underestimates the aerosol concentrations . following robock , this study suggests forest fire smoke , especially on continental scales , should be taken into account when forecasting surface temperature . however , weather forecasts in the mountainous region studied do not have good enough signal to noise levels to reveal the impact of the smoke on the forecasts .

abstractthe rim fire of 2013 , the third largest area burned by fire recorded in california history , is simulated by a climate model coupled with a sizeresolved aerosol model . modeled aerosol mass , number , and particle size distribution are within variability of data obtained from multipleairborne in situ measurements . simulations suggest that rim fire smoke may block 46% of sunlight energy reaching the surface , with a dimming efficiency around 120150 w m2 per unit aerosol optical depth in the midvisible at 13:0015:00 local time . underestimation of simulated smoke single scattering albedo at midvisible by 0.04 suggests that the model overestimates either the particle size or the absorption due to black carbon . this study shows that exceptional events like the 2013 rim fire can be simulated by a climate model with 1 resolution with overall good skill , although that resolution is still not sufficient to resolve the smoke peak near the source region .

Introduction Model Settings and Study Region Observational Data Sets Comparing Simulations With Observations of Rim Fire Smoke Radiative Effects of Rim Fire Smoke Discussions and Conclusions

forest fire smoke can cool the planet in the daytime by scattering sunlight [ robock , 1988 , 1991 ; westphal and toon , 1991 ] . robock used the difference between forecasted and observed temperatures to suggest that forest fires in canada during 1981 and 1982 , siberia in 1987 , as well as in yellowstone national park in 1988 , cooled the surface under the smoke by 1.5 to 7c in the daytime , but did not have an observable impact on nighttime temperatures . , this exceptional event provides a good opportunity to further quantify radiative forcing by forest fires using modern global climate modeling approaches constrained by both remote and in situ data . the rim fire started on 17 august and spread rapidly until 31 august 2013 due to warm ambient temperatures , high nearsurface wind speeds , and low relative humidity [ peterson et al . , multiple instruments on board the nasa dc8 aircraft provide a unique and rich data set on aerosol properties and chemical tracers in rim fire smoke . we use a climate model coupled with a sizeresolved aerosol model to simulate the rim fire smoke in order to examine if a relatively lowresolution model can correctly reproduce the physical and optical properties of rim fire smoke . in section 2 , we introduce the detailed modeling settings and emissions sources used ; in section 3 , we summarized observational data sets used in the study ; in section 4 , we evaluate the model performance on rim fire simulations ; in section 5 , we discuss the radiative impacts of rim fire smoke simulated by model ; in section 6 , we summarize the main findings of this study . physical and optical properties of rim fire smoke are simulated using the community earth system model , version 1 ( cesm1 ) coupled with a sectional aerosol microphysics model , the community aerosol and radiation model for atmospheres ( carma ) [ toon et al . the second group is an internal mixture of primary emitted organics , secondary organics , dust , sea salt , black carbon , and condensed sulfate . the core is composed of black carbon and dust , while the shell is composed of materials that are possibly in a liquid state including sulfate , organics , salt , and condensed water . at midvisible wavelengths , the refractive index of black carbon is assumed to be 1.75 0.443i and the index of the shell is assumed to be 1.43 + 0i according to hess et al . aerosol optical properties ( scattering coefficient , absorption coefficient , single scattering albedo , and asymmetry parameter ) are passed to cesm1 's rapid radiative transfer model for global climate model radiation model [ iacono et al . the optical properties vary spatially and temporally with dry particle size , relative humidity , black carbon amount , and dust amount [ yu et al . to better resolve the rim fire smoke , we conducted runs with 1 horizontal resolution instead of the 2 resolution used in yu et al . the rim fire smoke was introduced in the sixth year of the model run ( i.e. the nudging relaxes the model toward merra temperature and winds by 1% each time step ( i.e. emissions are tabulated in the qfed at 0.1 resolution , which we regrid to the model resolution of 1. qfed emissions for rim fires are evaluated and found not sufficient to resolve observed smoke amount we applied the correction factors generated by saide et al . table 1 lists the adjusted daily biomass burning emission rate ( g / m / d ) for organic aerosol ( oa ) and black carbon for the rim fire . the ratio of the daily emissions of oa to black carbon ( bc ) ranges from about 26 to 36 . adjusted emission rate ( kg s m ) between 37.75 to 38.15n and 120.3 to 119.05w the injection height of rim fire smoke measured by differential absorption laser / high spectral resolution lidar was 35 km above the ground , which is roughly between 700 to 500 hpa [ peterson et al . we put the rim fire emissions at five pressure levels of cesm between 712 and 581 hpa , with a peak at 618 hpa . the emissions are vertically distributed in a gaussian distribution with a median injection height at 618 hpa and a width of 25 hpa . the peak location ( around 618 hpa ) is consistent with the location of the highest measured organic concentration along the rim fire smoke plumes . we also examined an alternative approaches to inject the smoke near surface or higher than the observed smoke peak , and we found modeled smoke matches observation the best when we inject the smoke near 618 hpa . the modeled particle size distribution is controlled by the size distribution at time of emission , particle microphysical process ( e.g. the initial particle size distribution for smoke emissions is based on a daily mean size distribution retrieved by aerosol robotic network ( aeronet ) at university of nevadareno on 26 august 2013 when rim fire smoke heavily impacted the site . the model outputs aerosol mass , number , compositions , size , and optical properties along the dc8 flight track ( shown in figure 1 ) when and where the measurements are taken . table 2 lists aerosol properties used in this study and basic information of their instruments . aerosol properties and instruments used in this study las denotes tsi laser aerosol spectrometer . figure 1 shows the measured concentration of submicron oa along the flight tracks of the dc8 on 26 and 27 august as measured by the aerosol mass spectrometer ( ams ) [ dunlea et al . , we consider the smoke from california to montana with the highest concentration oa ( red dashed circle in figure 1 ) as the region of the smoke cloud , because it is most likely to have observable radiative effects due to its large aerosol concentration . figure 2 shows various aerosol properties in rim fire smoke observed by the ams , and the langley aerosol research group experiment ( large , 0.1 to 6.3 m in diameter ) , and as simulated by cesm / carma using the same aerosol size ranges . both model and observations suggest the effective radius ( around 0.14 m , measured by large laser aerosol spectrometer ) of smoke particles remains constant downwind , which is not shown in the figures in this paper . however , the lack of change in effective radius does suggest that no significant conversion of secondary organic aerosol or other gases to aerosols occurred as the smoke moved downwind . in addition observed ngstrm exponent ( ae ) of scattering ( 450 nm to 550 nm ) from large remains constantly along the smoke ( ranging from 1.9 to 2.2 , ae is derived from scattering coefficients measured by nephelometers ) . the smoke simulated in the model remains constant in altitude with limited variation , not much diurnal variations are shown in the model . the simulated oa mass concentration , particle number concentration , surface area concentration , and volume concentration in standard air averaged along the flight track within the dashed circle in figure 1 are within data variability ( 1 standard deviation ) . generally , the oa concentration from rim fire smoke peaks at around 600 hpa and decreases sharply by 23 orders of magnitude up to 400 hpa . the mean of the simulated oa concentration , and the other particle concentrations , is lower than the mean observed between 550 to 600 hpa , although they are still within the variability . it is possible that the concentrations are low because the 1 model is not able to resolve subgrid smoke plumes near the source region . it is also possible that the initial injection profile assumed from 700 to 500 hpa with a peak at 600 hpa is not completely correct . ( a ) oa concentration , ( b ) particle number density , ( c)aerosol surface area density , and ( d ) aerosol volume density of standard air simulated by carma ( shown in solid red lines ) and observed in seacrs ( show in dashed blue lines ) . the grey shadings denote temporal and spatial variability of the model ( 1 standard deviation ) . figure 3 shows aerosol extinction along rim fire smoke observed by large ( in blue dashed line [ chen et al . , as shown in figure 3 the model underestimates the aerosol extinction coefficient in the smoke region between 550 and 650 hpa . scattering is measured with dual integrating nephelometers operating at less than 40% and 80% relative humidity so that the extinctions are adjusted to the ambient humidity [ ziemba et al . , absorption is measured by a particle soot absorption photometer . for the region below 650 hpa , the simulations are within the variability of the observations . extinction coefficients at midvisible wavelength simulated by carma ( red ) and observed by large ( blue ) , crds ( green ) . the grey shading denotes temporal and spatial variability of model ( 1 standard deviation ) . the comparisons in figure 2 and figure 3 suggest that cesm / carma generally captures the location and physical properties of rim fire smoke , although the simulations may underestimate concentrations . the underestimation may be a consequence of the 1 resolution being inadequate to fully capture the denser parts of the smoke plume . both model and observation suggest that the ratio of oa to bc is quite large in the rim fire smoke . the data suggest that the ratio is about 4060 and the model about 3040 for pressures table 1 shows that the emission of primary oa from the fire is assumed to be 2636 times that of black carbon . showed that no net secondary organic aerosol formation was observed in the rim fire plume , consistent with observations for most other wildfire plumes studied from aircraft [ cubison et al . the comparison , thus , suggests that the initial injected oatobc ratio may be too low . the error bars denote variability ( standard deviation ) of observations ; the grey shading denotes data variability of model . ( right ) single scattering albedo ( ssa ) at midvisible wavelength simulated by carma ( red ) and observed by large ( blue ) . figure 4 ( right ) compares the simulated ssa in the rim fire smoke ( 0.91 ) with two sets of observations : one is humidified particle ssa measured by large [ ziemba et al . , 2013 ] , the other one is dry particle ssa measured by a combination of crds ( measure dry extinction coefficient ) and noaa aerosol photoacoustic absorption spectrometer ( pas , measure dry absorption coefficient ) . both measured ssa values are about 0.95 in the smoke region between 550 hpa and 700 hpa , which is larger than the modeled value ( 0.91 ) . as shown previously , we chose a relatively low value of the imaginary refractive index for bc , and we did not consider any absorption by brown carbon , which was present in this fire [ forrister et al . , both of these assumptions could bias the single scattering albedo high , rather than low as indicated by the observations . the single scattering albedo is likely too low in our simulations because the ratio of organic carbon to black carbon is about 25% too low , but larger particles could also reduce the ssa . the observed oatobc ratio declines above the rim fire smoke for pressures less than 550 hpa . the simulated ratio also declines to about 10 , and as a consequence the simulated single scattering albedo ( ssa ) at midvisible declines for pressures less than 550 hpa . using combined measurements of crds and pas , the observed ssa declines as low as 0.5 at 430 hpa ( not shown in figure 4 ) however , measured absorption coefficients above 500 hpa are close to the detection limit of pas ( 2 10 km ) . the lower ssa values at pressures below 550 hpa are partly due to the lower oa to bc ratio as shown in figure 4 ( left ) . figure 5 shows the modeled dust mass fraction for the size range between 0.2 and 3 m in diameter ( in dashed red lines ) ; modeled mass fractions for the size range between 0.1 and 17 m in diameter ( in dashed black lines ) ; in situ palms data ( particle analysis by laser mass spectrometry , detection limit : 0.22 m in diameter [ murphy et al . both model and observation suggest that dust mass fraction ( in the size range of 0.22 m ) is 1 to 5% in the upper troposphere ( 200 mb to 400 mb ) , while the model also suggests the total dust mass fraction could be as high as 820% . dust mass fraction : the red dashed line denotes simulated in carma for aerosol in the size range of 0.2 to 2 m in diameter ; the black dashed line denotes simulated in carma for aerosol in the size range of 0.1 to 17 m in diameter ; the blue line denotes observations from palms for the size range of 0.2 to 2 m in diameter . figure 6a shows the modis midvisible aerosol optical depth ( aod , deep blue algorithm [ sayer et al . near the source region , both modis and aeronet see a value about 1 at midvisible , while the model predicts a value of 0.6 . , one degree ) is not sufficient to resolve subgrid fire sources . downwind of the rim fire , modeled aod ( 0.30.6 ) , is close to observations . ( c ) net solar flux ( w m at midvisible ) at surface simulated in carma for the to rim fire smoke simulation minus the control , 20z22z of 27 august . ( d ) surface dimming efficiency for rim fire smoke for 20z22z of 27 august : surface dimming per aod of smoke ( w m per unit of midvisible aod ) . due to limited observation on 27 august , the aod data of university of nevadareno is taken at 23z of 27 august . to quantify the radiative impacts of rim fire smoke we conducted a control run with the same settings ( meteorology and initial conditions ) as in the base run but without black carbon and organic aerosols emitted in rim fire plumes . , 13:0015:00 local time of california ) from the run with rim fire smoke . the simulation suggests that rim fire smoke may prevent 46% of sunlight energy from reaching the surface . figure 6d illustrates the dimming efficiency ( defined as fsns difference per unit midvisible aod , w m per unit of aod ) calculated from the rim fire run and the control run ( 20z22z of 27 august ) . in the simulations the smoke is dimming the surface beneath it by 120140 w m per unit of midvisible aod this is consistent with the solar forcing efficiency of approximately 140 w m per unit midvisible aod measured by the broadband radiometers ( bbr ) and the spectrometer for skyscanning , sun tracking atmospheric research ( 4star ) on the dc8 as it flew gradient legs into and out of the smoke plume perpendicular to the smoke plume axis ( a. bucholtz , et al . , radiative forcing efficiencies and heating rates of forest fire smoke from the 2013 rim during seac4rs , submitted to journal of geophysical research , 2015 ) . the measured forcing efficiency of the smoke was derived from the slope of the net solar irradiance measured by the bbr versus the aod gradient measured by 4star . given high ssa observed ( 0.95 ) and modeled ( 0.91 ) , the surface dimming from rim fire smoke is mostly due to scattering rather than absorption of soot and brown carbon in the smoke . up to 1.7 k / d solar heating rate is shown between 600 and 650 hpa near the source region with denser smoke , and near local noon . in the far end of the smoke , the heating rate becomes noisy due to the less dense smoke and the large solar zenith angles as sampling occurred late in the afternoon . given the fact the model with 1 resolution underestimates aod near source region by a factor of 23 as shown in figure 6c , the peak solar heating rate might be several times higher than 1.7 k / d near the source region . absorption of brown carbon [ jacobson , 2014 ] is not modeled in this study , but the single scatter albedo of the simulated smoke is too low . simulated solar heating rate ( k / d ) difference between runs with and without rim fire smoke along the dc8 flight track from 21z on 26 august to 22z on 27 august . the rim fire of 2013 , which consumed the third largest area in california history , produced a dense smoke plume . we simulate this plume for 26 and 27 august , when the smoke extended from the active fires in the sierra nevada mountains near yosemite national park , to southern canada and the great lakes . on these days the nasa dc8 made a large number of observations of the smoke plume properties as part of the seacrs field program . our goal is to determine if a climate model , with relatively coarse resolution , can correctly reproduce the smoke properties , and the radiative impact of the smoke . in table 3 , we list some assumptions and limitations of the model in simulating smoke 's physical and optical properties . the major limitations come from the uncertainties of rim fire emissions and the model 's coarse resolution . uncertainties on injection height , initial size distribution , smoke 's density , and smoke 's aging process can affect the smoke plume mass budget , size distributions , and lifetime . in addition , the smoke optical properties assumed in the model are also directly related to the dimming forcing calculations . assumptions in simulating radiative impact of rim fire smoke observations suggest the initial smoke aerosol concentrations peak between 550 and 650 hpa . using 1 spatial resolution , carma is able to reproduce smoke oa mass concentration , particle number concentration , particle surface area concentration , particle volume concentration , and extinction coefficient within observed data variability , although the simulated mean values for all the parameters or just extinction tend to be biased low with respect to mean observed values . the simulated single scattering albedo ( 0.9 ) is too low compared with observations ( 0.95 ) . surprisingly the simulated single scattering albedo ( ssa ) at mid visible wavelength is lower in the background air above the smoke plumes than in them , due to higher simulated and observed black carbon mass fraction in the aerosols above the main smoke layer and possibly due to the presence of dust . both simulations and palms observations suggest that the dust mass fraction in the upper troposphere is a few percent for particles smaller than 2 m in diameter , while carma simulations also suggests the dust mass fraction in upper troposphere is 820% of total aerosol mass . the simulations suggest that scattering and absorption ( mostly scattering ) by the rim fire smoke reduced solar insolation at the surface at 20z22z on 27 august ( around local noon time ) by 2050 w m , which is roughly 46% of total solar radiation at the surface . the simulations also suggest that forest fire smoke may reduce surface solar flux with an efficiency of 120150 w m per unit aod . the peak of the simulated solar heating rate is 1.7 k / d , but the model may underestimate the heating rate by a factor of 23 especially near the source region because it underestimates the aerosol concentrations . following robock , this study suggests forest fire smoke , especially on continental scales , should be taken into account when forecasting surface temperature . however , weather forecasts in the mountainous region studied do not have good enough signal to noise levels to reveal the impact of the smoke on the forecasts .

it is mainly driven by peer pressure , homelessness , drug addiction , and poverty just to mention a few of the driving forces . in zimbabwe like most african countries , prostitution although not legal , it is still common among the poverty stricken communities . a lot of zimbabwean women are turning into prostitution in botswana and south africa due to poverty and unemployment in their home country . some individuals turn into prostitution to fund their drug / alcohol addiction behaviour [ 6 , 10 , 11 ] , which often leads to entrapment of women who prostitute in this sex - for - drugs lifestyle and others resort to drug / alcohol misuse to effectively sale their bodies without remorse [ 5 , 11 , 12 ] . clearly , drug / alcohol misuse can increase prostitution with less safe sex , which in turn leads to increase in sexually transmitted infections ( including hiv ) . prostitution associated with drug / alcohol misuse is among the main factors in the spread of hiv in the united states . similarly , in mauritus 75% of the prostitutes reported injecting drugs and 23% said they never used nonsterile injecting equipment which all pose a serious risk of hiv infection . among drug misusing prostitutes in mauritus who had been previously tested for hiv 13% were hiv positive , yet 68% of the prostitutes said they had not consistently used condoms during the previous three months . hiv prevalence among female sex workers ( prostitutes ) varies widely but in some countries it is more than 20 times higher than the hiv prevalence of the general population . a number of mathematical models have been developed to understand the role of social and behavioral processes in hiv transmission [ 1719 ] . the later developed a cross impact model to explore intentional transmission of hiv by nondisclosure of status in various risky situations . despite the fact that prostitution and hiv / aids are somehow linked , they have not been mathematically accounted for . here following ideas generated by bhunu and mushayabasa , the authors offer an indepth analysis of prostitution and hiv / aids transmission from the mathematical persipective . the rest of the manuscript is presented as follows . in section 2 , the model is presented and analysed . for this model the authors take a prostitute as anyone who buys sex to satisfy his / her own sexual desires or sells sex for material gain irregardless of that person 's gender . the model subdivides the population based on prostitution and hiv / aids status . these are susceptibles who are not prostitutes s n(t ) , susceptible prostitutes s p(t ) , and hiv positive people not yet displaying aids symptoms and are not prostitutes i h(t ) , hiv positive prostitutes not yet displaying aids symptoms i hp(t ) , hiv positive individuals who are not prostitutes and displaying aids symptoms a h(t ) , hiv positive prostitutes displaying aids symptoms a hp(t ) , and finaly aids patients on treatment a ht(t ) . it is assumed that susceptibles who are not prostitutes ( s n(t ) ) are recruited into the population through birth at a constant rate and that no person is born being a prostitute . individuals in different human subgroups suffer from natural death at a constant rate , which is proportional to the number in each class . we assume that interaction is homogoneous . although there are many causes of prostitution , here we dwell on peer pressure and poverty which are the main driving forces of prostitution among zimbabweans . the distinction between peer pressure and poverty as factors contributing to prostitution is not clear cut . individuals who are not prostitutes turn into prostitution due to poverty related peer pressure at rate p to move into the corresponding classes of prostitutes . however , it is worth mentioning here that aids patients who have lost hope of living and are poverty stricken acquire prostitution habits at rate p with < [ 0,1 ) signifying the reduced chances an aids patient has of attracting clients due to the on and off sickness . once they become prostitutes , they move into corresponding class of prostitutes displaying aids symptoms . we further assume that aids patients on treatment are nolonger prostitutes as they have been effectively counselled . some prostitutes especially those driven by poverty leave the prostitution trade after getting meaningful employment and/or getting into financially stable relationship at rate p to move into their corresponding nonprostituting classes . however , for those prostitutes in the aids stage of disease progression some are forced to leave prostitution due to sickness at rate s as they are not likely to be gainfully employed or get a stable financially secure relationship while they are always on and off the sick bed . susceptible nonprostitutes and prostitutes acquire hiv following sexual contact with an infected individual at rates ( 1 )(t ) and ( 1 ) h(t ) , respectively , to get into their corresponding hiv infected classes not yet showing aids symptoms ( i h(t ) and i hp(t ) ) . the role of condom use as a strategy to limit hiv infection is captured by ( 0,1 ) signifying that condom use offers some degree of protection against hiv infection and 1 signifies increased risk prostitutes have of contracting hiv infection as they often engage in less safe sex while they are under the influence alcohol and/or drugs . the force of infection for hiv h(t ) is given by ( 2)ht=hch(ih(t)+ihp(t)+h(ah(t)+ahp(t))+taht(t))n(t ) , where h is the probability of hiv transmission per sexual contact , c h is the effective contact rate for hiv infection to occur , and h > 1 models the fact that individuals in the aids stage and not on antiretroviral therapy are more infectious since the viral load is correlated with infectiousness . it is assumed that individuals on antiretroviral therapy transmit infection at the smallest rate t ( with 0 < t < 1 ) because of the fact that these individuals have very small viral load . it has been estimated by an analysis of longitudinal cohort data that antiretroviral therapy reduces per - partnership infectivity by as much as 60% ( so that t = 0.4 ) . hiv infected nonprostitutes and prostitutes progress to the aids stage of disease progression at rates n and p , respectively , with n p as prostitutes are more likely to experience multiple infections with other sexual transmitted infections , contributing to the more rapid deterioration of the immune system . we assume that antiretroviral therapy is given to aids individuals who are ill and have experienced aids - defining symptoms , or whose cd4 + t cell count is below 200/l , which is the recommended aids defining stage . thus , aids patients are assumed to get antiretroviral therapy at a constant rate . individuals in the aids stage and not yet on antiretroviral therapy experience aids related death at a rate > 0 and their corresponding parts on antiretroviral therapy eventually succumb to aids - induced mortality at a reduced rate with the parameter ( 0 < < 1 ) . unless stated otherwise , values for the parameters in the simulations are given in table 1 . based on the aforementioned , the following system of differential equations describes the dynamics of prostitution and drug ( alcohol ) misuse : ( 3)sn'(t)=(1)hsn(p+)sn+psp , ih'(t)=(1)hsnpih(+n)ih+pihp , ah'(t)=nihpah(++)ah+sahp , sp'(t)=psn(1)hsp(+p)sp , ihp'(t)=pih+(1)hsp(+p+p)ihp , ahp'(t)=pah+pihp(+++s)ahp , aht'(t)=ah+ahp(+)aht . in this section , we study some the basic results of solutions of model system ( 3 ) which are essential in the proofs of stability and persistence results . the nonnegative orthant + is positively invariant for model system ( 3 ) . the nonnegative orthant + is positively invariant for model system ( 3 ) . proofmodel system ( 3 ) can be written as x = ax + b with(4)a=a100p0001ha200p000na300s0p00a40000p01ha50000p0pa600000a7 , b=000000a1=p+(1)h+ , a2=p++n , a3=p+++,a4=(1)h++p , a5=+p+p , a6=+++s , a7=+.note that b 0 and a is a metzler matrix ( a matrix is whose off - diagonal entries are nonnegative ) which implies that system ( 3 ) is positively invariant in + . model system ( 3 ) can be written as x = ax + b with(4)a=a100p0001ha200p000na300s0p00a40000p01ha50000p0pa600000a7 , b=000000a1=p+(1)h+ , a2=p++n , a3=p+++,a4=(1)h++p , a5=+p+p , a6=+++s , a7=+.note that b 0 and a is a metzler matrix ( a matrix is whose off - diagonal entries are nonnegative ) which implies that system ( 3 ) is positively invariant in + . each nonnegative solution is bounded in l -norm by max{n(0 ) , /}. each nonnegative solution is bounded in l -norm by max{n(0 ) , /}. proofthe l -norm of each nonnegative solution n and it satisfies the inequality n n . solutions to the equation m = m are monotone increasing and bounded by / if m(0 ) < /. they are monotone decreasing and bounded above if m(0 ) /. since n m , the claim follows . the l -norm of each nonnegative solution n and it satisfies the inequality n n . solutions to the equation m = m are monotone increasing and bounded by / if m(0 ) < /. they are monotone decreasing and bounded above if m(0 ) /. since n m , the claim follows . the region = { ( s n , i h , a h , s n , i hp , a hp , a ht ) + : n max{n(0 ) , / } } is invariant and attracting for system ( 3 ) . the region = { ( s n , i h , a h , s n , i hp , a hp , a ht ) + : n max{n(0 ) , / } } is invariant and attracting for system ( 3 ) . thus , the model is mathematically and epidemiologically well - posed and it is sufficient to consider the dynamics of the flow generated by the system ( 3 ) in . theorem 4 . for every nonzero , nonnegative initial value , solutions of model system ( 3 ) exist for all times . for every nonzero , nonnegative initial value , solutions of model system ( 3 ) exist for all times . prooflocal existence of solutions follows from standard arguments , since the right hand side of system ( 3 ) is locally liptschitz continuous . local existence of solutions follows from standard arguments , since the right hand side of system ( 3 ) is locally liptschitz continuous . the disease - free equilibrium point of model system ( 3 ) is given by ( 5)e0=sn0,ih0,ah0,sp0,ihp0,ahp0,aht0=+pp++p,0,0,pp++p,0,0,0 . following van den driessche and watmough we have the reproduction number of the model as ( 6)rhp=hch1t+hm5pm1s+m2+p2+m4nph1m5m6m2+sm1+p++np + hchh2(1)(t+hm5)(pp(s+m2)+(p+m4)nm3)h1m5m6(m2+s)(m1(+p)+(+n)p ) + hchh2(1)(p+m3)h1(m1(+p)+(+n)p ) , with m 1 = p + + n , m 2 = p + + + , m 3 = + p + p , m 4 = + + + s , m 5 = + , m 6 = + + , h 1 = p + + p , and h 2 = h 1 p throughout the paper . here hp defines the number of secondary hiv cases generated by one hiv infected individual in community where prostitution is rife and condom use coupled with antiretroviral therapy is the intervention strategies available . theorem 5 follows from van den driessche and watmough theorem 2 . the disease - free equilibrium 0 is locally asymptotically stable for hp < 1 and unstable otherwise . the disease - free equilibrium 0 is locally asymptotically stable for hp < 1 and unstable otherwise . using a theorem from castillo - chavez et al . the disease - free equilibrium of system ( 3 ) is globally asymptotically stable provided that hp < 1 . the disease - free equilibrium of system ( 3 ) is globally asymptotically stable provided that hp < 1 . prooffollowing castillo - chavez et al . , we write system ( 3 ) in the form : ( 7)x't = fx , y , y'(t)=g(x , y ) , g(x,0)=0 , where x = ( s n , s p ) and y = ( i h , a h , i hp , a hp , a ht ) . here , x + denotes ( its components ) the number of uninfected individuals and y + denotes ( its components ) the number of infected individuals . the disease - free equilibrium is now denoted by 0 = ( x 0 , 0 ) , where x 0 = ( h 2/h 1 , p/h 1 ) . here , we have to prove that the two conditions ( h1 ) and ( h2 ) are met . ( 8)h1 for x't = fx,0 , x0 is globally asymptotically stableh2 gx , y = uyg^x , y , g^x , y0 , for x , y. consider f(x,0)=-(p+)sn+psppsn-(+p)sp,(9 ) ( 10)g^(x , y)=g1^(x , y)g2^(x , y)g3^(x , y)g4^(x , y)g5^(x , y)=(1)hch(ih+ihp+h(ah+ahp)taht)h2h1snn0(1)hch(ih+ihp+h(ah+ahp)taht)ph1spn00 . clearly , g2^(x , y)=g4^(x , y)=g5^(x , y)=0 and so we have to show that g1^(x , y ) and g3^(x , y ) are both positive . assume that statements in ( 11 ) are true , ( 11)i h2h1<snn , ( ii ) ph1<spn . from statements in ( 11 ) we have that ( 12)h2+ph1 < sn+spnn < sn+sd , since h2+ph1=1 , and a contradiction as statement ( 12 ) is not true . thus , ( 13)nsn+spi h2h1snn , ( ii ) ph1spn . therefore the disease - free equilibrium 0 is globally asymptotically stable . following castillo - chavez et al . , we write system ( 3 ) in the form : ( 7)x't = fx , y , y'(t)=g(x , y ) , g(x,0)=0 , where x = ( s n , s p ) and y = ( i h , a h , i hp , a hp , a ht ) . here , x + denotes ( its components ) the number of uninfected individuals and y + denotes ( its components ) the number of infected individuals . the disease - free equilibrium is now denoted by 0 = ( x 0 , 0 ) , where x 0 = ( h 2/h 1 , p/h 1 ) . here , we have to prove that the two conditions ( h1 ) and ( h2 ) are met . = fx,0 , x0 is globally asymptotically stableh2 gx , y = uyg^x , y , g^x , y0 , for x , y. consider f(x,0)=-(p+)sn+psppsn-(+p)sp,(9 ) ( 10)g^(x , y)=g1^(x , y)g2^(x , y)g3^(x , y)g4^(x , y)g5^(x , y)=(1)hch(ih+ihp+h(ah+ahp)taht)h2h1snn0(1)hch(ih+ihp+h(ah+ahp)taht)ph1spn00 . clearly , g2^(x , y)=g4^(x , y)=g5^(x , y)=0 and so we have to show that g1^(x , y ) and g3^(x , y ) are both positive . assume that statements in ( 11 ) are true , ( 11)i h2h1<snn , ( ii ) ph1<spn . from statements in ( 11 ) we have that ( 12)h2+ph1 < sn+spnn < sn+sd , since h2+ph1=1 , and a contradiction as statement ( 12 ) is not true . thus , ( 13)nsn+spi h2h1snn , ( ii ) ph1spn . thus , g^(x , y)0 . the utility of the basic reproductive number has been questioned , but we use it here as a threshold with the understanding that there may be further complexities in its application . here we look into the following scenarios . case 1i ( every person is not prostitute ) . in this case we have p = p = s = 0 , p = n , and = 1 and hp becomes ( 14)rh = lim(p,p,s,,p)(0,0,0,1,n)rhp=hch(1)+n1+n(t+hm5)(+1)m5m6 . to check the impact of antiretroviral therapy on a prostitution - free environment we take the partial derivative of h with respect to to obtain ( 15)rh=hchn1+1t+h+++m5m62(+n ) . equation ( 15 ) is negative only when 0 ( ( h 1) + ( h 1))/(1 t ) < . thus , as long as 0 ( ( h 1) + ( h 1))/(1 t ) < , then antretroviral therapy may be able to keep hiv infections in check in a prostitution - free environment where condom use is in place . however , for 0 < ( ( h 1) + ( h 1))/(1 t ) , the story is different as ( 15 ) will be positive , suggesting that the levels of antiretroviral therapy are not high enough to make a significant contribution to the to the reduction of hiv / aids cases . p = s , then ( 16)rq = limprhp=hch(1)+nntm5m6+nhm6 + 1,limrq=hch(1)+nnhm6 + 1 . clearly , q is decreasing function of bounded above by ( h c h(1 )/( + n))( n h / m 6 + 1 ) . this result suggests that antiretroviral therapy is more effective in communities with less levels of prostitution as there will be less chances of getting infected with different strains of hiv . thus , prostitution should be targetted as a way of controlling the spread of multiple strains of hiv . multiple strains of hiv may be difficult to keep in check using the commonly used antiretroviral therapy currently available . clearly lim1 hp = 0 suggests even in the presence of prostitution increase in the consistant and correct use of condoms will be able to keep hiv infections under control . this result suggests that safe sex ( condom use ) should always be encouraged especially in areas where prostitution is endemic . this scenario happens when ( s n , s p ) ( 0 , / ) and ( p , p , s , n)( , 0,0 , p ) so that hp becomes ph given by ( 17)rph = lim(p,p,s,n)(,0,0,p)rhp=0 . the fact that hp = 0 suggests if all people become prostitutes , then in the long term the entire population will be hiv infected ( therefore no new hiv infections ) as prostitutes often operate under the influence of drugs and/or alcohol resulting in inconstistancy in the use of condoms . furthermore , lim hp = suggests that increase in the levels prostitution enhances the risk of acquiring hiv . theoretically , the following hiv / aids endemic equilibria exist : ( i ) the prostitution free - endemic equilibrium , ( ii ) the prostitution only endemic equilibrium , and ( iii ) equilibria where both prostitutes and nonprostitutes coexist . however , the first two are trivial cases and therefore are not going to be discussed here , so only the endemic equilibrium where hiv is in a population with both prostitutes and nonprostitutes is considered . this equilibrium point in terms of the force of infection h is given by ( 18)e=(sn,ih,ah,sp,ihp,ahp,aht ) with the explicit expressions for s n , i h , a h , s p , i hp , a hp , and a ht being cumbersome to be written explicitly . the permanence of the disease destabilizes the disease - free equilibrium 0 since hp > 1 , and the coexistence of the prostitutes and nonprostitutes endemic equilibrium exists . lemma 7 . system ( 3 ) is uniformly persistent on . system ( 3 ) is uniformly persistent on . proofuniform persistence system of ( 3 ) implies there exists a constant > 0 such that any solution of ( 3 ) which starts in ( the interior of ) remains in . also , sn0,ih0,ah0,sp0,ihp , ahp0,aht0 , and ( 19)liminftsn(t ) , liminftih(t),liminftah(t ) , liminftsp(t),liminftihp(t ) , liminftahp(t),liminftaht(t ) . define the following korobeinikov - maini type lyapunov function ( 20)v(sn , ih , ah , sp , ihp , ahp , aht ) = ( snsnlnsn)+(ihihlnih)+(ahahlnah ) + ( spsplnsp)+(ihpihplnihp ) + ahpahplnahp+ahtahtlnaht , which is continuous for all ( s n , i h , a h , s p , i hp , a hp , a ht ) > 0 and satisfies ( cf . ) consequently , the endemic equilibrium is the only extremum and the global minimum of the function v + . also , v(s n , i h , a h , s p , i hp , a hp , a ht ) > 0 and v(s n , i h , a h , s p , i hp , a hp , a ht ) = 0 only at . thus , v(s n , i h , a h , s p , i hp , a hp , a ht ) is a lyapunov function . at equilibrium , = ( h ( 1 ) + p + )s n p s p , substituting this into the time derivative of v along the solution path of model system ( 3 ) , we have ( 22)v'=(snsn)sn'sn+(ihih)ih'ih+(ahah)ah'ah+(spsp)sp'sp+ihpihpihp'ihp+(ahpahp)ahp'ahp+(ahtaht)aht'ahtsnsn2sn+gsn , ih , ah , sp , ihp , ahp , aht , where g can be shown to be nonpositive using barbalat lemma . hence , v(s n , i h , a h , s p , i hp , a hp , a ht ) 0 with equality only at . the only invariant set in is the endemic equilibrium . thus , all solutions of ( 3 ) which intersect converge to the invariant ( singleton ) { } . therefore , from lyapunov - lasalle invariance principle , system ( 3 ) is uniformly persistent . uniform persistence system of ( 3 ) implies there exists a constant > 0 such that any solution of ( 3 ) which starts in ( the interior of ) remains in . also , sn0,ih0,ah0,sp0,ihp , ahp0,aht0 , and ( 19)liminftsn(t ) , liminftih(t),liminftah(t ) , liminftsp(t),liminftihp(t ) , liminftahp(t),liminftaht(t ) . define the following korobeinikov - maini type lyapunov function ( 20)v(sn , ih , ah , sp , ihp , ahp , aht ) = ( snsnlnsn)+(ihihlnih)+(ahahlnah ) + ( spsplnsp)+(ihpihplnihp ) + ahpahplnahp+ahtahtlnaht , which is continuous for all ( s n , i h , a h , s p , i hp , a hp , a ht ) > 0 and satisfies ( cf . ) consequently , the endemic equilibrium is the only extremum and the global minimum of the function v + . also , v(s n , i h , a h , s p , i hp , a hp , a ht ) > 0 and v(s n , i h , a h , s p , i hp , a hp , a ht ) = 0 only at . thus , v(s n , i h , a h , s p , i hp , a hp , a ht ) is a lyapunov function . at equilibrium , = ( h ( 1 ) + p + )s n p s p , substituting this into the time derivative of v along the solution path of model system ( 3 ) , we have ( 22)v'=(snsn)sn'sn+(ihih)ih'ih+(ahah)ah'ah+(spsp)sp'sp+ihpihpihp'ihp+(ahpahp)ahp'ahp+(ahtaht)aht'ahtsnsn2sn+gsn , ih , ah , sp , ihp , ahp , aht , where g can be shown to be nonpositive using barbalat lemma . hence , v(s n , i h , a h , s p , i hp , a hp , a ht ) 0 with equality only at . the only invariant set in is the endemic equilibrium . thus , all solutions of ( 3 ) which intersect converge to the invariant ( singleton ) { } . therefore , from lyapunov - lasalle invariance principle , system ( 3 ) is uniformly persistent . in this section , we make use of matlab to analyse model system ( 3 ) using model parameters in table 1 and the following initial conditions : s n(0 ) = 3 10 , s p(0 ) = 5 10 , i h(0 ) = 3 10 , i hp(0 ) = 2 10 , a h(0 ) = 10 , a hp(0 ) = 10 , and a ht(0 ) = 0 . in figure 2 , the possible effects prostitution has on hiv levels are noted by varying the percentages of prostitutes in the community in the presence of antiretroviral therapy and condom use . it is noted that an increase in the percentage of prostitutes results in an increase in the number of hiv infected people as prostitutes mostly operate under the influence of drugs and therefore have difficulties to properly use condoms . this suggests increase in employment creation coupled with counselling will be able to keep the prostitution in check as most people resort to prostitution due to poverty . this result somehow points to poverty as driving prostitution which in turn drives hiv in poor settings . thus , poverty eradication reduces prostitution which in turn results in a reduction in the number of hiv infections in the population . in figure 3 , it is shown that proper use of condoms will be able to keep hiv infections in check , even when prostitution exists in the population . this result suggests that encouraging the proper use of condoms should be encouraged especially in communities where prostitution is rife . it is important to note that proper use of condoms may be difficult especially among prostitutes as they often engage in sex while they are under the influence of drugs / alcohol . a mathematical model for the effect of prostitution on the transmission dynamics of hiv / aids is presented as a system of differential equations . the disease - free equilibrium point of the model is shown to be globally asymptotically stable when the corresponding reproduction number is less than unity . ( i ) increase in the levels of prostitution in a community enhances the chances of acquiring hiv if not infected and acquiring another hiv strain if already infected . ( ii ) antiretroviral therapy is more effective in communities with less prostitution as less prostitution corresponds to reduced chances of one getting infected with different strains of hiv . infection with different strains of hiv makes the hiv infected person worse as some strains may not respond to the antiretroviral therapy regimen available especially in resource - constrained settings . ( iii ) increase in the rates of proper condom use may be able to keep hiv infections even in communities where prostitution is rife . graphical representations clearly show that the number of hiv infected people and those displaying aids symptoms is higher in communities with more prostitutes than in communities with less prostitutes . this suggests that prostitution promotes hiv transmission , a result in total agreement with the analytical results . furthermore , graphical representations show increased reduction in levels of hiv infection when proper condom use is in place . thus , in the absence of monitored proper condom use which is the case in the world as sexual intercourse is a private thing , there is a strong need to control prostitution . even in the presence antiretroviral therapy , hiv / aids control is more effective in less - prostituting communities than in the prostituting communities . thus , prostitution may reduce the effectiveness of antiretroviral therapy by way of acquiring multiple strains of hiv as prostitutes often operate under the influence of alcohol / drugs and hence fail to engage in safe sex . hence , prostitution negatively affects hiv control and as long as hiv control is taken as a biomedical intervention only ; controlling hiv through antiretroviral therapy alone may not be successful in populations where prostitution is common . to put prostitution in check , there may be target poverty as reducing it will mean less people moving into prostitution as most are driven into it by poverty . it is worth mentioning here that the study presented here is not exhaustive , and it can be extended to incorporate alcohol / drug misuse which has always been associated with both prostitution and hiv / aids . the model may be further modified to assess the impact of poverty alleviation in hiv control .

hiv / aids has been somehow linked to prostitution for decades now . a mathematical model is presented to assess the link between prostitution and hiv transmission . the epidemic thresholds known as the reproduction numbers and equilibria for the model are determined and stabilities analyzed . analysis of the reproduction numbers suggests that hiv / aids control using antiretroviral therapy is more effective in the absence of prostitution . numerical simulations further show high levels of hiv / aids when percentage of prostitutes in the community is high . results from this study suggest that effectively controlling hiv / aids requires strategies that address both prostitution and hiv / aids transmission . addressing hiv / aids through condom use and antiretroviral therapy may not be enough to stem hiv / aids in the community as some drug / alcohol misusing prostitutes may not be able to negotiate for safe sex while they are in drunken stupor . furthermore , prostitutes are likely to get infected by different hiv strains some of which may be resistant to the antiretroviral therapy regimen in use .

1. Introduction 2. Model Description 3. Numerical Simulations 4. Discussion

it is mainly driven by peer pressure , homelessness , drug addiction , and poverty just to mention a few of the driving forces . some individuals turn into prostitution to fund their drug / alcohol addiction behaviour [ 6 , 10 , 11 ] , which often leads to entrapment of women who prostitute in this sex - for - drugs lifestyle and others resort to drug / alcohol misuse to effectively sale their bodies without remorse [ 5 , 11 , 12 ] . clearly , drug / alcohol misuse can increase prostitution with less safe sex , which in turn leads to increase in sexually transmitted infections ( including hiv ) . prostitution associated with drug / alcohol misuse is among the main factors in the spread of hiv in the united states . similarly , in mauritus 75% of the prostitutes reported injecting drugs and 23% said they never used nonsterile injecting equipment which all pose a serious risk of hiv infection . among drug misusing prostitutes in mauritus who had been previously tested for hiv 13% were hiv positive , yet 68% of the prostitutes said they had not consistently used condoms during the previous three months . hiv prevalence among female sex workers ( prostitutes ) varies widely but in some countries it is more than 20 times higher than the hiv prevalence of the general population . a number of mathematical models have been developed to understand the role of social and behavioral processes in hiv transmission [ 1719 ] . the later developed a cross impact model to explore intentional transmission of hiv by nondisclosure of status in various risky situations . despite the fact that prostitution and hiv / aids are somehow linked , they have not been mathematically accounted for . here following ideas generated by bhunu and mushayabasa , the authors offer an indepth analysis of prostitution and hiv / aids transmission from the mathematical persipective . the rest of the manuscript is presented as follows . in section 2 , the model is presented and analysed . the model subdivides the population based on prostitution and hiv / aids status . these are susceptibles who are not prostitutes s n(t ) , susceptible prostitutes s p(t ) , and hiv positive people not yet displaying aids symptoms and are not prostitutes i h(t ) , hiv positive prostitutes not yet displaying aids symptoms i hp(t ) , hiv positive individuals who are not prostitutes and displaying aids symptoms a h(t ) , hiv positive prostitutes displaying aids symptoms a hp(t ) , and finaly aids patients on treatment a ht(t ) . individuals in different human subgroups suffer from natural death at a constant rate , which is proportional to the number in each class . although there are many causes of prostitution , here we dwell on peer pressure and poverty which are the main driving forces of prostitution among zimbabweans . the distinction between peer pressure and poverty as factors contributing to prostitution is not clear cut . individuals who are not prostitutes turn into prostitution due to poverty related peer pressure at rate p to move into the corresponding classes of prostitutes . however , it is worth mentioning here that aids patients who have lost hope of living and are poverty stricken acquire prostitution habits at rate p with < [ 0,1 ) signifying the reduced chances an aids patient has of attracting clients due to the on and off sickness . once they become prostitutes , they move into corresponding class of prostitutes displaying aids symptoms . however , for those prostitutes in the aids stage of disease progression some are forced to leave prostitution due to sickness at rate s as they are not likely to be gainfully employed or get a stable financially secure relationship while they are always on and off the sick bed . susceptible nonprostitutes and prostitutes acquire hiv following sexual contact with an infected individual at rates ( 1 )(t ) and ( 1 ) h(t ) , respectively , to get into their corresponding hiv infected classes not yet showing aids symptoms ( i h(t ) and i hp(t ) ) . the role of condom use as a strategy to limit hiv infection is captured by ( 0,1 ) signifying that condom use offers some degree of protection against hiv infection and 1 signifies increased risk prostitutes have of contracting hiv infection as they often engage in less safe sex while they are under the influence alcohol and/or drugs . the force of infection for hiv h(t ) is given by ( 2)ht=hch(ih(t)+ihp(t)+h(ah(t)+ahp(t))+taht(t))n(t ) , where h is the probability of hiv transmission per sexual contact , c h is the effective contact rate for hiv infection to occur , and h > 1 models the fact that individuals in the aids stage and not on antiretroviral therapy are more infectious since the viral load is correlated with infectiousness . it is assumed that individuals on antiretroviral therapy transmit infection at the smallest rate t ( with 0 < t < 1 ) because of the fact that these individuals have very small viral load . it has been estimated by an analysis of longitudinal cohort data that antiretroviral therapy reduces per - partnership infectivity by as much as 60% ( so that t = 0.4 ) . hiv infected nonprostitutes and prostitutes progress to the aids stage of disease progression at rates n and p , respectively , with n p as prostitutes are more likely to experience multiple infections with other sexual transmitted infections , contributing to the more rapid deterioration of the immune system . we assume that antiretroviral therapy is given to aids individuals who are ill and have experienced aids - defining symptoms , or whose cd4 + t cell count is below 200/l , which is the recommended aids defining stage . thus , aids patients are assumed to get antiretroviral therapy at a constant rate . individuals in the aids stage and not yet on antiretroviral therapy experience aids related death at a rate > 0 and their corresponding parts on antiretroviral therapy eventually succumb to aids - induced mortality at a reduced rate with the parameter ( 0 < < 1 ) . unless stated otherwise , values for the parameters in the simulations are given in table 1 . based on the aforementioned , the following system of differential equations describes the dynamics of prostitution and drug ( alcohol ) misuse : ( 3)sn'(t)=(1)hsn(p+)sn+psp , ih'(t)=(1)hsnpih(+n)ih+pihp , ah'(t)=nihpah(++)ah+sahp , sp'(t)=psn(1)hsp(+p)sp , ihp'(t)=pih+(1)hsp(+p+p)ihp , ahp'(t)=pah+pihp(+++s)ahp , aht'(t)=ah+ahp(+)aht . in this section , we study some the basic results of solutions of model system ( 3 ) which are essential in the proofs of stability and persistence results . solutions to the equation m = m are monotone increasing and bounded by / if m(0 ) < /. they are monotone decreasing and bounded above if m(0 ) /. solutions to the equation m = m are monotone increasing and bounded by / if m(0 ) < /. they are monotone decreasing and bounded above if m(0 ) /. since n m , the claim follows . thus , the model is mathematically and epidemiologically well - posed and it is sufficient to consider the dynamics of the flow generated by the system ( 3 ) in . local existence of solutions follows from standard arguments , since the right hand side of system ( 3 ) is locally liptschitz continuous . following van den driessche and watmough we have the reproduction number of the model as ( 6)rhp=hch1t+hm5pm1s+m2+p2+m4nph1m5m6m2+sm1+p++np + hchh2(1)(t+hm5)(pp(s+m2)+(p+m4)nm3)h1m5m6(m2+s)(m1(+p)+(+n)p ) + hchh2(1)(p+m3)h1(m1(+p)+(+n)p ) , with m 1 = p + + n , m 2 = p + + + , m 3 = + p + p , m 4 = + + + s , m 5 = + , m 6 = + + , h 1 = p + + p , and h 2 = h 1 p throughout the paper . here hp defines the number of secondary hiv cases generated by one hiv infected individual in community where prostitution is rife and condom use coupled with antiretroviral therapy is the intervention strategies available . , we write system ( 3 ) in the form : ( 7)x't = fx , y , y'(t)=g(x , y ) , g(x,0)=0 , where x = ( s n , s p ) and y = ( i h , a h , i hp , a hp , a ht ) . ( 8)h1 for x't = fx,0 , x0 is globally asymptotically stableh2 gx , y = uyg^x , y , g^x , y0 , for x , y. consider f(x,0)=-(p+)sn+psppsn-(+p)sp,(9 ) ( 10)g^(x , y)=g1^(x , y)g2^(x , y)g3^(x , y)g4^(x , y)g5^(x , y)=(1)hch(ih+ihp+h(ah+ahp)taht)h2h1snn0(1)hch(ih+ihp+h(ah+ahp)taht)ph1spn00 . , we write system ( 3 ) in the form : ( 7)x't = fx , y , y'(t)=g(x , y ) , g(x,0)=0 , where x = ( s n , s p ) and y = ( i h , a h , i hp , a hp , a ht ) . thus , ( 13)nsn+spi h2h1snn , ( ii ) ph1spn . the utility of the basic reproductive number has been questioned , but we use it here as a threshold with the understanding that there may be further complexities in its application . to check the impact of antiretroviral therapy on a prostitution - free environment we take the partial derivative of h with respect to to obtain ( 15)rh=hchn1+1t+h+++m5m62(+n ) . thus , as long as 0 ( ( h 1) + ( h 1))/(1 t ) < , then antretroviral therapy may be able to keep hiv infections in check in a prostitution - free environment where condom use is in place . however , for 0 < ( ( h 1) + ( h 1))/(1 t ) , the story is different as ( 15 ) will be positive , suggesting that the levels of antiretroviral therapy are not high enough to make a significant contribution to the to the reduction of hiv / aids cases . this result suggests that antiretroviral therapy is more effective in communities with less levels of prostitution as there will be less chances of getting infected with different strains of hiv . thus , prostitution should be targetted as a way of controlling the spread of multiple strains of hiv . multiple strains of hiv may be difficult to keep in check using the commonly used antiretroviral therapy currently available . clearly lim1 hp = 0 suggests even in the presence of prostitution increase in the consistant and correct use of condoms will be able to keep hiv infections under control . this result suggests that safe sex ( condom use ) should always be encouraged especially in areas where prostitution is endemic . the fact that hp = 0 suggests if all people become prostitutes , then in the long term the entire population will be hiv infected ( therefore no new hiv infections ) as prostitutes often operate under the influence of drugs and/or alcohol resulting in inconstistancy in the use of condoms . furthermore , lim hp = suggests that increase in the levels prostitution enhances the risk of acquiring hiv . theoretically , the following hiv / aids endemic equilibria exist : ( i ) the prostitution free - endemic equilibrium , ( ii ) the prostitution only endemic equilibrium , and ( iii ) equilibria where both prostitutes and nonprostitutes coexist . this equilibrium point in terms of the force of infection h is given by ( 18)e=(sn,ih,ah,sp,ihp,ahp,aht ) with the explicit expressions for s n , i h , a h , s p , i hp , a hp , and a ht being cumbersome to be written explicitly . the permanence of the disease destabilizes the disease - free equilibrium 0 since hp > 1 , and the coexistence of the prostitutes and nonprostitutes endemic equilibrium exists . proofuniform persistence system of ( 3 ) implies there exists a constant > 0 such that any solution of ( 3 ) which starts in ( the interior of ) remains in . define the following korobeinikov - maini type lyapunov function ( 20)v(sn , ih , ah , sp , ihp , ahp , aht ) = ( snsnlnsn)+(ihihlnih)+(ahahlnah ) + ( spsplnsp)+(ihpihplnihp ) + ahpahplnahp+ahtahtlnaht , which is continuous for all ( s n , i h , a h , s p , i hp , a hp , a ht ) > 0 and satisfies ( cf . ) consequently , the endemic equilibrium is the only extremum and the global minimum of the function v + . hence , v(s n , i h , a h , s p , i hp , a hp , a ht ) 0 with equality only at . thus , all solutions of ( 3 ) which intersect converge to the invariant ( singleton ) { } . consequently , the endemic equilibrium is the only extremum and the global minimum of the function v + . thus , all solutions of ( 3 ) which intersect converge to the invariant ( singleton ) { } . in figure 2 , the possible effects prostitution has on hiv levels are noted by varying the percentages of prostitutes in the community in the presence of antiretroviral therapy and condom use . it is noted that an increase in the percentage of prostitutes results in an increase in the number of hiv infected people as prostitutes mostly operate under the influence of drugs and therefore have difficulties to properly use condoms . this suggests increase in employment creation coupled with counselling will be able to keep the prostitution in check as most people resort to prostitution due to poverty . thus , poverty eradication reduces prostitution which in turn results in a reduction in the number of hiv infections in the population . in figure 3 , it is shown that proper use of condoms will be able to keep hiv infections in check , even when prostitution exists in the population . this result suggests that encouraging the proper use of condoms should be encouraged especially in communities where prostitution is rife . it is important to note that proper use of condoms may be difficult especially among prostitutes as they often engage in sex while they are under the influence of drugs / alcohol . a mathematical model for the effect of prostitution on the transmission dynamics of hiv / aids is presented as a system of differential equations . the disease - free equilibrium point of the model is shown to be globally asymptotically stable when the corresponding reproduction number is less than unity . ( i ) increase in the levels of prostitution in a community enhances the chances of acquiring hiv if not infected and acquiring another hiv strain if already infected . ( ii ) antiretroviral therapy is more effective in communities with less prostitution as less prostitution corresponds to reduced chances of one getting infected with different strains of hiv . infection with different strains of hiv makes the hiv infected person worse as some strains may not respond to the antiretroviral therapy regimen available especially in resource - constrained settings . ( iii ) increase in the rates of proper condom use may be able to keep hiv infections even in communities where prostitution is rife . graphical representations clearly show that the number of hiv infected people and those displaying aids symptoms is higher in communities with more prostitutes than in communities with less prostitutes . this suggests that prostitution promotes hiv transmission , a result in total agreement with the analytical results . furthermore , graphical representations show increased reduction in levels of hiv infection when proper condom use is in place . thus , in the absence of monitored proper condom use which is the case in the world as sexual intercourse is a private thing , there is a strong need to control prostitution . even in the presence antiretroviral therapy , hiv / aids control is more effective in less - prostituting communities than in the prostituting communities . thus , prostitution may reduce the effectiveness of antiretroviral therapy by way of acquiring multiple strains of hiv as prostitutes often operate under the influence of alcohol / drugs and hence fail to engage in safe sex . hence , prostitution negatively affects hiv control and as long as hiv control is taken as a biomedical intervention only ; controlling hiv through antiretroviral therapy alone may not be successful in populations where prostitution is common . to put prostitution in check , there may be target poverty as reducing it will mean less people moving into prostitution as most are driven into it by poverty . it is worth mentioning here that the study presented here is not exhaustive , and it can be extended to incorporate alcohol / drug misuse which has always been associated with both prostitution and hiv / aids . the model may be further modified to assess the impact of poverty alleviation in hiv control .

in norway1 and most western societies,2 the major causes of long - term sick leave and work disability are chronic musculoskeletal and mental disorders . the numbers of disability pensioners are increasing and this represents a large economic burden for society . in addition , not being able to participate in the labor force has negative consequences for the individual . depression is the leading cause of disability3 and has a more negative prognosis for return to work ( rtw ) than other common mental health conditions.4,5 somatic symptoms and depression often coexist,6 and epidemiological findings support that there is a bidirectional association between depression and chronic musculoskeletal conditions.7 further , studies suggest that depression contributes to poor rtw in individuals referred to occupational rehabilitation , even controlling for pain severity.8 chronic pain is a common long - term condition that affects a person s physical and emotional functioning . approximately 20%50% of individuals with musculoskeletal conditions show evidence of elevated depressive symptoms , further suggesting that comorbid depression is associated with poorer rehabilitation outcomes.8,9 multidisciplinary rehabilitation approaches have been shifting away from a restricted biomedical model toward a multifactorial model of interrelating physical , psychological , and social / occupational factors,10 with emphasis on function and work ability , disability management , and psychosocial interventions . results support that participation in multidisciplinary rehabilitation programs increases quality of life and decreases anxiety and depression in employees with musculoskeletal disorders.11 several studies suggest that physical factors , such as improvements in strength , endurance , or flexibility , appear to have little relation to rtw rates after interdisciplinary treatment.12,13 other studies confirm that increased depressive symptoms are associated with decreased physical functioning.14,15 in a recent review , mcknight and kashdan16 stated that there is little research directly examining the relationship between depressive symptoms and physical ability . the extent to which employees with chronic musculoskeletal disorders and depressive symptoms successfully adapt back into their job roles after participating in a multidisciplinary rehabilitation program remains unclear . given the association between chronic pain , depression , and physical function,16,17 it is likely that rtw in persons with chronic musculoskeletal pain and comorbid depression would be associated with better physical functioning . the aim of our investigation was to assess whether various measurements of physical functioning among persons with chronic musculoskeletal pain and comorbid depression were positively associated with rtw after participating in a 57-week multidisciplinary rehabilitation program . participants with a long - term history of mixed chronic musculoskeletal pain were recruited by their general practitioners , who assigned them to a 57-week multidisciplinary rehabilitation program on the basis of interviews , observations , and clinical tests . inclusion criteria for participation in the rehabilitation program were that the participants had chronic musculoskeletal pain ( longer than 3 months ) and were receiving national insurance in the form of sickness benefit or rehabilitation benefit . in norway one can start drawing a retirement pension at the age of 62 . to ensure that none of the participants were in any initial phase of retirement , we excluded those aged 60 years and older . further , to assess any difference in physical function and return to work among those with comorbid depressive symptoms at the end of the rehabilitation , we included only those with high depressive symptoms measured by the hospital anxiety and depression scale ( hads ) . the participants provided written consent . to maintain the right to financial support in the form of sickness or rehabilitation benefits from the national health insurance office , an assessment of function and work capacity is required . general practitioners recruited participants into the 57-week rehabilitation program in cooperation with the national health insurance office , in order to obtain a function and work capacity evaluation as required . in cooperation with the public health services , national health insurance office , employment office , and the employer , an individually tailored rehabilitation program was conducted . the rehabilitation program included both individual counseling and group - based treatment , including physical components ( see table 1 ) . the program consisted of 1 ) a 5-week intensive period that the participants attended approximately 6 hours a day , 4 days a week , and 2 ) a follow - up period of 52 weeks that the participants attended approximately 6 hours a day , 13 days a week . symptoms of anxiety and depression were assessed using the hads , a self - rating scale commonly used for screening purposes in various clinical settings and in epidemiological studies.18 the symptom scale consists of seven items that cover anxiety and seven items that cover depression on a 4-point likert scale . individual items are scored from 03 or 30 , depending on the direction of the wording of the items . an optimal balance between sensitivity and specificity seems to be achieved when caseness is defined by a score of 8 on both the anxiety and depression subscales of the hads.18 four aspects of physical functioning ( muscle strength , endurance capacity , mobility , and balance ) and degree of physical pain were measured with single items on a visual analog scale ( vas ) . the use of vas in employees with musculoskeletal disorders is well - established , and single - item measures frequently perform almost as well as multi - item scales.19,20 the vas is a line 10 cm in length where agreement or disagreement represents a continuum of the specific physical function measured . the vas variables were individually measured and scored in millimeters ( 0100 ) . the participants were asked to rate the present condition / symptoms through the following questions : pain experience on a scale from 0100 where 0 is not troublesome at all and 100 is extremely troublesome , how troublesome do you experience the pain to be in everyday life ? muscle strength on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your muscle strength to be?21,22 endurance capacity on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your endurance capacity to be?21,22 mobility on a scale from 0100 where 0 is very limited flexibility and 100 is very flexible , how flexible do you consider your muscles and joints to be?21,22 balance on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your balance to be?21,22 physical fitness was measured through the validated coop / wonca charts23 by the following question : during the past two weeks , what was the hardest physical activity you could do for at least 2 minutes ? the answering categories consist of a 5-point ordinal scale ranging from 1 ( very heavy ) to 5 ( very light ) . age was used as a continuous variable , and marital status was measured as a dichotomous variable ( married / cohabiting versus unmarried / divorced ) . disability status was registered in the following categories : rehabilitation benefit , vocational rehabilitation , part time work / rehabilitation benefit , sick leave , part - time disability pension , disability pension , social assistance , students , or unemployed . active and passive strategies for work ability and work re - entry . participants who reported working on a part - time or full - time basis were categorized as active and coded as on their way into / in work ( rtw ) , as were individuals in job retraining or an education program . these latter two variables were categorized as active strategies by means of representing levels of functioning that in some ways mirror the work environment and bring forward work - related behavior in the participants eg , they have to go somewhere on a daily basis and keep functioning at a relatively consistent level . the participants still on sick leave , receiving disability pension , or applying for disability pension after completing the 57-week multidisciplinary rehabilitation program were categorized as passive and coded as on their way out / out of work ( non - rtw ) . the reason for this choice was that most participants in the rehabilitation program already had been on sick leave for several months before entering the 57-week rehabilitation program . thus it was very likely that those still on sick leave after 57 weeks were in the initial phase of the process of being granted a disability pension . the dependent variable rtw was self - reported and defined by being on their way into / in work . a student s t - test for independent samples ( continuous variables ) and chi - square test ( categorical variables ) were used to compare those who were returning to work and those who were not . bivariate associations between each domain of physical function and rtw were assessed through logistic regression models , estimating odds ratio ( or ) with 95% confidence intervals ( ci ) . ibm spss statistics 20 ( ibm corporation , armonk , ny , usa ) was used for the statistical analyses . participants with a long - term history of mixed chronic musculoskeletal pain were recruited by their general practitioners , who assigned them to a 57-week multidisciplinary rehabilitation program on the basis of interviews , observations , and clinical tests . inclusion criteria for participation in the rehabilitation program were that the participants had chronic musculoskeletal pain ( longer than 3 months ) and were receiving national insurance in the form of sickness benefit or rehabilitation benefit . in norway one can start drawing a retirement pension at the age of 62 . to ensure that none of the participants were in any initial phase of retirement , we excluded those aged 60 years and older . further , to assess any difference in physical function and return to work among those with comorbid depressive symptoms at the end of the rehabilitation , we included only those with high depressive symptoms measured by the hospital anxiety and depression scale ( hads ) . to maintain the right to financial support in the form of sickness or rehabilitation benefits from the national health insurance office , an assessment of function and work capacity is required . general practitioners recruited participants into the 57-week rehabilitation program in cooperation with the national health insurance office , in order to obtain a function and work capacity evaluation as required . in cooperation with the public health services , national health insurance office , employment office , and the employer , an individually tailored rehabilitation program was conducted . the rehabilitation program included both individual counseling and group - based treatment , including physical components ( see table 1 ) . the program consisted of 1 ) a 5-week intensive period that the participants attended approximately 6 hours a day , 4 days a week , and 2 ) a follow - up period of 52 weeks that the participants attended approximately 6 hours a day , 13 days a week . symptoms of anxiety and depression were assessed using the hads , a self - rating scale commonly used for screening purposes in various clinical settings and in epidemiological studies.18 the symptom scale consists of seven items that cover anxiety and seven items that cover depression on a 4-point likert scale . individual items are scored from 03 or 30 , depending on the direction of the wording of the items . an optimal balance between sensitivity and specificity seems to be achieved when caseness is defined by a score of 8 on both the anxiety and depression subscales of the hads.18 four aspects of physical functioning ( muscle strength , endurance capacity , mobility , and balance ) and degree of physical pain were measured with single items on a visual analog scale ( vas ) . the use of vas in employees with musculoskeletal disorders is well - established , and single - item measures frequently perform almost as well as multi - item scales.19,20 the vas is a line 10 cm in length where agreement or disagreement represents a continuum of the specific physical function measured . the participants were asked to rate the present condition / symptoms through the following questions : pain experience on a scale from 0100 where 0 is not troublesome at all and 100 is extremely troublesome , how troublesome do you experience the pain to be in everyday life ? muscle strength on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your muscle strength to be?21,22 endurance capacity on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your endurance capacity to be?21,22 mobility on a scale from 0100 where 0 is very limited flexibility and 100 is very flexible , how flexible do you consider your muscles and joints to be?21,22 balance on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your balance to be?21,22 physical fitness was measured through the validated coop / wonca charts23 by the following question : during the past two weeks , what was the hardest physical activity you could do for at least 2 minutes ? the answering categories consist of a 5-point ordinal scale ranging from 1 ( very heavy ) to 5 ( very light ) . age was used as a continuous variable , and marital status was measured as a dichotomous variable ( married / cohabiting versus unmarried / divorced ) . disability status was registered in the following categories : rehabilitation benefit , vocational rehabilitation , part time work / rehabilitation benefit , sick leave , part - time disability pension , disability pension , social assistance , students , or unemployed . active and passive strategies for work ability and work re - entry . participants who reported working on a part - time or full - time basis were categorized as active and coded as on their way into / in work ( rtw ) , as were individuals in job retraining or an education program . these latter two variables were categorized as active strategies by means of representing levels of functioning that in some ways mirror the work environment and bring forward work - related behavior in the participants eg , they have to go somewhere on a daily basis and keep functioning at a relatively consistent level . the participants still on sick leave , receiving disability pension , or applying for disability pension after completing the 57-week multidisciplinary rehabilitation program were categorized as passive and coded as on their way out / out of work ( non - rtw ) . the reason for this choice was that most participants in the rehabilitation program already had been on sick leave for several months before entering the 57-week rehabilitation program . thus it was very likely that those still on sick leave after 57 weeks were in the initial phase of the process of being granted a disability pension . the dependent variable rtw was self - reported and defined by being on their way into / in work . a student s t - test for independent samples ( continuous variables ) and chi - square test ( categorical variables ) were used to compare those who were returning to work and those who were not . bivariate associations between each domain of physical function and rtw were assessed through logistic regression models , estimating odds ratio ( or ) with 95% confidence intervals ( ci ) . ibm spss statistics 20 ( ibm corporation , armonk , ny , usa ) was used for the statistical analyses . the mean duration of pain prior to entering the rehabilitation program was 15 years ( ranging from 3 months to 36 years ) . most of the participants , 89.5% ( n=505 ) , reported pain in more than one location , with lower back , shoulders , and neck being the most common . to ensure that none of the participants were in any initial phase of retirement , we excluded those aged 60 years and older ( n=142 ) . further , to assess any difference in physical function and return to work among those with comorbid depressive symptoms at the end of the rehabilitation , we included only those with high depressive symptoms measured by the hospital anxiety and depression scale ( hads ) . the final sample consisted of 92 persons ( 21.7% of all participants < 60 years ) with chronic musculoskeletal disorders and comorbid depression under 60 years of age who had undergone the 57-week multidisciplinary rehabilitation program . of these , 70 were returning to work ( rtw ) and 22 were not ( non - rtw ) . all participants included in the present study had a hads depression score 8 at the end of the rehabilitation . group ( rtw ) and the on their way out / out of work group ( non - rtw ) in terms of age , marital status , level of depression above the cut - off value of 8 , anxiety level , and pain experience ( table 2 ) . muscle strength , endurance capacity , mobility , balance , and physical fitness were significantly better among those who were not returning to work . in the bivariate analyses , four of the five domains of physical functioning measured by vas ( muscle strength , mobility , balance , and physical fitness ) were significantly negatively associated with rtw , indicating that better physical functioning was associated with not returning to work ( table 3 ) . among those with better function on muscle strength ( or 0.61 , 95% ci 0.450.83 ) , mobility ( or 0.73 , 95% ci 0.570.95 ) , and balance ( or 0.67 , 95% ci 0.500.91 ) , the odds of being on an active work re - entry strategy were 23%39% lower compared with those with poorer physical function . the same pattern was seen for physical fitness measured by the coop / wonca charts ( or 1.75 , 95% 1.072.99 ) , indicating that the odds of returning to work were 75% lower among those who reported that they were able to perform hard physical activity for at least 2 minutes . in other words , each of these measures of physical functioning was worse among those reporting that they were on an active strategy for work re - entry . endurance capacity ( or 0.77 , 95% ci 0.581.03 ) did not reach any statistical association with rtw ; even so , the or of 0.77 ( p=0.076 ) indicated that this result was in line with the other results and it would probably have become statistically significant in a larger sample . in this study of persons with chronic musculoskeletal disorders and comorbid depression , we found higher levels of physical functioning to be significantly associated with non - rtw at the end of the rehabilitation period of 57 weeks . given that the multidisciplinary rehabilitation program in this study places a strong emphasis on function and work ability , our results are somewhat surprising . additionally , we have not been able to find other studies confirming our results or the opposite ; that individuals with chronic musculoskeletal disorders and comorbid depression returning to work have better physical function compared with those who are depressed not returning to work . in the present study , there were no differences in pain experience or severity of depression between those returning and those not returning to work . hence , there are no straightforward explanations of our findings of reduced odds of rtw among those with best physical functioning . in the following discussion we present two important aspects that may be relevant to our findings . physical limitations , physical ability and physical activity are all domains covering different aspects of human physical functioning . most studies on mental health and physical function suggest an inverse association between decreasing levels of depressive symptoms and increasing physical functioning . however , an exhaustive literature review showed that the association between depressive symptoms and physical functioning is unexpectedly weak.16 this may be due to low consensus on what physical measurement to use , and whether such measures should be performance - based or patient - reported . bean et al24 found that self - reported measures of physical function were associated with psychosocial factors such as depression , while performance - based measures were not . nevertheless , the former study was among elderly persons with mobility limitations and results can not be generalized to the sample in our study . an alternative explanation for the association found in the present study is that self - report of physical functioning may be influenced by psychological distress . despite participation in an occupational rehabilitation program , some employees may lack motivation to return to a certain workplace . in a qualitative study of disability pensioners with chronic back pain , magnussen et al25 found that some of the barriers related to rtw were earlier negative work experiences , poor self - judgment of work ability , and low self - esteem . further , many of the participants pointed to conditions at their former workplaces that they believed contributed to the disability process and that would make it difficult to return to work . hence , the experience of being granted a disability pension , which means not returning to work , might be followed by the feelings of relief and satisfaction , while those , by definition , on their way into work might be subjected to remaining in an unsafe and uncertain situation , both economically and occupationally . in turn , this emotional state may directly affect one s evaluation of physical functioning , despite being depressed . several studies support that the rtw is a multi - factorial process8 and that physical functioning should not be mixed up with occupational functioning . occupational functioning is more than being able to perform certain tasks at work,26 as it also involves non job - specific behaviors such as cooperation and dedication . thus , occupational functioning can be related to factors at the individual level , at the group level , and at the organizational level . nevertheless , according to the literature most psychosocial interventions to achieve optimal rehabilitation outcomes in employees with musculoskeletal pain have focused on risk factors within the individual ( eg , pain catastrophizing and expectancies ) and not on relevant factors at the workplace , such as interpersonal conflicts or job stress ( at the group or institutional level).27 in the vast majority of observational studies on factors related to the rtw process , as in this study as well , psychosocial interventions did not follow common evidence - based guidelines or treatment protocols.27,28 in turn , this makes any evaluation of treatment outcome after rehabilitation difficult , especially regarding assessment of interventions that focus on work - related factors outside of the individual.27 further , depression affects several areas related to occupational functioning , such as absenteeism , education , and employment opportunities . depressed workers also report more conflicts and negative social outcomes.16 in a qualitative study of factors relevant in the assessment of the rtw process of employees on long - term sickness absence due to a depressive disorder , muijzer et al4 found that physical functioning , attitudes , competence , and environmental factors such as employer employee relationship have implications for the rtw process . the study was , however , based on focus - group interviews with labor experts working at the social insurance institute in the netherlands . in a recent swedish study , sjstrm et al9 evaluated a 7-week multidisciplinary rehabilitation program in patients with musculoskeletal pain , with emphasis on mental symptoms measured by the hads . they found that , even if stress levels improved , those with full - time sick leave showed no changes in anxiety and depression levels during the 2-year study period . according to the authors , the results indicate that persons with musculoskeletal pain and comorbid psychological distress may require rehabilitation with different content than that given to employees with low psychological symptoms . in a recent systematic review of factors associated with work participation and work functioning in depressed workers , lagerveld et al29 found that work - related and personal factors were less frequently studied . further , the authors stated that gaps in knowledge exist on factors predictive of work participation and work functioning in depressed workers . these results illustrate that the rtw process among depressed employees with musculoskeletal conditions is multifactorial and not merely a result of physical functioning or pain severity . epidemiological studies from the nord - trndelag health study in norway suggest that approximately one - third of individuals reporting somatic health problems also have anxiety disorder and/or depression30 and that a statistical relationship exists between anxiety , depression , and functional somatic symptoms , independent of age and sex.6 at the same time , medical examination of patients with chronic musculoskeletal pain often fails to demonstrate any organic diseases.15 in a norwegian population - based cohort study , mykletun et al31 found anxiety and depression to be strongly and independently associated with disability pensions granted for physical conditions and diagnoses , suggesting that administrative data may have underestimated the contribution of mental disorders to the awarding of disability pensions . the latter findings are partly supported by a recently published swedish study28 of biopsychosocial functioning and assessment of the ability to work in 174 patients on long - term sick leave due to chronic musculoskeletal pain . all patients underwent orthopedic assessment of physical function and ability to work ; 83 were referred for psychiatric evaluation of diagnosis and function . the final evaluation involved the orthopedist and the psychiatrist working together as a team to form a mutual opinion of the patients ` functional , physical , and psychological abilities in relation to occupational demands and the prognosis for returning to work . interestingly , the main cause of sick leave changed from a somatic diagnosis to a psychiatric diagnosis in 69% of patients . of these , 33% were considered not able to return to work . according to the authors,28 the study sheds light on the limitations related to the assessment of ability to work , because physicians , lacking an established medical protocol , evaluate the patient according to their own experience . inappropriate assessment of work ability leads to the wrong choice of interventions , which may employ and maintain unethical practices regarding the rtw process in employees with chronic musculoskeletal pain and comorbid depression . depression and pain share biological pathways and neurotransmitters , which has implications for the treatment . additionally , psychiatric disorders in patients with chronic musculoskeletal pain have commonly been undiagnosed.32 the combination of depression and pain is associated with worse clinical outcomes than either condition alone . thus , a treatment model that incorporates treatment of both depression and pain seems necessary for more optimal outcomes.33 pain is also a subjective sensory experience that is closely associated with affect , cognition , and aspects of the employee s social environment . thus , it is possible that depressive symptoms may act both as a cause and as a consequence of chronic musculoskeletal pain and absence from work . further , it is possible , although not necessarily the case , that work - related factors are significantly involved in this complex process . according to the literature,16 few studies exist on the bidirectional relationship between depression and occupational functioning . in a recent evaluation of a swedish 7-week rehabilitation program , sjstrm et al9 followed 60 participants with musculoskeletal pain for a period of up to 2 years . they found that the ten participants with full - time sick leave ( absence from work for medical reasons ) during the study period showed improved stress levels but no change in anxiety and depression levels ( assessed by the hads ) . this group also had higher pain ratings and higher subjective disability ratings during the rehabilitation program than did those with no sick leave or those on part - time sick leave . the study illustrates that mental health problems and musculoskeletal pain are interrelated and that traditional multidisciplinary rehabilitation may not meet any special needs of this selected group . all data are self- reported ; thus , it is not possible to confirm whether participants actually did return to work or not . given the use of perceived measures , one might argue that it is uncertain whether the results are indicative of physical ability or rather perception of ability . further , we had no information on medical diagnoses or daily use of prescribed drugs . still , as we found no difference in pain experience between those returning to work and those not returning to work , it is unlikely that the better physical functioning was due to the experience of less pain among those not returning to work . the hads is useful in the assessment of symptoms of depression and anxiety ; however , it is not validated as a diagnostic tool for clinical diagnoses in accordance with the international classification of diseases and the diagnostic and statistical manual of mental disorders , fourth edition.34 the influence of occupational history , such as if the participants were unemployed or if they held a temporary or a permanent occupation position , may influence the results , but these factors were not taken into account in our study . in addition , the cross - sectional design of this study makes it impossible to make any inference on causality . however , the main focus of this investigation was to assess the association between physical function and rtw among participants with chronic musculoskeletal pain with elevated depressive symptoms at the end of a multidisciplinary rehabilitation program . nevertheless , 22% of those younger than 60 years participating in this 57-week multidisciplinary rehabilitation program had elevated depressive symptoms at the end of the program . it is possible that these participants constitute a marginalized group consisting of those who have had the longest duration of musculoskeletal pain and the longest sick leave prior to their participation in the multidisciplinary program . this , however , gives reason to speculate whether the evaluation of the ability to work may not have taken mental health status into account prior to or during participation in the multidisciplinary rehabilitation program . further , and in line with the suggestions from sjstrm et al,9 it is possible that this selected group of individuals with elevated depressive symptoms may require rehabilitation with different content other than what is usually offered through a traditional multidisciplinary rehabilitation program . physical limitations , physical ability and physical activity are all domains covering different aspects of human physical functioning . most studies on mental health and physical function suggest an inverse association between decreasing levels of depressive symptoms and increasing physical functioning . however , an exhaustive literature review showed that the association between depressive symptoms and physical functioning is unexpectedly weak.16 this may be due to low consensus on what physical measurement to use , and whether such measures should be performance - based or patient - reported . bean et al24 found that self - reported measures of physical function were associated with psychosocial factors such as depression , while performance - based measures were not . nevertheless , the former study was among elderly persons with mobility limitations and results can not be generalized to the sample in our study . an alternative explanation for the association found in the present study is that self - report of physical functioning may be influenced by psychological distress . despite participation in an occupational rehabilitation program , some employees may lack motivation to return to a certain workplace . in a qualitative study of disability pensioners with chronic back pain , magnussen et al25 found that some of the barriers related to rtw were earlier negative work experiences , poor self - judgment of work ability , and low self - esteem . further , many of the participants pointed to conditions at their former workplaces that they believed contributed to the disability process and that would make it difficult to return to work . hence , the experience of being granted a disability pension , which means not returning to work , might be followed by the feelings of relief and satisfaction , while those , by definition , on their way into work might be subjected to remaining in an unsafe and uncertain situation , both economically and occupationally . in turn , this emotional state may directly affect one s evaluation of physical functioning , despite being depressed . several studies support that the rtw is a multi - factorial process8 and that physical functioning should not be mixed up with occupational functioning . occupational functioning is more than being able to perform certain tasks at work,26 as it also involves non job - specific behaviors such as cooperation and dedication . thus , occupational functioning can be related to factors at the individual level , at the group level , and at the organizational level . nevertheless , according to the literature most psychosocial interventions to achieve optimal rehabilitation outcomes in employees with musculoskeletal pain have focused on risk factors within the individual ( eg , pain catastrophizing and expectancies ) and not on relevant factors at the workplace , such as interpersonal conflicts or job stress ( at the group or institutional level).27 in the vast majority of observational studies on factors related to the rtw process , as in this study as well , psychosocial interventions did not follow common evidence - based guidelines or treatment protocols.27,28 in turn , this makes any evaluation of treatment outcome after rehabilitation difficult , especially regarding assessment of interventions that focus on work - related factors outside of the individual.27 further , depression affects several areas related to occupational functioning , such as absenteeism , education , and employment opportunities . depressed workers also report more conflicts and negative social outcomes.16 in a qualitative study of factors relevant in the assessment of the rtw process of employees on long - term sickness absence due to a depressive disorder , muijzer et al4 found that physical functioning , attitudes , competence , and environmental factors such as employer the study was , however , based on focus - group interviews with labor experts working at the social insurance institute in the netherlands . in a recent swedish study , sjstrm et al9 evaluated a 7-week multidisciplinary rehabilitation program in patients with musculoskeletal pain , with emphasis on mental symptoms measured by the hads . they found that , even if stress levels improved , those with full - time sick leave showed no changes in anxiety and depression levels during the 2-year study period . according to the authors , the results indicate that persons with musculoskeletal pain and comorbid psychological distress may require rehabilitation with different content than that given to employees with low psychological symptoms . in a recent systematic review of factors associated with work participation and work functioning in depressed workers , lagerveld further , the authors stated that gaps in knowledge exist on factors predictive of work participation and work functioning in depressed workers . these results illustrate that the rtw process among depressed employees with musculoskeletal conditions is multifactorial and not merely a result of physical functioning or pain severity . epidemiological studies from the nord - trndelag health study in norway suggest that approximately one - third of individuals reporting somatic health problems also have anxiety disorder and/or depression30 and that a statistical relationship exists between anxiety , depression , and functional somatic symptoms , independent of age and sex.6 at the same time , medical examination of patients with chronic musculoskeletal pain often fails to demonstrate any organic diseases.15 in a norwegian population - based cohort study , mykletun et al31 found anxiety and depression to be strongly and independently associated with disability pensions granted for physical conditions and diagnoses , suggesting that administrative data may have underestimated the contribution of mental disorders to the awarding of disability pensions . the latter findings are partly supported by a recently published swedish study28 of biopsychosocial functioning and assessment of the ability to work in 174 patients on long - term sick leave due to chronic musculoskeletal pain . all patients underwent orthopedic assessment of physical function and ability to work ; 83 were referred for psychiatric evaluation of diagnosis and function . the final evaluation involved the orthopedist and the psychiatrist working together as a team to form a mutual opinion of the patients ` functional , physical , and psychological abilities in relation to occupational demands and the prognosis for returning to work . interestingly , the main cause of sick leave changed from a somatic diagnosis to a psychiatric diagnosis in 69% of patients . of these , 33% were considered not able to return to work . according to the authors,28 the study sheds light on the limitations related to the assessment of ability to work , because physicians , lacking an established medical protocol , evaluate the patient according to their own experience . inappropriate assessment of work ability leads to the wrong choice of interventions , which may employ and maintain unethical practices regarding the rtw process in employees with chronic musculoskeletal pain and comorbid depression . depression and pain share biological pathways and neurotransmitters , which has implications for the treatment . additionally , psychiatric disorders in patients with chronic musculoskeletal pain have commonly been undiagnosed.32 the combination of depression and pain is associated with worse clinical outcomes than either condition alone . thus , a treatment model that incorporates treatment of both depression and pain seems necessary for more optimal outcomes.33 pain is also a subjective sensory experience that is closely associated with affect , cognition , and aspects of the employee s social environment . thus , it is possible that depressive symptoms may act both as a cause and as a consequence of chronic musculoskeletal pain and absence from work . further , it is possible , although not necessarily the case , that work - related factors are significantly involved in this complex process . according to the literature,16 few studies exist on the bidirectional relationship between depression and occupational functioning . in a recent evaluation of a swedish 7-week rehabilitation program , sjstrm et al9 followed 60 participants with musculoskeletal pain for a period of up to 2 years . they found that the ten participants with full - time sick leave ( absence from work for medical reasons ) during the study period showed improved stress levels but no change in anxiety and depression levels ( assessed by the hads ) . this group also had higher pain ratings and higher subjective disability ratings during the rehabilitation program than did those with no sick leave or those on part - time sick leave . the study illustrates that mental health problems and musculoskeletal pain are interrelated and that traditional multidisciplinary rehabilitation may not meet any special needs of this selected group . all data are self- reported ; thus , it is not possible to confirm whether participants actually did return to work or not . given the use of perceived measures , one might argue that it is uncertain whether the results are indicative of physical ability or rather perception of ability . further , we had no information on medical diagnoses or daily use of prescribed drugs . still , as we found no difference in pain experience between those returning to work and those not returning to work , it is unlikely that the better physical functioning was due to the experience of less pain among those not returning to work . the hads is useful in the assessment of symptoms of depression and anxiety ; however , it is not validated as a diagnostic tool for clinical diagnoses in accordance with the international classification of diseases and the diagnostic and statistical manual of mental disorders , fourth edition.34 the influence of occupational history , such as if the participants were unemployed or if they held a temporary or a permanent occupation position , may influence the results , but these factors were not taken into account in our study . in addition , the cross - sectional design of this study makes it impossible to make any inference on causality . however , the main focus of this investigation was to assess the association between physical function and rtw among participants with chronic musculoskeletal pain with elevated depressive symptoms at the end of a multidisciplinary rehabilitation program . nevertheless , 22% of those younger than 60 years participating in this 57-week multidisciplinary rehabilitation program had elevated depressive symptoms at the end of the program . it is possible that these participants constitute a marginalized group consisting of those who have had the longest duration of musculoskeletal pain and the longest sick leave prior to their participation in the multidisciplinary program . this , however , gives reason to speculate whether the evaluation of the ability to work may not have taken mental health status into account prior to or during participation in the multidisciplinary rehabilitation program . further , and in line with the suggestions from sjstrm et al,9 it is possible that this selected group of individuals with elevated depressive symptoms may require rehabilitation with different content other than what is usually offered through a traditional multidisciplinary rehabilitation program . the findings of higher physical functioning among depressed employees not returning to work after participating in a multidisciplinary rehabilitation program in this study illustrate that self - reported physical functioning does not act as a proxy for occupational functioning in the rtw process . according to the literature , there is a need for more research on the bidirectional association between depression and occupational functioning . further , recent studies suggest that there is a need for standardized procedures in the evaluation of the ability to work and that this procedure needs to take mental health status into account . longitudinal studies with the use of different registries ( employment history , sick leave , and use of prescribed drugs ) should preferably be used in the evaluation of factors promoting the rtw process among people with chronic musculoskeletal disorders and poor mental health .

backgroundthe aim of this investigation was to assess whether measures of physical functioning after multidisciplinary rehabilitation are associated with return to work among individuals with chronic musculoskeletal pain conditions and comorbid depressive symptoms.methodsincluded were 92 employees with chronic musculoskeletal disorders who had participated in a 57- week multidisciplinary rehabilitation program . their ages ranged from 2559 years . the hospital anxiety and depression scale was used to assess depressive symptoms . different aspects of physical functioning ( muscle strength , mobility , endurance capacity , and balance ) were measured with single - item visual analog scales , and physical fitness was measured with the validated coop / wonca charts . being on active work strategies , such as receiving rehabilitation benefit / vocational rehabilitation or being reported partly or completely fit , was defined as on their way into / in work . cross - sectional associations were measured using logistic regression models , estimating odds ratio with 95% confidence intervals.resultsthere were no differences between the on their way into / in work group ( n=70 ) and the on their way out / out of work group ( n=22 ) regarding age , sex , or levels of anxiety or pain . surprisingly , regression analyses showed that those with higher levels of physical functioning had significantly lower odds of returning to work.conclusionthe findings of an inverse relationship between self - reported physical function and returning to work in this sample illustrate that the return - to - work process among employees with chronic musculoskeletal pain and comorbid depressive symptoms is multifactorial and influenced by factors other than physical functioning at the individual level . further research , especially longitudinal studies , is needed to assess the occupational trajectories among employees with chronic musculoskeletal pain and comorbid depressive symptoms after participation in a multidisciplinary rehabilitation program .

Introduction Materials and methods Participants The multidisciplinary rehabilitation program Hospital Anxiety and Depression scale Physical function assessments Return to work Statistical analyses Results Discussion Physical functioning versus occupational functioning Somatic versus mental health as barriers to RTW Conclusion

depression is the leading cause of disability3 and has a more negative prognosis for return to work ( rtw ) than other common mental health conditions.4,5 somatic symptoms and depression often coexist,6 and epidemiological findings support that there is a bidirectional association between depression and chronic musculoskeletal conditions.7 further , studies suggest that depression contributes to poor rtw in individuals referred to occupational rehabilitation , even controlling for pain severity.8 chronic pain is a common long - term condition that affects a person s physical and emotional functioning . approximately 20%50% of individuals with musculoskeletal conditions show evidence of elevated depressive symptoms , further suggesting that comorbid depression is associated with poorer rehabilitation outcomes.8,9 multidisciplinary rehabilitation approaches have been shifting away from a restricted biomedical model toward a multifactorial model of interrelating physical , psychological , and social / occupational factors,10 with emphasis on function and work ability , disability management , and psychosocial interventions . results support that participation in multidisciplinary rehabilitation programs increases quality of life and decreases anxiety and depression in employees with musculoskeletal disorders.11 several studies suggest that physical factors , such as improvements in strength , endurance , or flexibility , appear to have little relation to rtw rates after interdisciplinary treatment.12,13 other studies confirm that increased depressive symptoms are associated with decreased physical functioning.14,15 in a recent review , mcknight and kashdan16 stated that there is little research directly examining the relationship between depressive symptoms and physical ability . the extent to which employees with chronic musculoskeletal disorders and depressive symptoms successfully adapt back into their job roles after participating in a multidisciplinary rehabilitation program remains unclear . given the association between chronic pain , depression , and physical function,16,17 it is likely that rtw in persons with chronic musculoskeletal pain and comorbid depression would be associated with better physical functioning . the aim of our investigation was to assess whether various measurements of physical functioning among persons with chronic musculoskeletal pain and comorbid depression were positively associated with rtw after participating in a 57-week multidisciplinary rehabilitation program . participants with a long - term history of mixed chronic musculoskeletal pain were recruited by their general practitioners , who assigned them to a 57-week multidisciplinary rehabilitation program on the basis of interviews , observations , and clinical tests . inclusion criteria for participation in the rehabilitation program were that the participants had chronic musculoskeletal pain ( longer than 3 months ) and were receiving national insurance in the form of sickness benefit or rehabilitation benefit . further , to assess any difference in physical function and return to work among those with comorbid depressive symptoms at the end of the rehabilitation , we included only those with high depressive symptoms measured by the hospital anxiety and depression scale ( hads ) . an optimal balance between sensitivity and specificity seems to be achieved when caseness is defined by a score of 8 on both the anxiety and depression subscales of the hads.18 four aspects of physical functioning ( muscle strength , endurance capacity , mobility , and balance ) and degree of physical pain were measured with single items on a visual analog scale ( vas ) . the use of vas in employees with musculoskeletal disorders is well - established , and single - item measures frequently perform almost as well as multi - item scales.19,20 the vas is a line 10 cm in length where agreement or disagreement represents a continuum of the specific physical function measured . muscle strength on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your muscle strength to be?21,22 endurance capacity on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your endurance capacity to be?21,22 mobility on a scale from 0100 where 0 is very limited flexibility and 100 is very flexible , how flexible do you consider your muscles and joints to be?21,22 balance on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your balance to be?21,22 physical fitness was measured through the validated coop / wonca charts23 by the following question : during the past two weeks , what was the hardest physical activity you could do for at least 2 minutes ? participants who reported working on a part - time or full - time basis were categorized as active and coded as on their way into / in work ( rtw ) , as were individuals in job retraining or an education program . the participants still on sick leave , receiving disability pension , or applying for disability pension after completing the 57-week multidisciplinary rehabilitation program were categorized as passive and coded as on their way out / out of work ( non - rtw ) . the dependent variable rtw was self - reported and defined by being on their way into / in work . a student s t - test for independent samples ( continuous variables ) and chi - square test ( categorical variables ) were used to compare those who were returning to work and those who were not . bivariate associations between each domain of physical function and rtw were assessed through logistic regression models , estimating odds ratio ( or ) with 95% confidence intervals ( ci ) . participants with a long - term history of mixed chronic musculoskeletal pain were recruited by their general practitioners , who assigned them to a 57-week multidisciplinary rehabilitation program on the basis of interviews , observations , and clinical tests . inclusion criteria for participation in the rehabilitation program were that the participants had chronic musculoskeletal pain ( longer than 3 months ) and were receiving national insurance in the form of sickness benefit or rehabilitation benefit . further , to assess any difference in physical function and return to work among those with comorbid depressive symptoms at the end of the rehabilitation , we included only those with high depressive symptoms measured by the hospital anxiety and depression scale ( hads ) . an optimal balance between sensitivity and specificity seems to be achieved when caseness is defined by a score of 8 on both the anxiety and depression subscales of the hads.18 four aspects of physical functioning ( muscle strength , endurance capacity , mobility , and balance ) and degree of physical pain were measured with single items on a visual analog scale ( vas ) . the use of vas in employees with musculoskeletal disorders is well - established , and single - item measures frequently perform almost as well as multi - item scales.19,20 the vas is a line 10 cm in length where agreement or disagreement represents a continuum of the specific physical function measured . muscle strength on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your muscle strength to be?21,22 endurance capacity on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your endurance capacity to be?21,22 mobility on a scale from 0100 where 0 is very limited flexibility and 100 is very flexible , how flexible do you consider your muscles and joints to be?21,22 balance on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your balance to be?21,22 physical fitness was measured through the validated coop / wonca charts23 by the following question : during the past two weeks , what was the hardest physical activity you could do for at least 2 minutes ? participants who reported working on a part - time or full - time basis were categorized as active and coded as on their way into / in work ( rtw ) , as were individuals in job retraining or an education program . the participants still on sick leave , receiving disability pension , or applying for disability pension after completing the 57-week multidisciplinary rehabilitation program were categorized as passive and coded as on their way out / out of work ( non - rtw ) . the dependent variable rtw was self - reported and defined by being on their way into / in work . a student s t - test for independent samples ( continuous variables ) and chi - square test ( categorical variables ) were used to compare those who were returning to work and those who were not . bivariate associations between each domain of physical function and rtw were assessed through logistic regression models , estimating odds ratio ( or ) with 95% confidence intervals ( ci ) . further , to assess any difference in physical function and return to work among those with comorbid depressive symptoms at the end of the rehabilitation , we included only those with high depressive symptoms measured by the hospital anxiety and depression scale ( hads ) . the final sample consisted of 92 persons ( 21.7% of all participants < 60 years ) with chronic musculoskeletal disorders and comorbid depression under 60 years of age who had undergone the 57-week multidisciplinary rehabilitation program . group ( rtw ) and the on their way out / out of work group ( non - rtw ) in terms of age , marital status , level of depression above the cut - off value of 8 , anxiety level , and pain experience ( table 2 ) . muscle strength , endurance capacity , mobility , balance , and physical fitness were significantly better among those who were not returning to work . in the bivariate analyses , four of the five domains of physical functioning measured by vas ( muscle strength , mobility , balance , and physical fitness ) were significantly negatively associated with rtw , indicating that better physical functioning was associated with not returning to work ( table 3 ) . among those with better function on muscle strength ( or 0.61 , 95% ci 0.450.83 ) , mobility ( or 0.73 , 95% ci 0.570.95 ) , and balance ( or 0.67 , 95% ci 0.500.91 ) , the odds of being on an active work re - entry strategy were 23%39% lower compared with those with poorer physical function . the same pattern was seen for physical fitness measured by the coop / wonca charts ( or 1.75 , 95% 1.072.99 ) , indicating that the odds of returning to work were 75% lower among those who reported that they were able to perform hard physical activity for at least 2 minutes . in this study of persons with chronic musculoskeletal disorders and comorbid depression , we found higher levels of physical functioning to be significantly associated with non - rtw at the end of the rehabilitation period of 57 weeks . given that the multidisciplinary rehabilitation program in this study places a strong emphasis on function and work ability , our results are somewhat surprising . additionally , we have not been able to find other studies confirming our results or the opposite ; that individuals with chronic musculoskeletal disorders and comorbid depression returning to work have better physical function compared with those who are depressed not returning to work . most studies on mental health and physical function suggest an inverse association between decreasing levels of depressive symptoms and increasing physical functioning . however , an exhaustive literature review showed that the association between depressive symptoms and physical functioning is unexpectedly weak.16 this may be due to low consensus on what physical measurement to use , and whether such measures should be performance - based or patient - reported . bean et al24 found that self - reported measures of physical function were associated with psychosocial factors such as depression , while performance - based measures were not . in a qualitative study of disability pensioners with chronic back pain , magnussen et al25 found that some of the barriers related to rtw were earlier negative work experiences , poor self - judgment of work ability , and low self - esteem . hence , the experience of being granted a disability pension , which means not returning to work , might be followed by the feelings of relief and satisfaction , while those , by definition , on their way into work might be subjected to remaining in an unsafe and uncertain situation , both economically and occupationally . nevertheless , according to the literature most psychosocial interventions to achieve optimal rehabilitation outcomes in employees with musculoskeletal pain have focused on risk factors within the individual ( eg , pain catastrophizing and expectancies ) and not on relevant factors at the workplace , such as interpersonal conflicts or job stress ( at the group or institutional level).27 in the vast majority of observational studies on factors related to the rtw process , as in this study as well , psychosocial interventions did not follow common evidence - based guidelines or treatment protocols.27,28 in turn , this makes any evaluation of treatment outcome after rehabilitation difficult , especially regarding assessment of interventions that focus on work - related factors outside of the individual.27 further , depression affects several areas related to occupational functioning , such as absenteeism , education , and employment opportunities . in a recent swedish study , sjstrm et al9 evaluated a 7-week multidisciplinary rehabilitation program in patients with musculoskeletal pain , with emphasis on mental symptoms measured by the hads . these results illustrate that the rtw process among depressed employees with musculoskeletal conditions is multifactorial and not merely a result of physical functioning or pain severity . epidemiological studies from the nord - trndelag health study in norway suggest that approximately one - third of individuals reporting somatic health problems also have anxiety disorder and/or depression30 and that a statistical relationship exists between anxiety , depression , and functional somatic symptoms , independent of age and sex.6 at the same time , medical examination of patients with chronic musculoskeletal pain often fails to demonstrate any organic diseases.15 in a norwegian population - based cohort study , mykletun et al31 found anxiety and depression to be strongly and independently associated with disability pensions granted for physical conditions and diagnoses , suggesting that administrative data may have underestimated the contribution of mental disorders to the awarding of disability pensions . all patients underwent orthopedic assessment of physical function and ability to work ; 83 were referred for psychiatric evaluation of diagnosis and function . inappropriate assessment of work ability leads to the wrong choice of interventions , which may employ and maintain unethical practices regarding the rtw process in employees with chronic musculoskeletal pain and comorbid depression . however , the main focus of this investigation was to assess the association between physical function and rtw among participants with chronic musculoskeletal pain with elevated depressive symptoms at the end of a multidisciplinary rehabilitation program . nevertheless , 22% of those younger than 60 years participating in this 57-week multidisciplinary rehabilitation program had elevated depressive symptoms at the end of the program . further , and in line with the suggestions from sjstrm et al,9 it is possible that this selected group of individuals with elevated depressive symptoms may require rehabilitation with different content other than what is usually offered through a traditional multidisciplinary rehabilitation program . most studies on mental health and physical function suggest an inverse association between decreasing levels of depressive symptoms and increasing physical functioning . however , an exhaustive literature review showed that the association between depressive symptoms and physical functioning is unexpectedly weak.16 this may be due to low consensus on what physical measurement to use , and whether such measures should be performance - based or patient - reported . bean et al24 found that self - reported measures of physical function were associated with psychosocial factors such as depression , while performance - based measures were not . in a qualitative study of disability pensioners with chronic back pain , magnussen et al25 found that some of the barriers related to rtw were earlier negative work experiences , poor self - judgment of work ability , and low self - esteem . hence , the experience of being granted a disability pension , which means not returning to work , might be followed by the feelings of relief and satisfaction , while those , by definition , on their way into work might be subjected to remaining in an unsafe and uncertain situation , both economically and occupationally . nevertheless , according to the literature most psychosocial interventions to achieve optimal rehabilitation outcomes in employees with musculoskeletal pain have focused on risk factors within the individual ( eg , pain catastrophizing and expectancies ) and not on relevant factors at the workplace , such as interpersonal conflicts or job stress ( at the group or institutional level).27 in the vast majority of observational studies on factors related to the rtw process , as in this study as well , psychosocial interventions did not follow common evidence - based guidelines or treatment protocols.27,28 in turn , this makes any evaluation of treatment outcome after rehabilitation difficult , especially regarding assessment of interventions that focus on work - related factors outside of the individual.27 further , depression affects several areas related to occupational functioning , such as absenteeism , education , and employment opportunities . depressed workers also report more conflicts and negative social outcomes.16 in a qualitative study of factors relevant in the assessment of the rtw process of employees on long - term sickness absence due to a depressive disorder , muijzer et al4 found that physical functioning , attitudes , competence , and environmental factors such as employer the study was , however , based on focus - group interviews with labor experts working at the social insurance institute in the netherlands . these results illustrate that the rtw process among depressed employees with musculoskeletal conditions is multifactorial and not merely a result of physical functioning or pain severity . epidemiological studies from the nord - trndelag health study in norway suggest that approximately one - third of individuals reporting somatic health problems also have anxiety disorder and/or depression30 and that a statistical relationship exists between anxiety , depression , and functional somatic symptoms , independent of age and sex.6 at the same time , medical examination of patients with chronic musculoskeletal pain often fails to demonstrate any organic diseases.15 in a norwegian population - based cohort study , mykletun et al31 found anxiety and depression to be strongly and independently associated with disability pensions granted for physical conditions and diagnoses , suggesting that administrative data may have underestimated the contribution of mental disorders to the awarding of disability pensions . the latter findings are partly supported by a recently published swedish study28 of biopsychosocial functioning and assessment of the ability to work in 174 patients on long - term sick leave due to chronic musculoskeletal pain . all patients underwent orthopedic assessment of physical function and ability to work ; 83 were referred for psychiatric evaluation of diagnosis and function . inappropriate assessment of work ability leads to the wrong choice of interventions , which may employ and maintain unethical practices regarding the rtw process in employees with chronic musculoskeletal pain and comorbid depression . however , the main focus of this investigation was to assess the association between physical function and rtw among participants with chronic musculoskeletal pain with elevated depressive symptoms at the end of a multidisciplinary rehabilitation program . nevertheless , 22% of those younger than 60 years participating in this 57-week multidisciplinary rehabilitation program had elevated depressive symptoms at the end of the program . further , and in line with the suggestions from sjstrm et al,9 it is possible that this selected group of individuals with elevated depressive symptoms may require rehabilitation with different content other than what is usually offered through a traditional multidisciplinary rehabilitation program . the findings of higher physical functioning among depressed employees not returning to work after participating in a multidisciplinary rehabilitation program in this study illustrate that self - reported physical functioning does not act as a proxy for occupational functioning in the rtw process . longitudinal studies with the use of different registries ( employment history , sick leave , and use of prescribed drugs ) should preferably be used in the evaluation of factors promoting the rtw process among people with chronic musculoskeletal disorders and poor mental health .

this study compared the use of two different topical antibiotics ( besifloxacin and gatifloxacin , each administered three times daily for 7 days ) in 33 neonatal subjects with bacterial conjunctivitis , a condition for which there are little published data in this age group.high rates of clinical resolution were observed with both antibiotics ; however , bacterial eradication occurred earlier with besifloxacin.both antibiotics were well tolerated in this small group of neonates and there were no adverse events with besifloxacin treatment . neonatal conjunctivitis is an acute condition characterized by conjunctival erythema , swelling , and mucopurulent discharge occurring within the first 30 days of life [ 13 ] . while viruses are a common cause of pediatric conjunctivitis , studies conducted in north america that included children < 5 years of age with purulent conjunctival discharge identified a bacterial pathogen in 6580% of cultures [ 46 ] . the american academy of ophthalmology recommends conjunctival cultures be taken from all cases of suspected neonatal bacterial conjunctivitis . conjunctivitis in a newborn can pose a risk for the development of secondary ocular infections , including endophthalmitis and keratitis ( in cases of gonococcal etiology ) [ 1 , 8 ] , permanent eye damage , and even blindness , although the latter is rare in industrialized countries . systemic complications such as pneumonitis , meningitis , and septicemia are also possible [ 1 , 2 , 8 ] . the recommended management of suspected bacterial neonatal conjunctivitis includes the use of systemic and/or topical antibiotics [ 1 , 4 ] . topical fluoroquinolones are often preferred for the treatment of bacterial conjunctivitis in the general population due to their rapid bactericidal activity , broad spectrum of activity , and low toxicity [ 9 , 10 ] . although some studies have included neonates within a broader population [ 1114 ] , to our knowledge there are no published reports of topical fluoroquinolones used specifically in neonatal conjunctivitis , with the exception of a single study published in abstract form only . besifloxacin is a topical fluoroquinolone and represents the first chloro - fluoroquinolone developed specifically for ophthalmic use . the broad spectrum activity of besifloxacin includes potent in vitro activity against drug - resistant strains such as ciprofloxacin - resistant , methicillin - resistant s. aureus ( mrsa ) and methicillin - resistant coagulase - negative staphylococcus species [ 1619 ] . besifloxacin ophthalmic suspension 0.6% ( besivance ; bausch & lomb , tampa , fl , usa ) is approved by the us fda for the treatment of bacterial conjunctivitis , with a recommended dosing regimen of three times daily for 7 days ; this indication is based on clinical studies conducted in patients 1 year of age . a prior analysis of pediatric ( ages 117 ; n = 815 ) subgroup data from three bacterial conjunctivitis studies in patients of all ages demonstrated significant and high rates of clinical resolution ( 88.1% ) and bacterial eradication ( 82.8% ) with besifloxacin treatment at day 8 or 9 ( visit 3 ) compared with vehicle ( p 0.009 ) . rates of clinical resolution and bacterial eradication were similar between besifloxacin and moxifloxacin treatment groups for patients aged 117 at all visits ( p = ns ) . among subjects aged 1 year , there was statistically significant bacterial eradication at day 5 1 ( visit 2 ) compared with vehicle ( p = 0.04 ) . the current study was designed to evaluate the safety and efficacy of besifloxacin ophthalmic suspension 0.6% compared with gatifloxacin ophthalmic solution 0.3% ( zymar ; allergan , irvine , ca , usa ) when administered three times daily for 7 days in neonates with bacterial conjunctivitis . gatifloxacin was selected as an active comparator because it has an antibacterial action similar to besifloxacin , they both inhibit bacterial dna gyrase and topoisomerase iv , and because it was previously reported to be safe when administered three times daily in neonates . this multicenter , randomized , double - masked , active - controlled , parallel group study ( nct01330355 ) was initiated at 11 sites in the us between may 2011 and october 2012 . eligible subjects were aged 31 days , with a clinical diagnosis of acute bacterial conjunctivitis in one or both eyes , and a severity of grade 1 for both conjunctival discharge and conjunctival hyperemia in the same eye ( each rated on a scale of 0 [ absent / normal ] to 3 [ severe ] ) . subjects with suspected fungal , protozoal , or viral etiology in either eye , evidence of chemical or physical trauma to either eye or ocular adnexa , and subjects with corneal infiltrates or ulcer in either eye were excluded . use of systemic or topical non - prophylactic antimicrobial therapy within 96 h of day 1 ( baseline ) , or expected use of such during the study period , was not allowed . subjects who had received topical antimicrobial therapy for routine prophylaxis ( e.g. at the time of birth ) could be enrolled 24 h or more after the last application of antibiotic prophylaxis . systemic or topical antimicrobials were not allowed to be used by the breastfeeding mother or wet nurse . subjects were also excluded if they required concomitant use of ophthalmic ( either eye ) or systemic corticosteroids , systemic non - steroidal anti - inflammatory drugs ( nsaids ) , systemic antihistamines , or ocular immunosuppressants . the study was conducted in accordance with good clinical practice ( as described in the international conference on harmonisation guidelines ) , applicable local regulations , and the ethical principles in the declaration of helsinki . the protocol was approved by the institutional review boards associated with individual study sites , and written informed consent was obtained by each subject s parent or legally authorized representative prior to study participation . subjects who met the eligibility criteria had an initial eye examination that included an assessment of ocular signs , and had a conjunctival swab taken for culture from the affected eye(s ) . subjects were then randomly assigned in a 1:1 ratio , according to a computer - generated randomization list , to receive besifloxacin or gatifloxacin instilled in the affected eye(s ) three times daily for 7 days . the investigator , the subject s parent / authorized representative , and all study personnel involved in study conduct and monitoring were masked to the study treatment identity . masking was accomplished by replacing the commercial labeling on besifloxacin and gatifloxacin bottles with identical investigational labels and packaging them in identical kit boxes . a designee at each study site was given responsibility for dispensing / collecting study materials to subjects . the first dose of study medication was instilled in the clinic following the initial eye examination and conjunctival culture . parents / guardians were instructed to continue administration of study treatment at approximately 6 h intervals . both remaining doses on day 1 were to be administered , even if the resulting intervals were shorter than 6 h. following the day 1/start of treatment visit , subjects returned to the clinic at visit 3 ( day 4 1 ) and visit 5 ( day 8 or 9 ) for clinical assessment of ocular signs and culture of the affected eye(s ) . vital signs and body weight measurements were taken , an ocular examination of both eyes was performed , and ocular and non - ocular adverse events ( aes ) were recorded . ocular examinations consisted of assessment of light perception , eyelid edema , conjunctival chemosis , the pupillary reflex , and the red reflex test , as well as clinical examination of the eyelid ( other than eyelid edema ) , the conjunctiva ( other than conjunctival discharge , conjunctival hyperemia , and conjunctival chemosis ) , and the cornea . all assessments were performed in both eyes , with the exception of microbial cultures , which were taken from baseline - affected eyes only . in addition to the clinic visits , parents / guardians were asked to complete a telephone contact at visits 2 ( day 2 ) and 4 ( day 6 or 7 ) to confirm their compliance with dosing and to solicit aes . at each visit , samples were obtained from the conjunctival cul - de - sac of baseline - affected eye(s ) using a sterile swab , and the swabs were sent to covance central laboratory services , inc . subjects were considered culture - positive or culture - confirmed if the bacterial colony count for a particular species ( in colony forming units per ml ; cfu / ml ) equaled or exceeded the threshold value for that species on the cagle list , as modified by leibowitz [ 24 , 25 ] . for isolates that met / exceeded the bacterial threshold , minimum inhibitory concentration ( mic ) testing was performed for besifloxacin and comparator antibacterial agents following procedures recommended by the clinical and laboratory standards institute . primary endpoints included clinical resolution ( binary outcome [ yes / no ] , with clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia ; severity grade = 0 at visit 5 ) and rates of ocular and non - ocular treatment - emergent aes ( teaes ) . ocular discharge was rated on a scale of 0 ( absent ) , 1 ( mild ) , 2 ( moderate ) , and 3 ( severe ) , and conjunctival hyperemia was rated on a scale of 0 ( normal ) , 1 ( mild ) , 2 ( moderate ) , and 3 ( severe ) . secondary endpoints included clinical resolution at visit 3 and bacterial eradication at visits 3 and 5 in culture - positive eyes . bacterial eradication ( binary outcome [ yes / no ] ) was defined as the absence of all ocular bacterial species that were present at or above threshold at visit 1 . all teaes ( ocular and non - ocular ) observed by the investigator or reported by the subject s parent / guardian were recorded using the medical dictionary for regulatory activities ( version 15.1 ) body system and preferred terms , and characterized as mild , moderate , or severe . secondary safety outcomes included the results of ocular examinations , physical examination , and vital signs . ocular examinations included light perception assessment ( present or absent ) , eyelid edema and conjunctival chemosis ( each rated on a scale from 0 = none to 3 = severe ) , and pupillary reflex and red reflex test ( rated as normal or abnormal ) . with the exception of the endpoint of individual or species - specific bacterial eradication , one eye per subject was designated as the study eye for analysis of efficacy endpoints . in subjects with bilateral conjunctivitis , the study eye was the eye with the highest combined sum of ratings ( i.e. severity ) for ocular discharge and conjunctival hyperemia at baseline . for cases in which baseline severity ratings were equal for both eyes , the right eye was designated as the study eye . in the analyses by individual bacterial species , non - study eyes ( i.e. fellow eyes ) could contribute data provided the severity of conjunctivitis in that eye met the inclusion criteria and the bacterial species that was at or above threshold in that eye at baseline was different from the species cultured from the study eye . the primary population for the efficacy analysis was the intent - to - treat ( itt ) population , which included all randomized subjects . additional analyses , including all analyses on bacterial endpoints , were performed in the modified itt ( mitt ) population , which was defined as subjects in the itt population with baseline bacterial culture at or above threshold for any accepted ocular bacterial species . the safety population included all subjects who received at least one dose of study drug as part of the protocol and who had at least one post - treatment safety assessment . efficacy findings for baseline - designated study eyes and treated fellow eyes were summarized using descriptive statistics , with missing data imputed using the last observation carried forward . differences between treatments in clinical resolution and bacterial eradication were evaluated for baseline - designated study eyes only using the asymptotic pearson chi - square test . all analyses were performed using statistical analysis software version 9.1 ( sas institute , inc . , the study initially sought to enroll 200 subjects ( 100 per treatment group ) to obtain 100 culture - positive subjects , but was terminated early due to a low enrollment rate unrelated to safety or efficacy concerns . this multicenter , randomized , double - masked , active - controlled , parallel group study ( nct01330355 ) was initiated at 11 sites in the us between may 2011 and october 2012 . eligible subjects were aged 31 days , with a clinical diagnosis of acute bacterial conjunctivitis in one or both eyes , and a severity of grade 1 for both conjunctival discharge and conjunctival hyperemia in the same eye ( each rated on a scale of 0 [ absent / normal ] to 3 [ severe ] ) . subjects with suspected fungal , protozoal , or viral etiology in either eye , evidence of chemical or physical trauma to either eye or ocular adnexa , and subjects with corneal infiltrates or ulcer in either eye were excluded . use of systemic or topical non - prophylactic antimicrobial therapy within 96 h of day 1 ( baseline ) , or expected use of such during the study period , was not allowed . subjects who had received topical antimicrobial therapy for routine prophylaxis ( e.g. at the time of birth ) could be enrolled 24 h or more after the last application of antibiotic prophylaxis . systemic or topical antimicrobials were not allowed to be used by the breastfeeding mother or wet nurse . subjects were also excluded if they required concomitant use of ophthalmic ( either eye ) or systemic corticosteroids , systemic non - steroidal anti - inflammatory drugs ( nsaids ) , systemic antihistamines , or ocular immunosuppressants . the study was conducted in accordance with good clinical practice ( as described in the international conference on harmonisation guidelines ) , applicable local regulations , and the ethical principles in the declaration of helsinki . the protocol was approved by the institutional review boards associated with individual study sites , and written informed consent was obtained by each subject s parent or legally authorized representative prior to study participation . subjects who met the eligibility criteria had an initial eye examination that included an assessment of ocular signs , and had a conjunctival swab taken for culture from the affected eye(s ) . subjects were then randomly assigned in a 1:1 ratio , according to a computer - generated randomization list , to receive besifloxacin or gatifloxacin instilled in the affected eye(s ) three times daily for 7 days . the investigator , the subject s parent / authorized representative , and all study personnel involved in study conduct and monitoring were masked to the study treatment identity . masking was accomplished by replacing the commercial labeling on besifloxacin and gatifloxacin bottles with identical investigational labels and packaging them in identical kit boxes . a designee at each study site was given responsibility for dispensing / collecting study materials to subjects . the first dose of study medication was instilled in the clinic following the initial eye examination and conjunctival culture . parents / guardians were instructed to continue administration of study treatment at approximately 6 h intervals . both remaining doses on day 1 were to be administered , even if the resulting intervals were shorter than 6 h. following the day 1/start of treatment visit , subjects returned to the clinic at visit 3 ( day 4 1 ) and visit 5 ( day 8 or 9 ) for clinical assessment of ocular signs and culture of the affected eye(s ) . vital signs and body weight measurements were taken , an ocular examination of both eyes was performed , and ocular and non - ocular adverse events ( aes ) were recorded . ocular examinations consisted of assessment of light perception , eyelid edema , conjunctival chemosis , the pupillary reflex , and the red reflex test , as well as clinical examination of the eyelid ( other than eyelid edema ) , the conjunctiva ( other than conjunctival discharge , conjunctival hyperemia , and conjunctival chemosis ) , and the cornea . all assessments were performed in both eyes , with the exception of microbial cultures , which were taken from baseline - affected eyes only . in addition to the clinic visits , parents / guardians were asked to complete a telephone contact at visits 2 ( day 2 ) and 4 ( day 6 or 7 ) to confirm their compliance with dosing and to solicit aes . at each visit , samples were obtained from the conjunctival cul - de - sac of baseline - affected eye(s ) using a sterile swab , and the swabs were sent to covance central laboratory services , inc . subjects were considered culture - positive or culture - confirmed if the bacterial colony count for a particular species ( in colony forming units per ml ; cfu / ml ) equaled or exceeded the threshold value for that species on the cagle list , as modified by leibowitz [ 24 , 25 ] . for isolates that met / exceeded the bacterial threshold , minimum inhibitory concentration ( mic ) testing was performed for besifloxacin and comparator antibacterial agents following procedures recommended by the clinical and laboratory standards institute . primary endpoints included clinical resolution ( binary outcome [ yes / no ] , with clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia ; severity grade = 0 at visit 5 ) and rates of ocular and non - ocular treatment - emergent aes ( teaes ) . ocular discharge was rated on a scale of 0 ( absent ) , 1 ( mild ) , 2 ( moderate ) , and 3 ( severe ) , and conjunctival hyperemia was rated on a scale of 0 ( normal ) , 1 ( mild ) , 2 ( moderate ) , and 3 ( severe ) . secondary endpoints included clinical resolution at visit 3 and bacterial eradication at visits 3 and 5 in culture - positive eyes . bacterial eradication ( binary outcome [ yes / no ] ) was defined as the absence of all ocular bacterial species that were present at or above threshold at visit 1 . all teaes ( ocular and non - ocular ) observed by the investigator or reported by the subject s parent / guardian were recorded using the medical dictionary for regulatory activities ( version 15.1 ) body system and preferred terms , and characterized as mild , moderate , or severe . secondary safety outcomes included the results of ocular examinations , physical examination , and vital signs . ocular examinations included light perception assessment ( present or absent ) , eyelid edema and conjunctival chemosis ( each rated on a scale from 0 = none to 3 = severe ) , and pupillary reflex and red reflex test ( rated as normal or abnormal ) . with the exception of the endpoint of individual or species - specific bacterial eradication , one eye per subject was designated as the study eye for analysis of efficacy endpoints . in subjects with bilateral conjunctivitis , the study eye was the eye with the highest combined sum of ratings ( i.e. severity ) for ocular discharge and conjunctival hyperemia at baseline . for cases in which baseline severity ratings were equal for both eyes , the right eye was designated as the study eye . in the analyses by individual bacterial species , non - study eyes ( i.e. fellow eyes ) could contribute data provided the severity of conjunctivitis in that eye met the inclusion criteria and the bacterial species that was at or above threshold in that eye at baseline was different from the species cultured from the study eye . the primary population for the efficacy analysis was the intent - to - treat ( itt ) population , which included all randomized subjects . additional analyses , including all analyses on bacterial endpoints , were performed in the modified itt ( mitt ) population , which was defined as subjects in the itt population with baseline bacterial culture at or above threshold for any accepted ocular bacterial species . the safety population included all subjects who received at least one dose of study drug as part of the protocol and who had at least one post - treatment safety assessment . efficacy findings for baseline - designated study eyes and treated fellow eyes were summarized using descriptive statistics , with missing data imputed using the last observation carried forward . differences between treatments in clinical resolution and bacterial eradication were evaluated for baseline - designated study eyes only using the asymptotic pearson chi - square test . all analyses were performed using statistical analysis software version 9.1 ( sas institute , inc . , the study initially sought to enroll 200 subjects ( 100 per treatment group ) to obtain 100 culture - positive subjects , but was terminated early due to a low enrollment rate unrelated to safety or efficacy concerns . a total of 33 subjects ( besifloxacin , n = 16 ; gatifloxacin , n = 17 ) , all neonatal ( < 28 days ) , were enrolled at seven clinical sites and comprised the itt population . of the 33 enrolled subjects , 32 ( 97% ) completed the study ; one subject randomized to gatifloxacin withdrew consent and discontinued . twenty - two subjects ( besifloxacin , n = 13 ; gatifloxacin , n = 9 ) had culture - positive conjunctivitis in at least one eye and were included in the mitt population . overall , the mean ( standard deviation ) age of subjects was 15.5 days ( 6.0 ) and 15.7 days ( 5.3 ) , and 57.6% and 54.5% of subjects in the itt and mitt populations , respectively , were female.table 1demographic characteristicsitt populationmitt populationbesifloxacin [ n = 16]gatifloxacin [ n = 17]besifloxacin [ n = 13]gatifloxacin [ n = 9]age , days mean ( sd)15.8 ( 6.39)15.2 ( 5.75)15.9 ( 6.01)15.4 ( 4.48 ) min , max6 , 265 , 256 , 2511 , 25gender male4 ( 25.0)10 ( 58.8)3 ( 23.1)7 ( 77.8 ) female12 ( 75.0)7 ( 41.2)10 ( 76.9)2 ( 22.2)race asian1 ( 6.3)1 ( 5.9)1 ( 7.7)1 ( 11.1 ) black / african american01 ( 5.9)01 ( 11.1 ) white12 ( 75.0)12 ( 70.6)10 ( 76.9)6 ( 66.7 ) other3 ( 18.8)3 ( 17.6)2 ( 15.4)1 ( 11.1)data are expressed as n ( % ) unless otherwise specified itt intent - to - treat , mitt modified intent - to - treat , sd standard deviation , min minimum , max maximum demographic characteristics data are expressed as n ( % ) unless otherwise specified itt intent - to - treat , mitt modified intent - to - treat , sd standard deviation , min minimum , max maximum table 2 presents bacterial pathogens above threshold isolated at baseline from all culture - positive eyes , along with the mic of besifloxacin and gatifloxacin for these isolates . a total of 50 bacterial isolates meeting threshold criteria for pathogenicity in bacterial conjunctivitis were identified , and most were gram - positive . the most common gram - positive bacterial species cultured were streptococcus mitis , staphylococcus epidermidis , and staphylococcus aureus , while the most common gram - negative bacterial species cultured was moraxella catarrhalis . overall , the mic or range of mics ( in cases of more than one isolate ) for besifloxacin appeared lower to those noted with gatifloxacin for gram - positive organisms ; for gram - negative organisms , besifloxacin mics were higher than or equal to those observed with gatifloxacin.table 2bacterial species above the threshold criteria for pathogenicity isolated at baseline , and minimum inhibitory concentrations of besifloxacin and gatifloxacin for those isolatesbacterial speciesno . of isolates minimum inhibitory concentrations ( g / ml)besifloxacingatifloxacingram - positive380.01580.0664 cdc coryneform group g10.030.06 enterococcus faecalis 10.250.5 lactococcus garvieae 10.50.5 staphylococcus aureus 60.0150.060.060.25 staphylococcus epidermidis 90.0380.0664 staphylococcus hominis 40.060.120.25 staphylococcus warneri 10.120.25 streptococcus mitis group110.0620.2532 streptococcus salivarius group40.060.120.120.5gram - negative120.0340.031 chryseobacterium indologenes 210.5 chryseobacterium species 141 elizabethkingia meningoseptica 110.25 haemophilus influenzae 10.030.03 leclercia adecarboxylata 10.060.03 moraxella catarrhalis 40.060.120.030.06 serratia marcescens 10.50.25 wautersiella falsenii 10.50.5 cfu colony - forming units , cdc centers for disease control threshold criteria were 1000 cfu / ml for the cdc coryneform group g ; 100 cfu / ml for staphylococcus spp ( except s. aureus ) ; 10 cfu / ml for e. faecalis , l. garvieae , s. aureus , streptococcus spp , and m. catarrhalis ; 1 cfu / ml for gram - negative species ( except m. catarrhalis ) number of times a specific bacterial species was isolated at or above threshold at baseline from study eyes or treated fellow eyes bacterial species above the threshold criteria for pathogenicity isolated at baseline , and minimum inhibitory concentrations of besifloxacin and gatifloxacin for those isolates cfu colony - forming units , cdc centers for disease control threshold criteria were 1000 cfu / ml for the cdc coryneform group g ; 100 cfu / ml for staphylococcus spp ( except s. aureus ) ; 10 cfu / ml for e. faecalis , l. garvieae , s. aureus , streptococcus spp , and m. catarrhalis ; 1 cfu / ml for gram - negative species ( except m. catarrhalis ) number of times a specific bacterial species was isolated at or above threshold at baseline from study eyes or treated fellow eyes table 3 presents clinical resolution rates at visits 3 and 5 . in the itt population , clinical resolution at visit 5 ( day 8 or 9 ; primary efficacy endpoint ) was observed in 75.0% of study eyes treated with besifloxacin , compared with 70.6% of study eyes treated with gatifloxacin ( p = 0.78 ) . in the mitt population , clinical resolution at visit 5 was observed in 84.6% of besifloxacin - treated study eyes compared with 77.8% of gatifloxacin - treated study eyes ( p = 0.68 ) . at visit 3 ( day 4 1 ) , clinical resolution rates were 18.8 vs. 29.4% ( itt population ) and 7.7 vs. 33.3% ( mitt population ) in besifloxacin- and gatifloxacin - treated eyes , respectively ( p 0.13 ) . in treated fellow eyes , patterns in clinical resolution rates between treatment groups appeared similar to those observed in study eyes at both visit 3 and visit 5 ( statistical analyses not performed).table 3clinical resolution at visits 3 and 5 ( locf)baseline - designated study eye [ n / n ( % ) ] fellow treated eye [ n / n ( % ) ] besifloxacingatifloxacin p value besifloxacingatifloxacinvisit 3 ( day 4 1 ) itt population3/16 ( 18.8)5/17 ( 29.4)0.481/8 ( 12.5)2/5 ( 40.0 ) mitt population1/13 ( 7.7)3/9 ( 33.3)0.130/5 ( 0.0)2/3 ( 66.7)visit 5 ( day 8 or 9 ) itt population12/16 ( 75.0)12/17 ( 70.6)0.785/8 ( 62.5)2/5 ( 40.0 ) mitt population11/13 ( 84.6)7/9 ( 77.8)0.684/5 ( 80.0)2/3 ( 66.7 ) itt intent - to - treat , mitt modified intent - to - treat , locf last observation carried forward clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia pearson chi - square test ; locf primary outcome visit ( study eye only ) clinical resolution at visits 3 and 5 ( locf ) itt intent - to - treat , mitt modified intent - to - treat , locf last observation carried forward clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia pearson chi - square test ; locf primary outcome visit ( study eye only ) figure 1 presents bacterial eradication rates at visits 3 and 5 for culture - positive , baseline - designated study eyes ( mitt population ) . there was a significant difference between treatments in favor of besifloxacin at visit 3 ( 84.6 vs. 44.4% ; p = 0.0467 ) but not at visit 5 ( 92.3 vs. 88.9% : p = 0.7839 ) . among treated fellow eyes , bacterial eradication at visit 3 was achieved in three of five eyes treated with besifloxacin and two of three eyes treated with gatifloxacin ; at visit 5 , bacterial eradication occurred in four of five eyes treated with besifloxacin and all eyes ( 3/3 ) treated with gatifloxacin.fig . 1bacterial eradication rates at visits 3 and 5 ( culture - positive , baseline - designated study eyes , modified intent - to - treat population , last observation carried forward ) . p values from the pearson chi - square test bacterial eradication rates at visits 3 and 5 ( culture - positive , baseline - designated study eyes , modified intent - to - treat population , last observation carried forward ) . p values from the pearson chi - square test table 4 presents bacterial eradication data for individual bacterial species at visits 3 and 5 . as indicated earlier , treated fellow eyes could contribute data if the bacterial species isolated from that eye was different from that cultured from the baseline - designated study eye . at visit 3 , 88.9% ( 16/18 ) of gram - positive organisms were eradicated in besifloxacin - treated eyes , compared with 46.2% ( 6/16 ) in gatifloxacin - treated eyes . the percentage of all culture - positive treated eyes showing eradication of gram - negative organisms by visit 3 was 100% ( 6/6 ) in the besifloxacin group and 75.0% ( 3/4 ) in the gatifloxacin group . at visit 5 , all species were eradicated , with the only exceptions being staphylococcus epidermidis in a gatifloxacin - treated eye and staphylococcus hominis in a besifloxacin - treated eye . however , the bacterial count for these isolates was observed to be reduced to below threshold levels , with no new species present in both cases.table 4bacterial eradication by species at visits 3 and 5 , mitt population ( locf)bacterial speciesspecies - specific study eye [ n / n]besifloxacingatifloxacinvisit 3visit 5visit 3visit 5gram - positive16/1817/186/1312/13 cdc coryneform group g1/11/1 enterococcus faecalis 1/11/1 lactococcus garvieae 1/11/1 staphylococcus aureus 3/33/31/22/2 staphylococcus epidermidis 2/22/22/54/5 staphylococcus hominis 1/21/20/11/1 staphylococcus warneri 1/11/1 streptococcus mitis group5/66/62/33/3 streptococcus salivarius group2/22/20/11/1gram - negative6/66/63/44/4 chryseobacterium indologenes 1/11/1 chryseobacterium species 1/11/1 elizabethkingia meningoseptica 1/11/1 haemophilus influenzae 1/11/1 leclercia adecarboxylata 1/11/1 moraxella catarrhalis 2/22/21/11/1 serratia marcescens 0/11/1 wautersiella falsenii 1/11/1 mitt modified intent - to - treat , locf last observation carried forward , cdc centers for disease control bacterial eradication by species at visits 3 and 5 , mitt population ( locf ) mitt modified intent - to - treat , locf last observation carried forward , cdc centers for disease control study eyes had a mean exposure of 6.94 ( 0.25 ) and 6.65 ( 1.46 ) days in the besifloxacin and gatifloxacin treatment groups , respectively . the mean exposure for all treated eyes ( i.e. sum of study eye and treated fellow eye exposure ) was 10.38 ( 3.52 ) and 8.71 ( 3.60 ) eye - days for the besifloxacin and gatifloxacin treatment groups , respectively . no teaes , either ocular or non - ocular , were reported in either study or fellow eyes of subjects in the besifloxacin treatment group . in the gatifloxacin treatment group , a total of six aes ( five non - ocular , one ocular ) were reported . the one ocular ae consisted of mild bacterial conjunctivitis in an initially untreated fellow eye , which occurred after visit 1 . the five non - ocular aes occurring in four subjects included abdominal pain , irritability , rhinorrhea , acne infantile , and dermatitis . there were no meaningful findings noted for vital sign measurements or results of ocular or physical examinations performed during the study . in addition , there were no cases of severe eyelid edema or severe chemosis at any visit , and all cases of eyelid edema and chemosis resolved at visit 5 . pupillary reflex , red reflex test , and light perception were normal throughout the study . no major findings were identified on eyelid and conjunctival examinations in either treatment group , and all corneal examinations were normal . a total of 33 subjects ( besifloxacin , n = 16 ; gatifloxacin , n = 17 ) , all neonatal ( < 28 days ) , were enrolled at seven clinical sites and comprised the itt population . of the 33 enrolled subjects , 32 ( 97% ) completed the study ; one subject randomized to gatifloxacin withdrew consent and discontinued . twenty - two subjects ( besifloxacin , n = 13 ; gatifloxacin , n = 9 ) had culture - positive conjunctivitis in at least one eye and were included in the mitt population . overall , the mean ( standard deviation ) age of subjects was 15.5 days ( 6.0 ) and 15.7 days ( 5.3 ) , and 57.6% and 54.5% of subjects in the itt and mitt populations , respectively , were female.table 1demographic characteristicsitt populationmitt populationbesifloxacin [ n = 16]gatifloxacin [ n = 17]besifloxacin [ n = 13]gatifloxacin [ n = 9]age , days mean ( sd)15.8 ( 6.39)15.2 ( 5.75)15.9 ( 6.01)15.4 ( 4.48 ) min , max6 , 265 , 256 , 2511 , 25gender male4 ( 25.0)10 ( 58.8)3 ( 23.1)7 ( 77.8 ) female12 ( 75.0)7 ( 41.2)10 ( 76.9)2 ( 22.2)race asian1 ( 6.3)1 ( 5.9)1 ( 7.7)1 ( 11.1 ) black / african american01 ( 5.9)01 ( 11.1 ) white12 ( 75.0)12 ( 70.6)10 ( 76.9)6 ( 66.7 ) other3 ( 18.8)3 ( 17.6)2 ( 15.4)1 ( 11.1)data are expressed as n ( % ) unless otherwise specified itt intent - to - treat , mitt modified intent - to - treat , sd standard deviation , min minimum , max maximum demographic characteristics data are expressed as n ( % ) unless otherwise specified itt intent - to - treat , mitt modified intent - to - treat , sd standard deviation , min minimum , max maximum table 2 presents bacterial pathogens above threshold isolated at baseline from all culture - positive eyes , along with the mic of besifloxacin and gatifloxacin for these isolates . a total of 50 bacterial isolates meeting threshold criteria for pathogenicity in bacterial conjunctivitis were identified , and most were gram - positive . the most common gram - positive bacterial species cultured were streptococcus mitis , staphylococcus epidermidis , and staphylococcus aureus , while the most common gram - negative bacterial species cultured was moraxella catarrhalis . overall , the mic or range of mics ( in cases of more than one isolate ) for besifloxacin appeared lower to those noted with gatifloxacin for gram - positive organisms ; for gram - negative organisms , besifloxacin mics were higher than or equal to those observed with gatifloxacin.table 2bacterial species above the threshold criteria for pathogenicity isolated at baseline , and minimum inhibitory concentrations of besifloxacin and gatifloxacin for those isolatesbacterial speciesno . of isolates minimum inhibitory concentrations ( g / ml)besifloxacingatifloxacingram - positive380.01580.0664 cdc coryneform group g10.030.06 enterococcus faecalis 10.250.5 lactococcus garvieae 10.50.5 staphylococcus aureus 60.0150.060.060.25 staphylococcus epidermidis 90.0380.0664 staphylococcus hominis 40.060.120.25 staphylococcus warneri 10.120.25 streptococcus mitis group110.0620.2532 streptococcus salivarius group40.060.120.120.5gram - negative120.0340.031 chryseobacterium indologenes 210.5 chryseobacterium species 141 elizabethkingia meningoseptica 110.25 haemophilus influenzae 10.030.03 leclercia adecarboxylata 10.060.03 moraxella catarrhalis 40.060.120.030.06 serratia marcescens 10.50.25 wautersiella falsenii 10.50.5 cfu colony - forming units , cdc centers for disease control threshold criteria were 1000 cfu / ml for the cdc coryneform group g ; 100 cfu / ml for staphylococcus spp ( except s. aureus ) ; 10 cfu / ml for e. faecalis , l. garvieae , s. aureus , streptococcus spp , and m. catarrhalis ; 1 cfu / ml for gram - negative species ( except m. catarrhalis ) number of times a specific bacterial species was isolated at or above threshold at baseline from study eyes or treated fellow eyes bacterial species above the threshold criteria for pathogenicity isolated at baseline , and minimum inhibitory concentrations of besifloxacin and gatifloxacin for those isolates cfu colony - forming units , cdc centers for disease control threshold criteria were 1000 cfu / ml for the cdc coryneform group g ; 100 cfu / ml for staphylococcus spp ( except s. aureus ) ; 10 cfu / ml for e. faecalis , l. garvieae , s. aureus , streptococcus spp , and m. catarrhalis ; 1 cfu / ml for gram - negative species ( except m. catarrhalis ) number of times a specific bacterial species was isolated at or above threshold at baseline from study eyes or treated fellow eyes table 3 presents clinical resolution rates at visits 3 and 5 . in the itt population , clinical resolution at visit 5 ( day 8 or 9 ; primary efficacy endpoint ) was observed in 75.0% of study eyes treated with besifloxacin , compared with 70.6% of study eyes treated with gatifloxacin ( p = 0.78 ) . in the mitt population , clinical resolution at visit 5 was observed in 84.6% of besifloxacin - treated study eyes compared with 77.8% of gatifloxacin - treated study eyes ( p = 0.68 ) . at visit 3 ( day 4 1 ) , clinical resolution rates were 18.8 vs. 29.4% ( itt population ) and 7.7 vs. 33.3% ( mitt population ) in besifloxacin- and gatifloxacin - treated eyes , respectively ( p 0.13 ) . in treated fellow eyes , patterns in clinical resolution rates between treatment groups appeared similar to those observed in study eyes at both visit 3 and visit 5 ( statistical analyses not performed).table 3clinical resolution at visits 3 and 5 ( locf)baseline - designated study eye [ n / n ( % ) ] fellow treated eye [ n / n ( % ) ] besifloxacingatifloxacin p value besifloxacingatifloxacinvisit 3 ( day 4 1 ) itt population3/16 ( 18.8)5/17 ( 29.4)0.481/8 ( 12.5)2/5 ( 40.0 ) mitt population1/13 ( 7.7)3/9 ( 33.3)0.130/5 ( 0.0)2/3 ( 66.7)visit 5 ( day 8 or 9 ) itt population12/16 ( 75.0)12/17 ( 70.6)0.785/8 ( 62.5)2/5 ( 40.0 ) mitt population11/13 ( 84.6)7/9 ( 77.8)0.684/5 ( 80.0)2/3 ( 66.7 ) itt intent - to - treat , mitt modified intent - to - treat , locf last observation carried forward clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia pearson chi - square test ; locf primary outcome visit ( study eye only ) clinical resolution at visits 3 and 5 ( locf ) itt intent - to - treat , mitt modified intent - to - treat , locf last observation carried forward clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia pearson chi - square test ; locf primary outcome visit ( study eye only ) figure 1 presents bacterial eradication rates at visits 3 and 5 for culture - positive , baseline - designated study eyes ( mitt population ) . there was a significant difference between treatments in favor of besifloxacin at visit 3 ( 84.6 vs. 44.4% ; p = 0.0467 ) but not at visit 5 ( 92.3 vs. 88.9% : p = 0.7839 ) . among treated fellow eyes , bacterial eradication at visit 3 was achieved in three of five eyes treated with besifloxacin and two of three eyes treated with gatifloxacin ; at visit 5 , bacterial eradication occurred in four of five eyes treated with besifloxacin and all eyes ( 3/3 ) treated with gatifloxacin.fig . 1bacterial eradication rates at visits 3 and 5 ( culture - positive , baseline - designated study eyes , modified intent - to - treat population , last observation carried forward ) . p values from the pearson chi - square test bacterial eradication rates at visits 3 and 5 ( culture - positive , baseline - designated study eyes , modified intent - to - treat population , last observation carried forward ) . p values from the pearson chi - square test table 4 presents bacterial eradication data for individual bacterial species at visits 3 and 5 . as indicated earlier , treated fellow eyes could contribute data if the bacterial species isolated from that eye was different from that cultured from the baseline - designated study eye . at visit 3 , 88.9% ( 16/18 ) of gram - positive organisms were eradicated in besifloxacin - treated eyes , compared with 46.2% ( 6/16 ) in gatifloxacin - treated eyes . the percentage of all culture - positive treated eyes showing eradication of gram - negative organisms by visit 3 was 100% ( 6/6 ) in the besifloxacin group and 75.0% ( 3/4 ) in the gatifloxacin group . at visit 5 , all species were eradicated , with the only exceptions being staphylococcus epidermidis in a gatifloxacin - treated eye and staphylococcus hominis in a besifloxacin - treated eye . however , the bacterial count for these isolates was observed to be reduced to below threshold levels , with no new species present in both cases.table 4bacterial eradication by species at visits 3 and 5 , mitt population ( locf)bacterial speciesspecies - specific study eye [ n / n]besifloxacingatifloxacinvisit 3visit 5visit 3visit 5gram - positive16/1817/186/1312/13 cdc coryneform group g1/11/1 enterococcus faecalis 1/11/1 lactococcus garvieae 1/11/1 staphylococcus aureus 3/33/31/22/2 staphylococcus epidermidis 2/22/22/54/5 staphylococcus hominis 1/21/20/11/1 staphylococcus warneri 1/11/1 streptococcus mitis group5/66/62/33/3 streptococcus salivarius group2/22/20/11/1gram - negative6/66/63/44/4 chryseobacterium indologenes 1/11/1 chryseobacterium species 1/11/1 elizabethkingia meningoseptica 1/11/1 haemophilus influenzae 1/11/1 leclercia adecarboxylata 1/11/1 moraxella catarrhalis 2/22/21/11/1 serratia marcescens 0/11/1 wautersiella falsenii 1/11/1 mitt modified intent - to - treat , locf last observation carried forward , cdc centers for disease control bacterial eradication by species at visits 3 and 5 , mitt population ( locf ) mitt modified intent - to - treat , locf last observation carried forward , cdc centers for disease control table 3 presents clinical resolution rates at visits 3 and 5 . in the itt population , clinical resolution at visit 5 ( day 8 or 9 ; primary efficacy endpoint ) was observed in 75.0% of study eyes treated with besifloxacin , compared with 70.6% of study eyes treated with gatifloxacin ( p = 0.78 ) . in the mitt population , clinical resolution at visit 5 was observed in 84.6% of besifloxacin - treated study eyes compared with 77.8% of gatifloxacin - treated study eyes ( p = 0.68 ) . at visit 3 ( day 4 1 ) , clinical resolution rates were 18.8 vs. 29.4% ( itt population ) and 7.7 vs. 33.3% ( mitt population ) in besifloxacin- and gatifloxacin - treated eyes , respectively ( p 0.13 ) . in treated fellow eyes , patterns in clinical resolution rates between treatment groups appeared similar to those observed in study eyes at both visit 3 and visit 5 ( statistical analyses not performed).table 3clinical resolution at visits 3 and 5 ( locf)baseline - designated study eye [ n / n ( % ) ] fellow treated eye [ n / n ( % ) ] besifloxacingatifloxacin p value besifloxacingatifloxacinvisit 3 ( day 4 1 ) itt population3/16 ( 18.8)5/17 ( 29.4)0.481/8 ( 12.5)2/5 ( 40.0 ) mitt population1/13 ( 7.7)3/9 ( 33.3)0.130/5 ( 0.0)2/3 ( 66.7)visit 5 ( day 8 or 9 ) itt population12/16 ( 75.0)12/17 ( 70.6)0.785/8 ( 62.5)2/5 ( 40.0 ) mitt population11/13 ( 84.6)7/9 ( 77.8)0.684/5 ( 80.0)2/3 ( 66.7 ) itt intent - to - treat , mitt modified intent - to - treat , locf last observation carried forward clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia pearson chi - square test ; locf primary outcome visit ( study eye only ) clinical resolution at visits 3 and 5 ( locf ) itt intent - to - treat , mitt modified intent - to - treat , locf last observation carried forward clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia pearson chi - square test ; locf primary outcome visit ( study eye only ) figure 1 presents bacterial eradication rates at visits 3 and 5 for culture - positive , baseline - designated study eyes ( mitt population ) . there was a significant difference between treatments in favor of besifloxacin at visit 3 ( 84.6 vs. 44.4% ; p = 0.0467 ) but not at visit 5 ( 92.3 vs. 88.9% : p = 0.7839 ) . among treated fellow eyes , bacterial eradication at visit 3 was achieved in three of five eyes treated with besifloxacin and two of three eyes treated with gatifloxacin ; at visit 5 , bacterial eradication occurred in four of five eyes treated with besifloxacin and all eyes ( 3/3 ) treated with gatifloxacin.fig . 1bacterial eradication rates at visits 3 and 5 ( culture - positive , baseline - designated study eyes , modified intent - to - treat population , last observation carried forward ) . p values from the pearson chi - square test bacterial eradication rates at visits 3 and 5 ( culture - positive , baseline - designated study eyes , modified intent - to - treat population , last observation carried forward ) . p values from the pearson chi - square test table 4 presents bacterial eradication data for individual bacterial species at visits 3 and 5 . as indicated earlier , treated fellow eyes could contribute data if the bacterial species isolated from that eye was different from that cultured from the baseline - designated study eye . at visit 3 , 88.9% ( 16/18 ) of gram - positive organisms were eradicated in besifloxacin - treated eyes , compared with 46.2% ( 6/16 ) in gatifloxacin - treated eyes . the percentage of all culture - positive treated eyes showing eradication of gram - negative organisms by visit 3 was 100% ( 6/6 ) in the besifloxacin group and 75.0% ( 3/4 ) in the gatifloxacin group . at visit 5 , all species were eradicated , with the only exceptions being staphylococcus epidermidis in a gatifloxacin - treated eye and staphylococcus hominis in a besifloxacin - treated eye . however , the bacterial count for these isolates was observed to be reduced to below threshold levels , with no new species present in both cases.table 4bacterial eradication by species at visits 3 and 5 , mitt population ( locf)bacterial speciesspecies - specific study eye [ n / n]besifloxacingatifloxacinvisit 3visit 5visit 3visit 5gram - positive16/1817/186/1312/13 cdc coryneform group g1/11/1 enterococcus faecalis 1/11/1 lactococcus garvieae 1/11/1 staphylococcus aureus 3/33/31/22/2 staphylococcus epidermidis 2/22/22/54/5 staphylococcus hominis 1/21/20/11/1 staphylococcus warneri 1/11/1 streptococcus mitis group5/66/62/33/3 streptococcus salivarius group2/22/20/11/1gram - negative6/66/63/44/4 chryseobacterium indologenes 1/11/1 chryseobacterium species 1/11/1 elizabethkingia meningoseptica 1/11/1 haemophilus influenzae 1/11/1 leclercia adecarboxylata 1/11/1 moraxella catarrhalis 2/22/21/11/1 serratia marcescens 0/11/1 wautersiella falsenii 1/11/1 mitt modified intent - to - treat , locf last observation carried forward , cdc centers for disease control bacterial eradication by species at visits 3 and 5 , mitt population ( locf ) mitt modified intent - to - treat , locf last observation carried forward , cdc centers for disease control study eyes had a mean exposure of 6.94 ( 0.25 ) and 6.65 ( 1.46 ) days in the besifloxacin and gatifloxacin treatment groups , respectively . the mean exposure for all treated eyes ( i.e. sum of study eye and treated fellow eye exposure ) was 10.38 ( 3.52 ) and 8.71 ( 3.60 ) eye - days for the besifloxacin and gatifloxacin treatment groups , respectively . no teaes , either ocular or non - ocular , were reported in either study or fellow eyes of subjects in the besifloxacin treatment group . in the gatifloxacin treatment group , a total of six aes ( five non - ocular , one ocular ) were reported . the one ocular ae consisted of mild bacterial conjunctivitis in an initially untreated fellow eye , which occurred after visit 1 . the five non - ocular aes occurring in four subjects included abdominal pain , irritability , rhinorrhea , acne infantile , and dermatitis . there were no meaningful findings noted for vital sign measurements or results of ocular or physical examinations performed during the study . in addition , there were no cases of severe eyelid edema or severe chemosis at any visit , and all cases of eyelid edema and chemosis resolved at visit 5 . pupillary reflex , red reflex test , and light perception were normal throughout the study . no major findings were identified on eyelid and conjunctival examinations in either treatment group , and all corneal examinations were normal . in this report , we describe the results of a double - masked , multicenter study comparing besifloxacin ophthalmic suspension 0.6% with gatifloxacin ophthalmic solution 0.3% in 33 neonatal subjects with bacterial conjunctivitis . other than a study published in abstract form only , this is the first published study evaluating the safety and efficacy of two topical fluoroquinolones in neonatal patients with bacterial conjunctivitis . both besifloxacin and gatifloxacin appeared to be well - tolerated in this group of neonates . no teaes were reported in the besifloxacin group , and the six aes in the gatifloxacin group were not considered related to treatment . the percentages of eyes showing clinical resolution were not statistically different between the besifloxacin and gatifloxacin groups at either visit 3 ( day 4 1 ) or visit 5 ( day 8 or 9 ) . however , the proportion of eyes showing bacterial eradication was significantly higher in the besifloxacin group at visit 3 , almost double that of the gatifloxacin group . besifloxacin has previously been shown to have more rapid in vitro bactericidal activity compared with gatifloxacin , possibly evidenced in the current study by the higher rate of bacterial eradication at visit 3 in the besifloxacin group . however , by visit 5 the two treatment groups showed similarly high percentages of bacterial eradication , likely due to the action of the antibacterial in conjunction with the host immune response in this self - limited condition . bacterial eradication findings with besifloxacin were consistent with rates observed at treatment completion in several previous clinical studies [ 2830 ] . the range of mics for cultured pathogens at baseline differed somewhat between besifloxacin and gatifloxacin . besifloxacin mic ranges appeared better ( i.e. lower ) than gatifloxacin mic ranges for gram - positive pathogens , while gatifloxacin mics were similar or slightly better to besifloxacin mics for gram - negative organisms . the greatest differences in in vitro susceptibility between the two antibacterials was noted among the streptococcus mitis group , for which besifloxacin mics were 4- to 16-fold lower than those for gatifloxacin . yet , by visit 5 , all s. mitis organisms were found to have been eradicated in both treatment groups , indicating that in vitro data may not always predict in vivo efficacy when antibacterial drugs are used topically in ocular infections . at visit 5 , each treatment group had one species - specific eye in which a bacterial species failed to be eradicated ( s. epidermidis in a gatifloxacin - treated eye and s. hominis in a besifloxacin - treated eye ) ; however , the bacterial count for these species was reduced compared with visit 3 . very little published data on the use of topical fluoroquinolones in neonates with bacterial conjunctivitis are available . in a study published in abstract form only , 142 culture - positive patients < 31 days of age received either moxifloxacin or ciprofloxacin three times daily for 4 days ; at the test - of - cure visit ( day 9 ) , clinical cure was 80% for both treatments , and microbiological eradication was 92% versus 87% for moxifloxacin and ciprofloxacin , respectively . both medications were well tolerated , with no treatment - related serious aes or treatment - related changes in ocular and cardiovascular examination parameters . in an analysis of safety data from five separate studies of moxifloxacin for the treatment of bacterial conjunctivitis , the incidences of common aes among subjects < 28 days of age ( n = 100 ) were generally similar to or lower than the incidences observed in other age groups , and there were no serious aes in newborns . the findings from a study comparing gatifloxacin ( n = 84 ) and moxifloxacin ( n = 86 ) in neonates with conjunctivitis have been reported on the clinicaltrials.gov website . rates of non - serious aes ( all conjunctivitis or conjunctivitis bacterial ) were 13.1% with gatifloxacin and 9.3% with moxifloxacin ; one serious ae ( pyrexia ) was reported in the moxifloxacin group . the major limitation of the current study was the small sample size and resulting lack of statistical power , a consequence of early study termination for low enrollment , unrelated to safety concerns or efficacy findings . despite the smaller - than - planned population , the findings presented are of interest simply because of the paucity of published data on topical fluoroquinolone use in neonates . further studies of adequate sample size are required to confirm the favorable efficacy and safety data observed in this study and to add to the generalizability of the findings . antibiotic efficacy against mrsa is increasingly being recognized as a critical issue in the management of ocular bacterial infections overall and among newborns [ 31 , 32 ] . notably , in vitro susceptibility studies conducted over recent years have demonstrated potent activity of besifloxacin against ocular mrsa isolates compared with other antibiotics , including other fluoroquinolones [ 19 , 33 ] . if mrsa isolates had been recovered in the current neonatal study , potential differences between besifloxacin and gatifloxacin may have been more apparent . finally , the lack of a vehicle control group did not allow for an efficacy comparison for either fluoroquinolone versus no active treatment . in this study of neonatal subjects with bacterial conjunctivitis , the rates of clinical resolution and bacterial eradication were high and were similar in eyes treated with besifloxacin compared with gatifloxacin after 7 days , while bacterial eradiation appeared to be more rapid with besifloxacin use . while larger studies are warranted to confirm these findings , both treatments were well - tolerated and no safety concerns were noted in this small study . christine m. sanfilippo , catherine m. allaire , heleen h. decory are employees of bausch & lomb , inc . all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards .

purposethe aim of this study was to evaluate the safety and efficacy of topical besifloxacin ophthalmic suspension 0.6% compared with gatifloxacin ophthalmic solution 0.3% in the treatment of bacterial conjunctivitis in neonates.methodsthis was a multicenter , randomized , double - masked , parallel group study . subjects 31 days of age with severity grade 1 ( scale 03 ) for both conjunctival discharge and conjunctival hyperemia were randomized to besifloxacin or gatifloxacin instilled three times daily for 7 days , and completed five study visits ( three clinic visits and two phone calls ) . primary endpoints included clinical resolution ( absence of both conjunctival discharge and conjunctival hyperemia ) at visit 5 ( day 8 or 9 ) and ocular and non - ocular treatment - emergent adverse events ( aes ) . bacterial eradication was a secondary endpoint.resultsthirty-three subjects were included in the intent - to - treat ( itt ) population . all were aged < 28 days , with a mean ( standard deviation ) age of 15.5 days ( 6.0 ) , and 57.6% were female . twenty - two subjects had culture - confirmed conjunctivitis in at least one eye ( modified itt [ mitt ] population ) , most often with gram - positive bacteria . visit 5 clinical resolution and bacterial eradication rates were comparable among besifloxacin- and gatifloxacin - treated study eyes ( clinical resolution : 12/16 [ 75.0% ] vs. 12/17 [ 70.6% ] for the itt population , and 11/13 [ 84.6% ] vs. 7/9 [ 77.8% ] for the mitt population ; bacterial eradication : 12/13 [ 92.3% ] vs. 8/9 [ 88.9% ] for the mitt population , respectively ) . no aes were reported in the besifloxacin treatment group , and aes reported in the gatifloxacin group were considered not treatment-related.conclusionsin this small study in neonates , both besifloxacin and gatifloxacin appeared effective and safe in the treatment of bacterial conjunctivitis . larger studies are warranted .

Key Points Background Materials and Methods Study Design and Procedures Outcomes Statistical Analyses Results Subjects and Baseline Pathogens Efficacy Clinical Resolution Bacterial Eradication Safety Discussion Conclusions Conflict of interest Ethical approval

besifloxacin ophthalmic suspension 0.6% ( besivance ; bausch & lomb , tampa , fl , usa ) is approved by the us fda for the treatment of bacterial conjunctivitis , with a recommended dosing regimen of three times daily for 7 days ; this indication is based on clinical studies conducted in patients 1 year of age . the current study was designed to evaluate the safety and efficacy of besifloxacin ophthalmic suspension 0.6% compared with gatifloxacin ophthalmic solution 0.3% ( zymar ; allergan , irvine , ca , usa ) when administered three times daily for 7 days in neonates with bacterial conjunctivitis . eligible subjects were aged 31 days , with a clinical diagnosis of acute bacterial conjunctivitis in one or both eyes , and a severity of grade 1 for both conjunctival discharge and conjunctival hyperemia in the same eye ( each rated on a scale of 0 [ absent / normal ] to 3 [ severe ] ) . primary endpoints included clinical resolution ( binary outcome [ yes / no ] , with clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia ; severity grade = 0 at visit 5 ) and rates of ocular and non - ocular treatment - emergent aes ( teaes ) . eligible subjects were aged 31 days , with a clinical diagnosis of acute bacterial conjunctivitis in one or both eyes , and a severity of grade 1 for both conjunctival discharge and conjunctival hyperemia in the same eye ( each rated on a scale of 0 [ absent / normal ] to 3 [ severe ] ) . primary endpoints included clinical resolution ( binary outcome [ yes / no ] , with clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia ; severity grade = 0 at visit 5 ) and rates of ocular and non - ocular treatment - emergent aes ( teaes ) . twenty - two subjects ( besifloxacin , n = 13 ; gatifloxacin , n = 9 ) had culture - positive conjunctivitis in at least one eye and were included in the mitt population . overall , the mean ( standard deviation ) age of subjects was 15.5 days ( 6.0 ) and 15.7 days ( 5.3 ) , and 57.6% and 54.5% of subjects in the itt and mitt populations , respectively , were female.table 1demographic characteristicsitt populationmitt populationbesifloxacin [ n = 16]gatifloxacin [ n = 17]besifloxacin [ n = 13]gatifloxacin [ n = 9]age , days mean ( sd)15.8 ( 6.39)15.2 ( 5.75)15.9 ( 6.01)15.4 ( 4.48 ) min , max6 , 265 , 256 , 2511 , 25gender male4 ( 25.0)10 ( 58.8)3 ( 23.1)7 ( 77.8 ) female12 ( 75.0)7 ( 41.2)10 ( 76.9)2 ( 22.2)race asian1 ( 6.3)1 ( 5.9)1 ( 7.7)1 ( 11.1 ) black / african american01 ( 5.9)01 ( 11.1 ) white12 ( 75.0)12 ( 70.6)10 ( 76.9)6 ( 66.7 ) other3 ( 18.8)3 ( 17.6)2 ( 15.4)1 ( 11.1)data are expressed as n ( % ) unless otherwise specified itt intent - to - treat , mitt modified intent - to - treat , sd standard deviation , min minimum , max maximum demographic characteristics data are expressed as n ( % ) unless otherwise specified itt intent - to - treat , mitt modified intent - to - treat , sd standard deviation , min minimum , max maximum table 2 presents bacterial pathogens above threshold isolated at baseline from all culture - positive eyes , along with the mic of besifloxacin and gatifloxacin for these isolates . in the itt population , clinical resolution at visit 5 ( day 8 or 9 ; primary efficacy endpoint ) was observed in 75.0% of study eyes treated with besifloxacin , compared with 70.6% of study eyes treated with gatifloxacin ( p = 0.78 ) . in the mitt population , clinical resolution at visit 5 was observed in 84.6% of besifloxacin - treated study eyes compared with 77.8% of gatifloxacin - treated study eyes ( p = 0.68 ) . at visit 3 ( day 4 1 ) , clinical resolution rates were 18.8 vs. 29.4% ( itt population ) and 7.7 vs. 33.3% ( mitt population ) in besifloxacin- and gatifloxacin - treated eyes , respectively ( p 0.13 ) . in treated fellow eyes , patterns in clinical resolution rates between treatment groups appeared similar to those observed in study eyes at both visit 3 and visit 5 ( statistical analyses not performed).table 3clinical resolution at visits 3 and 5 ( locf)baseline - designated study eye [ n / n ( % ) ] fellow treated eye [ n / n ( % ) ] besifloxacingatifloxacin p value besifloxacingatifloxacinvisit 3 ( day 4 1 ) itt population3/16 ( 18.8)5/17 ( 29.4)0.481/8 ( 12.5)2/5 ( 40.0 ) mitt population1/13 ( 7.7)3/9 ( 33.3)0.130/5 ( 0.0)2/3 ( 66.7)visit 5 ( day 8 or 9 ) itt population12/16 ( 75.0)12/17 ( 70.6)0.785/8 ( 62.5)2/5 ( 40.0 ) mitt population11/13 ( 84.6)7/9 ( 77.8)0.684/5 ( 80.0)2/3 ( 66.7 ) itt intent - to - treat , mitt modified intent - to - treat , locf last observation carried forward clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia pearson chi - square test ; locf primary outcome visit ( study eye only ) clinical resolution at visits 3 and 5 ( locf ) itt intent - to - treat , mitt modified intent - to - treat , locf last observation carried forward clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia pearson chi - square test ; locf primary outcome visit ( study eye only ) figure 1 presents bacterial eradication rates at visits 3 and 5 for culture - positive , baseline - designated study eyes ( mitt population ) . however , the bacterial count for these isolates was observed to be reduced to below threshold levels , with no new species present in both cases.table 4bacterial eradication by species at visits 3 and 5 , mitt population ( locf)bacterial speciesspecies - specific study eye [ n / n]besifloxacingatifloxacinvisit 3visit 5visit 3visit 5gram - positive16/1817/186/1312/13 cdc coryneform group g1/11/1 enterococcus faecalis 1/11/1 lactococcus garvieae 1/11/1 staphylococcus aureus 3/33/31/22/2 staphylococcus epidermidis 2/22/22/54/5 staphylococcus hominis 1/21/20/11/1 staphylococcus warneri 1/11/1 streptococcus mitis group5/66/62/33/3 streptococcus salivarius group2/22/20/11/1gram - negative6/66/63/44/4 chryseobacterium indologenes 1/11/1 chryseobacterium species 1/11/1 elizabethkingia meningoseptica 1/11/1 haemophilus influenzae 1/11/1 leclercia adecarboxylata 1/11/1 moraxella catarrhalis 2/22/21/11/1 serratia marcescens 0/11/1 wautersiella falsenii 1/11/1 mitt modified intent - to - treat , locf last observation carried forward , cdc centers for disease control bacterial eradication by species at visits 3 and 5 , mitt population ( locf ) mitt modified intent - to - treat , locf last observation carried forward , cdc centers for disease control study eyes had a mean exposure of 6.94 ( 0.25 ) and 6.65 ( 1.46 ) days in the besifloxacin and gatifloxacin treatment groups , respectively . twenty - two subjects ( besifloxacin , n = 13 ; gatifloxacin , n = 9 ) had culture - positive conjunctivitis in at least one eye and were included in the mitt population . overall , the mean ( standard deviation ) age of subjects was 15.5 days ( 6.0 ) and 15.7 days ( 5.3 ) , and 57.6% and 54.5% of subjects in the itt and mitt populations , respectively , were female.table 1demographic characteristicsitt populationmitt populationbesifloxacin [ n = 16]gatifloxacin [ n = 17]besifloxacin [ n = 13]gatifloxacin [ n = 9]age , days mean ( sd)15.8 ( 6.39)15.2 ( 5.75)15.9 ( 6.01)15.4 ( 4.48 ) min , max6 , 265 , 256 , 2511 , 25gender male4 ( 25.0)10 ( 58.8)3 ( 23.1)7 ( 77.8 ) female12 ( 75.0)7 ( 41.2)10 ( 76.9)2 ( 22.2)race asian1 ( 6.3)1 ( 5.9)1 ( 7.7)1 ( 11.1 ) black / african american01 ( 5.9)01 ( 11.1 ) white12 ( 75.0)12 ( 70.6)10 ( 76.9)6 ( 66.7 ) other3 ( 18.8)3 ( 17.6)2 ( 15.4)1 ( 11.1)data are expressed as n ( % ) unless otherwise specified itt intent - to - treat , mitt modified intent - to - treat , sd standard deviation , min minimum , max maximum demographic characteristics data are expressed as n ( % ) unless otherwise specified itt intent - to - treat , mitt modified intent - to - treat , sd standard deviation , min minimum , max maximum table 2 presents bacterial pathogens above threshold isolated at baseline from all culture - positive eyes , along with the mic of besifloxacin and gatifloxacin for these isolates . in the itt population , clinical resolution at visit 5 ( day 8 or 9 ; primary efficacy endpoint ) was observed in 75.0% of study eyes treated with besifloxacin , compared with 70.6% of study eyes treated with gatifloxacin ( p = 0.78 ) . in the mitt population , clinical resolution at visit 5 was observed in 84.6% of besifloxacin - treated study eyes compared with 77.8% of gatifloxacin - treated study eyes ( p = 0.68 ) . at visit 3 ( day 4 1 ) , clinical resolution rates were 18.8 vs. 29.4% ( itt population ) and 7.7 vs. 33.3% ( mitt population ) in besifloxacin- and gatifloxacin - treated eyes , respectively ( p 0.13 ) . in treated fellow eyes , patterns in clinical resolution rates between treatment groups appeared similar to those observed in study eyes at both visit 3 and visit 5 ( statistical analyses not performed).table 3clinical resolution at visits 3 and 5 ( locf)baseline - designated study eye [ n / n ( % ) ] fellow treated eye [ n / n ( % ) ] besifloxacingatifloxacin p value besifloxacingatifloxacinvisit 3 ( day 4 1 ) itt population3/16 ( 18.8)5/17 ( 29.4)0.481/8 ( 12.5)2/5 ( 40.0 ) mitt population1/13 ( 7.7)3/9 ( 33.3)0.130/5 ( 0.0)2/3 ( 66.7)visit 5 ( day 8 or 9 ) itt population12/16 ( 75.0)12/17 ( 70.6)0.785/8 ( 62.5)2/5 ( 40.0 ) mitt population11/13 ( 84.6)7/9 ( 77.8)0.684/5 ( 80.0)2/3 ( 66.7 ) itt intent - to - treat , mitt modified intent - to - treat , locf last observation carried forward clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia pearson chi - square test ; locf primary outcome visit ( study eye only ) clinical resolution at visits 3 and 5 ( locf ) itt intent - to - treat , mitt modified intent - to - treat , locf last observation carried forward clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia pearson chi - square test ; locf primary outcome visit ( study eye only ) figure 1 presents bacterial eradication rates at visits 3 and 5 for culture - positive , baseline - designated study eyes ( mitt population ) . in the itt population , clinical resolution at visit 5 ( day 8 or 9 ; primary efficacy endpoint ) was observed in 75.0% of study eyes treated with besifloxacin , compared with 70.6% of study eyes treated with gatifloxacin ( p = 0.78 ) . at visit 3 ( day 4 1 ) , clinical resolution rates were 18.8 vs. 29.4% ( itt population ) and 7.7 vs. 33.3% ( mitt population ) in besifloxacin- and gatifloxacin - treated eyes , respectively ( p 0.13 ) . in treated fellow eyes , patterns in clinical resolution rates between treatment groups appeared similar to those observed in study eyes at both visit 3 and visit 5 ( statistical analyses not performed).table 3clinical resolution at visits 3 and 5 ( locf)baseline - designated study eye [ n / n ( % ) ] fellow treated eye [ n / n ( % ) ] besifloxacingatifloxacin p value besifloxacingatifloxacinvisit 3 ( day 4 1 ) itt population3/16 ( 18.8)5/17 ( 29.4)0.481/8 ( 12.5)2/5 ( 40.0 ) mitt population1/13 ( 7.7)3/9 ( 33.3)0.130/5 ( 0.0)2/3 ( 66.7)visit 5 ( day 8 or 9 ) itt population12/16 ( 75.0)12/17 ( 70.6)0.785/8 ( 62.5)2/5 ( 40.0 ) mitt population11/13 ( 84.6)7/9 ( 77.8)0.684/5 ( 80.0)2/3 ( 66.7 ) itt intent - to - treat , mitt modified intent - to - treat , locf last observation carried forward clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia pearson chi - square test ; locf primary outcome visit ( study eye only ) clinical resolution at visits 3 and 5 ( locf ) itt intent - to - treat , mitt modified intent - to - treat , locf last observation carried forward clinical resolution defined as the absence of both conjunctival discharge and conjunctival hyperemia pearson chi - square test ; locf primary outcome visit ( study eye only ) figure 1 presents bacterial eradication rates at visits 3 and 5 for culture - positive , baseline - designated study eyes ( mitt population ) . however , the bacterial count for these isolates was observed to be reduced to below threshold levels , with no new species present in both cases.table 4bacterial eradication by species at visits 3 and 5 , mitt population ( locf)bacterial speciesspecies - specific study eye [ n / n]besifloxacingatifloxacinvisit 3visit 5visit 3visit 5gram - positive16/1817/186/1312/13 cdc coryneform group g1/11/1 enterococcus faecalis 1/11/1 lactococcus garvieae 1/11/1 staphylococcus aureus 3/33/31/22/2 staphylococcus epidermidis 2/22/22/54/5 staphylococcus hominis 1/21/20/11/1 staphylococcus warneri 1/11/1 streptococcus mitis group5/66/62/33/3 streptococcus salivarius group2/22/20/11/1gram - negative6/66/63/44/4 chryseobacterium indologenes 1/11/1 chryseobacterium species 1/11/1 elizabethkingia meningoseptica 1/11/1 haemophilus influenzae 1/11/1 leclercia adecarboxylata 1/11/1 moraxella catarrhalis 2/22/21/11/1 serratia marcescens 0/11/1 wautersiella falsenii 1/11/1 mitt modified intent - to - treat , locf last observation carried forward , cdc centers for disease control bacterial eradication by species at visits 3 and 5 , mitt population ( locf ) mitt modified intent - to - treat , locf last observation carried forward , cdc centers for disease control study eyes had a mean exposure of 6.94 ( 0.25 ) and 6.65 ( 1.46 ) days in the besifloxacin and gatifloxacin treatment groups , respectively . in a study published in abstract form only , 142 culture - positive patients < 31 days of age received either moxifloxacin or ciprofloxacin three times daily for 4 days ; at the test - of - cure visit ( day 9 ) , clinical cure was 80% for both treatments , and microbiological eradication was 92% versus 87% for moxifloxacin and ciprofloxacin , respectively .

there have been many attempts over the past decades for automating cancer grading in tissue , most notably in breast and prostate tissue , where the standard scoring systems in use are the elston and gleason grading systems , respectively . more recently , high classification rates were obtained for the simple case of discriminating between low - grade and high - grade cancer in prostate tissue [ 47 ] . there have been attempts in [ 4 , 5 ] at performing the classification task and extracting a large and diverse feature set including color , texture , morphometric , fractal , and wavelet features . often this is followed by a feature reduction method such as sequential forward feature selection as in or similar greedy algorithms , which , though being suboptimal approaches , are motivated by the fact that the feature set is large and a brute force or branch - and - bound method may become intractable or computationally inefficient . often a main factor that limits automated classification lies not in the choice of classifier but in the choice of feature set . the discriminative ability of a classifier is limited by the extent to which the classes themselves are separate in feature space . for well - represented classes , the intrinsic overlap and proximity of the classes in feature space the selection of a large number of different types of features is common in practice and is often an indication of lack of knowledge as to what features exactly have discriminative power ; instead it reflects speculation over which features may prove useful or may have a contributing role . however , choosing a plethora of features , whether informative or not , increases the dimensionality of the feature space and often exposes the classification task to the peaking phenomenon . furthermore , this shifts the burden of the problem toward feature selection or extraction which is often difficult to solve in a manner that is true to the final objective ( i.e. , the final classification rate ) , and this is due to computational limitations and the prevalence of either suboptimal criteria or criteria that are often not aligned with the final objective . it is therefore important to select a discriminative set of features that is able to separate among the different classes . one reason lies in the difficulty of translating the experience and observations of the human expert , that is , the trained pathologist , into well - defined features that can be extracted automatically from the image . moreover , due to the complexity of the tissue structure and subjectivity of the grading process , especially among the intermediate grades , there is no clear consensus as to which features or combination of features is to be used consistently . upon deeper examination , we find that experts ' rules tend to eventually branch out into increasingly complicated conditions and exceptions . there is therefore a problem in identifying features explicitly , and moreover even when such features have been suggested by pathologists , the complexity and variability of the images and tissue structures in addition to variables relating to stain absorption can still obstruct the extraction of such features in a reliable manner that allows for automation . in general , there is a sensitive balance between overadapting to the complexity of the problem on the one hand and weakly accounting for it on the other hand such as the case when extracting global texture features without taking into account any knowledge of tissue architecture . both of these extreme approaches may lead to inadequate results and an inability to generalize well . in the approach that we propose , we avoid the explicit extraction of structure properties ( such as nuclei shape , glandular unit shape , and thickness of epithelium layer ) beyond a rough decomposition of images into a few classes based on the staining . yet , the method is still strongly founded on the architecture of glandular tissue ( such as breast or prostate ) and relies upon detecting glandular lumen and tissue components as a starting point . we use sequential region expansion to sample the space around lumen regions in the form of rings and preserve the statistics and component ratios within these rings in order to describe and represent these regions in an implicit manner . during the progression of cancer into advanced stages , when a glandular unit transforms into cribriform shape or splits into multiple lumen regions , such a phenomenon should be detected by the method due to the unusual presence of lumen and other structures in the outer sampling rings which reflects on the shape of the extracted profile curve and consequently on its classification and labeling . as opposed to most local neighborhood sampling or bag of features methods that either are patch - based or result in orderless , histogram - based features [ 10 , 11 ] , the method we propose does the sampling around a given ( lumen ) region as opposed to a pixel , while preserving the region 's boundary shape and encoding spatial distance from it . the contributions of our work can be stated as follows.we present a new approach to encode features in complex tissue images such as prostate and breast . the approach called statistical proximity sampling relies on a method of boundary expansion around lumen regions ; it uses rings or neighborhood strips around these regions while preserving the boundary shapes.the method is able to simultaneously encode the relative quantitative proportions of each tissue type around a lumen region as well as the spatial distribution of these proportions from the central lumen region , resulting in highly descriptive and discriminative features.combining this neighborhood - based feature description with multiple - instance learning , we are able to represent complex images in an efficient and information - preserving manner , which is more consequential than representing an entire image with a single feature vector . we present a new approach to encode features in complex tissue images such as prostate and breast . the approach called statistical proximity sampling relies on a method of boundary expansion around lumen regions ; it uses rings or neighborhood strips around these regions while preserving the boundary shapes . the method is able to simultaneously encode the relative quantitative proportions of each tissue type around a lumen region as well as the spatial distribution of these proportions from the central lumen region , resulting in highly descriptive and discriminative features . combining this neighborhood - based feature description with multiple - instance learning , we are able to represent complex images in an efficient and information - preserving manner , which is more consequential than representing an entire image with a single feature vector . to highlight the context of our work , we briefly describe below the elston and gleason grading systems and how our method relates to some important aspects of these . tubularity , where the presence of glandular tissue in the sample is given a score from 1 to 3 , ranging , respectively , from healthy tissue ( prevalently glandular ) to solid tumors ( scarcely glandular ) . the second component is nuclear pleomorphism and is concerned with nuclear size , shape , and chromatin texture ; this attribute is also assigned a score from 1 to 3 depending on the morphological irregularities of nuclei . the third component of grading is mitotic activity which corresponds to growth rate and is determined by counting dividing cells , ranging from a low cell count ( score 1 ) to a high cell count ( score 3 ) . the final , high - level elston grade is then derived by summing up the individual scores from the three parts : a sum of 35 points is defined as elston grade i , 6 - 7 as grade ii , and 8 - 9 as grade iii . analogously , gleason grading for prostate is based on five patterns , which are highly dependent on tissue architecture and the description of glandular units . the patterns from 1 to 5 are described by how glands alter form while transitioning from small , well - defined , and closely packed units , corresponding to well - differentiated carcinoma ( pattern 1 ) , to larger glandular units with increased interglandular distances , corresponding to moderately differentiated carcinoma ( pattern 2 ) , until the glands are no longer recognizable and cells start to invade surrounding tissue in neoplastic clumps . in pattern 5 , thus , in conclusion , both gleason grading and the first component of elston grading are based on patterns that are defined by the amount and architecture of glandular units and tubules present in the tissue sample . the ability to identify tubules and glandular structures is an essential requirement for both grading systems . while there is a lot of work on identifying nuclear pleomorphism and mitotic count , as most recently in , our contribution in this paper is to propose a new effective way of extracting information concerning glandular architecture , which is directly related to the first component in elston grading and is an essential part of gleason grading . in particular , what we present in this paper is a method that enables us to distinguish between images with tubular structures , denoted by c1 , and images lacking tubular structures , denoted by c0 , where these images are taken from both healthy and cancerous breast tissue , since we want to be able to identify tubules in both healthy and cancer tissue samples . the grading of cancerous tissue of glandular organs such as prostate and breast is to a large extent based on the tissue architecture around the glandular lumen regions . in previous work [ 13 , 14 ] , we have presented automated methods for color decomposition and pattern - based image segmentation that result in density or probability maps , one per stained tissue type . in the current work , we present a method that uses such types of maps as input for deriving a set of features based on statistically sampling the neighborhood of lumen regions . our method proceeds in the following manner.a tissue image is softly classified into a set of k probability maps using any method such as color decomposition ( see ) or a pattern analysis approach ( see ) ; an example is shown in figure 1 , where the k tissue types correspond to lumen , epithelium , nuclei , and stroma.starting from the lumen regions , each region is separately dilated by a square structuring element in sequential unit steps forming a set of rings or annuli around the original lumen space ( see figure 2 ) . these rings are regarded as neighborhood strips from which we will gather statistics on the quantity and location of surrounding tissue types . the boundary shape is preserved within a reasonable number of sampling rings.within each ring , we compute the proportions of the different tissue types ( lumen , epithelium , nuclei , and stroma ) using the derived probability maps . thus , for each ring , we obtain a vector of length k. for instance , a vector such as [ 0.2 , 0.4 , 0.1 , 0.3 ] in a given ring indicates that the relative proportions of lumen , epithelium , nuclei , and stroma are 20% , 40% , 10% , and 30% , respectively.the vectors obtained in step ( 3 ) are stacked , forming a single vector of length r k , where r is the number of rings used , that is , neighborhood strips . thus each lumen region from step ( 1 ) will be represented by such a feature vector of length r k. an example of these vectors is shown in figure 4 , where there are 4 lumen regions and consequently 4 such feature vectors plotted using different colors.we present each image as a bag or collection of feature vectors corresponding to lumen regions in the image . thus , we use multiple - instance learning to represent an image using a collection of feature vectors and perform the classification of each image based on its contents . we also use the bag dissimilarity approach to decouple the classification task from the multiple - instance formulation , allowing us to use any type of classifier without difficulty . a tissue image is softly classified into a set of k probability maps using any method such as color decomposition ( see ) or a pattern analysis approach ( see ) ; an example is shown in figure 1 , where the k tissue types correspond to lumen , epithelium , nuclei , and stroma . starting from the lumen regions , each region is separately dilated by a square structuring element in sequential unit steps forming a set of rings or annuli around the original lumen space ( see figure 2 ) . these rings are regarded as neighborhood strips from which we will gather statistics on the quantity and location of surrounding tissue types . the boundary shape is preserved within a reasonable number of sampling rings . within each ring , we compute the proportions of the different tissue types ( lumen , epithelium , nuclei , and stroma ) using the derived probability maps . thus , for each ring , we obtain a vector of length k. for instance , a vector such as [ 0.2 , 0.4 , 0.1 , 0.3 ] in a given ring indicates that the relative proportions of lumen , epithelium , nuclei , and stroma are 20% , 40% , 10% , and 30% , respectively . the vectors obtained in step ( 3 ) are stacked , forming a single vector of length r k , where r is the number of rings used , that is , neighborhood strips . thus each lumen region from step ( 1 ) will be represented by such a feature vector of length r k. an example of these vectors is shown in figure 4 , where there are 4 lumen regions and consequently 4 such feature vectors plotted using different colors . we present each image as a bag or collection of feature vectors corresponding to lumen regions in the image . thus , we use multiple - instance learning to represent an image using a collection of feature vectors and perform the classification of each image based on its contents . we also use the bag dissimilarity approach to decouple the classification task from the multiple - instance formulation , allowing us to use any type of classifier without difficulty . in step ( 2 ) , it is possible to use dilations with larger steps or with a larger structuring element when deriving the rings . this would make the collected statistics less noisy but would also decrease the spatial resolution of the collected data ( analogous to the effect of applying a moving average filter ) . we begin by explaining the method through an example for the case of prostate tissue . figure 1 shows a cross - section of prostate tissue that has been stained with a sirius - hematoxylin stain combination along with the resulting image decomposition into four probability maps which represent in this case the classes corresponding to lumen , epithelium , nuclei , and stromal regions . note that the decomposition method used for prostate tissue follows from our previous work in . the proximity sampling method takes as input the probability maps generated from the decomposition , regardless of which method was employed for the latter . the image selected for decomposition in figure 1 is a cropped image of size 183 339 and was chosen to contain only a small number of lumen regions so that the number of feature profiles that follow remains tractable for display . the probability maps were automatically rearranged according to a descending order of mean intensity value . this allows the automatic selection of the lumen class as the first image in this ordered sequence . in what follows , we discuss in detail how the main feature vector of the statistical proximity sampling method is obtained . the method proceeds by statistically sampling the neighborhood around each lumen in terms of class component quantities . by sequential dilation of the lumen region and subtraction of the preceding area , we obtain concentric rings or annuli progressing spatially away from the lumen in either inward , outward , or both directions , extending the lumen shape ( see figure 2 ) . within each ring , the fraction of each class component , that is , lumen , epithelium , nuclei , and stroma , is computed as a ratio of the sum of class posterior probabilities within the ring to the total area of the ring . these are then concatenated into a vector with k = 4 parts , where k is the number of classes . the number of dilations or rings we have used in this case for illustration was 30 . this creates a profile of how these class quantities are changing spatially as one moves away from the lumen within its neighborhood . as cancer progresses from benign to malignant , the different grades of cancer are expected to exhibit different patterns in terms of the quantities and order of these class components around the lumen which would result in different profile curves sampled from these rings . the shape of the curve captures spatial ( order ) information and represents statistical quantification of the classes . figure 3 shows lumen regions extracted from the corresponding posterior map and labeled according to their 4-connected neighborhood ; that is , pixels that are adjacent diagonally are not considered neighbors . figure 4 shows the profile curves obtained , one for each of the four lumen regions . the first 30 elements represent the changing amount of neighboring lumen within those rings ; the second part consisting of another 30 elements represents that of epithelium , the third part that of nuclei , and the fourth that of stroma . to validate and understand what the curves represent , one should compare the profile of each lumen to its spatial neighborhood shown in figure 3 . the colors shown in figure 4 have been set to match those lumen regions shown in figure 3 . for example , the cyan curve represents the cyan colored lumen region . from figure 3 , we notice that the sampling rings should contain a considerable fraction of lumen due to the large neighboring lumen region shown in red color . similar analysis follows for the other parts of the curve in which one can see how each class component varies as one moves away from the lumen region . the fourth part of the curve is particularly easy to notice since there is no stromal component close enough to the cyan luminal region . alternatively , in order to show how the different luminal regions compare to each other in terms of the spatial composition of their proximities , we replot the curves of figure 4 such that the proportions of the four different tissue types ( lumen , epithelium , nuclei , and stroma ) across the rings are shown in a relative frequency pie chart for each luminal region separately . this is illustrated in figure 5 , where each subplot represents a luminal region indicated by the color of the central rectangle , and we note here that each of these exhibits a different profile . we note that the previous example was based on the spectral decomposition of tissue that was specifically stained ( using sirius - hematoxylin ) in order to express the different relevant tissue components ( see ) . however , in order to illustrate that the proposed concept is robust and generally applicable , we have also applied it to images of breast cancer tissues from the human protein atlas database which are stained using hematoxylin - eosin + dab to visualize general background tissue structures and specific proteins . as an example , we show a cropped image region of size 362 450 selected from the case figure 6 shows the decomposition of the image into four classes corresponding to lumen , stroma , nuclei , and dab . figure 7 shows four selected lumen regions from the posterior map of the lumen class in order to display their respective feature curves as based on the proximity sampling method described above . finally , figure 8 shows the profile curves for this example , where the number of sampling rings around each lumen was set to 10 . in a similar manner to the previous example , several detailed conclusions may be drawn from these figures ; however the most general one is that these feature curves capture the statistical distribution of the classes around each lumen region and may therefore be used to classify those regions . to test our method , we used a dataset consisting of images of breast tissue sections obtained from the human protein atlas project , where every image has been assigned a malignancy grade by an expert . the assigned class labels denoted by c0 and c1 are associated with the tubule - based elston grading , where the main factor is the absence ( c0 ) or presence ( c1 ) of milk ducts in the tissue . note that all microscopy images of the given dataset were acquired under the same magnification level of 40 . the dataset consists of tissue sections containing cells , glands , and luminal regions , and the proximity sampling method we have proposed applies in general also for images of tissue types that contain similar structures in living organisms . in the dataset , an image may contain several lumen regions . a feature vector is derived for each of these lumen regions using the proximity sampling method described . the aim is to train a classifier on the labeled , that is , graded , images in order to predict the label of a new image automatically . thus , there is an inherent relation between the formulation of this problem and multiple - instance learning . in the context of the latter , the feature vectors derived from the lumen regions in an image may be regarded as instances or objects and the image itself as a bag or compound object consisting of one or more instances . the instances themselves are not labeled , but rather only the bag carries a label , which in this case is the tubule - based grade assigned by the pathologists . . some of the instances in a bag may be less important in contributing to the bag label , whereas one or more may be key instances , belonging to the so - called concept , that significantly define the bag label . for example , an image may contain one gland unit that characterizes a grade 3 cancer region , in addition to several noncontributing , background lumen regions . in such a case , one of the instances belongs to the concept that contributes to the grade 3 label . the multiple - instance learning approach is more flexible than standard classification approaches in that the representation allows us to encode more information from a single image by considering it as a collection of feature vectors rather than encoding the entire image by a single feature vector . images of real life objects ( such as tissue sections ) often contain a lot of important subregions with different characteristics and may be therefore too complex to be represented by a single feature vector . the multiple - instance representation is highly informative in this situation since it encodes information from different regions in an image , each of which may contribute to the final grade or label of the image as a whole . however , the classification task that ensues becomes more complex as a classifier is trained and optimized over the dataset . therefore , in order not to add complexity to the construction of a classifier and preserve the flexibility of the task , we follow the bag dissimilarity approach described in , which does not attempt to locate a concept but rather uses a similarity measure across bags , which are seen as sets of instances . the dissimilarities computed between the bags become the new features , and this allows us to construct any classifier in this new feature space , thus decoupling the original multiple - instance problem from the classification task itself . moreover , the bag dissimilarity approach allows us to consider multiclass data , that is , data with several grades , whereas in the traditional multiple - instance learning problem only two classes , namely , a positive and a negative class , are considered at any given time , and a one - against - one or one - against - all approach is often used in the classification of multiclass situations . insofar , we have presented a new vectorial proximity - based feature for describing tissue architecture around glands , and we proceed in the next section to demonstrate its usefulness as a feature descriptor . however , in order to evaluate whether more conventional scalar features add any information to the new feature , we have implemented four classical , well - known scalar features that are simple to compute from each lumen region . the first is the size of the region , while the other measures relate to its shape and are the bending energy , area - to - perimeter ratio , and convexity ratio . bending energy is defined around the lumen perimeter based on the chain code sequence and is given by eb = p=1(p ) , where (p ) is a smoothed version of the curvature signal (p ) = tan((yc(p ) yc(p 1))/(xc(p ) xc(p 1 ) ) ) , where ( xc(p 1 ) , yc(p 1 ) ) and ( xc(p ) , yc(p ) ) are two consecutive points of the curvature . the minimum value is 2/r and is attained for a circle of radius r. this feature is an indicator of convexity / concavity of the lumen boundary . the area - to - perimeter ratio is defined as ( 4a)/p , where p is the perimeter and a the area of the lumen region . convexity is defined as alumen / aconvex hull , where alumen is the area of the lumen region and aconvex hull is the area covered by the convex hull encompassing the lumen region . this ratio is in the range and is closer to 1 when the lumen shape is convex and closer to 0 when highly irregular such as the case of cribriform grade 3 - 4 glandular units in prostate tissue , for instance . these lumen shape features are then compared with the main proximity feature vector , and classification results are presented in section 3 . for each image in the dataset , we have used our proximity - based feature method to obtain a set of descriptive features . then we used multiple - instance learning to represent each image as a bag of instances and transform the feature space into a dissimilarity space by computing the distances among the different bags . the mil toolbox and various classifiers were used for this purpose [ 19 , 20 ] . our dissimilarity matrix is computed among the bags based on the linear assignment distance measured between sets [ 15 , 21 ] . the dataset is then randomly split into a training set and a test set , and cross - validation procedures are used throughout . note that ten sampling rings were used for the statistical proximity sampling method throughout all cases resulting in 40-element feature vectors for the case of the breast dataset since the number of classes was four using the hematoxylin - eosin + dab stain . figure 10 shows the classification rates and classifier learning curves using only the features derived by the statistical proximity sampling method . note that the parameters for the support vector classifier and k - nearest neighbor classifier were optimized using leave - one - out cross - validation over a training set comprising randomly 25% of the original dataset . the 10-fold cross - validation rates for all classifiers over the remaining test set are then computed . the highest classification rates were obtained using the k - nearest neighbor classifier and the support vector classifier with 93.4% and 94.2% correct classification for the breast dataset , respectively . note that assigning misclassification costs for different classes may be set as desired through the regularization parameter of svc if needed . for comparison , a similar procedure was applied , however , using only the 4 classical lumen shape features that were described in section 2.3 . although we do not explore unsupervised methods for malignancy grading in this paper , we would like to highlight the possibility of applying a clustering - based approach coupled with an information criterion for classifying images . we demonstrate in what follows how clustering may be applied to classify instances in the absence of bag labels . in other words , we attempt to identify and locate key clusters or groups of instances forming main clusters . a bag label ( i.e. , image grade ) may then be obtained using a voting scheme over the cluster labels of the instances that belong to it . to study whether there may be an inherent number of clusters in the data possibly due to a certain fixed number of neighborhood descriptions that tend to recur around lumen regions we clustered the breast dataset using the gaussian mixture model ( gmm ) several times with a varying number of clusters k ranging from 1 to 10 , and we computed the bic values as defined by bic = 2ln(l ) + kln(n ) , where n is the number of objects in the data , l is the likelihood value of the mixture fit , and k is the number of clusters . note that the mixture model was initialized randomly 10 times for each value of k. as the number of clusters increases , we expect the log - likelihood to increase monotonically ; however the bic measure also includes the model parameters into the tradeoff , which in this case is the number of clusters k. the optimal mixture model would have a high log - likelihood yet at a lowest possible complexity k. a plot of the bic values versus the number of clusters is shown in figure 11 . we deduce in this case that for the breast dataset the optimal number of clusters at which the bic curve attains a minimum is 2 . this result does not necessarily imply that there are 2 clusters in the data in an absolute sense . when using the akaike information criterion ( aic ) , we note in figure 11 that the optimal number of clusters at which the curve attains a minimum becomes 4 , since aic in this case penalizes model complexity less heavily than bic and thus results in the selection of a larger model . conclusively , this might suggest that a model selection of 2 , 3 , or 4 classes in this case is a reasonable choice . we have presented a general and simple method for statistically describing the distribution of glandular structures around lumen regions . the method makes use of sampling based on an iterative region expansion procedure that preserves the shape of the lumen areas . one advantage of this approach is that , by analyzing the neighborhoods of lumen regions and preserving the spatial and statistical information in these proximities , we avoid the need to extract explicit features concerning the underlying tissue structures themselves . the result is a set of feature vectors containing spatial and statistical information that may be used to describe regions in tissue images for a large variety of purposes , among which is tubule - based grading , as we have demonstrated in this paper . the input required for the method can be either a set of probability or binary maps derived from soft or crisp classification regardless of the supervised or unsupervised method ( e.g. , [ 13 , 14 ] ) used to generate these maps . the method is also robust and its dependence on the quality of these maps is minimal since the approach does not attempt to derive any precise cellular or subcellular features , which would require accurate image segmentation . due to the natural complexity of biological tissue and the grading process , we have avoided the single feature vector based representation used in standard pattern recognition . automated grading was instead done using a bag dissimilarity approach while treating the problem in a similar manner to multiple - instance learning . since images of tissue sections often contain various spatial subregions which may have completely different properties and characteristics , such an approach is more capable of encoding the diverse content and level of information represented in these images . classification results using cross - validation have shown that the statistical proximity sampling method presented is able to provide a set of discriminative features for tubule - based cancer grading . a possible drawback of the dissimilarity approach we have used in our classification is that although the classification task itself is accomplished and the diagnosis is automated , no single concept however , alternative multiple - instance learning methods that are based on the notion of finding a concept may be used for this purpose if needed . the advantage of identifying a concept is that it becomes then possible to visually map the concept or its instances back to the corresponding regions in the image . the results obtained for the hpa dataset in this paper are meant to illustrate the potential of our approach in feature extraction and grading and its prospect for further extended studies over large datasets and possible combination with complementary approaches that address other aspects of grading ( such as nuclear pleomorphism and mitotic count ) , possibly leading to applications in the clinical context . a comprehensive automated system that would be able to eventually assign high - level grading akin to that by pathologists would undoubtedly have to incorporate , in addition to the work described in this paper , methods that are designed to address nuclear pleomorphism and mitotic count ( as most recently in ) . the final aim is to aid pathologists in the malignancy grading of cancer . as a first step towards that goal , we have in this paper addressed tubule - based grading , which contributes to one of the three components for malignancy grading under the ellis - elston system and which is also considered an important factor in gleason grading .

due to the complexity of biological tissue and variations in staining procedures , features that are based on the explicit extraction of properties from subglandular structures in tissue images may have difficulty generalizing well over an unrestricted set of images and staining variations . we circumvent this problem by an implicit representation that is both robust and highly descriptive , especially when combined with a multiple instance learning approach to image classification . the new feature method is able to describe tissue architecture based on glandular structure . it is based on statistically representing the relative distribution of tissue components around lumen regions , while preserving spatial and quantitative information , as a basis for diagnosing and analyzing different areas within an image . we demonstrate the efficacy of the method in extracting discriminative features for obtaining high classification rates for tubular formation in both healthy and cancerous tissue , which is an important component in gleason and tubule - based elston grading . the proposed method may be used for glandular classification , also in other tissue types , in addition to general applicability as a region - based feature descriptor in image analysis where the image represents a bag with a certain label ( or grade ) and the region - based feature vectors represent instances .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

there have been many attempts over the past decades for automating cancer grading in tissue , most notably in breast and prostate tissue , where the standard scoring systems in use are the elston and gleason grading systems , respectively . more recently , high classification rates were obtained for the simple case of discriminating between low - grade and high - grade cancer in prostate tissue [ 47 ] . however , choosing a plethora of features , whether informative or not , increases the dimensionality of the feature space and often exposes the classification task to the peaking phenomenon . furthermore , this shifts the burden of the problem toward feature selection or extraction which is often difficult to solve in a manner that is true to the final objective ( i.e. , the final classification rate ) , and this is due to computational limitations and the prevalence of either suboptimal criteria or criteria that are often not aligned with the final objective . it is therefore important to select a discriminative set of features that is able to separate among the different classes . one reason lies in the difficulty of translating the experience and observations of the human expert , that is , the trained pathologist , into well - defined features that can be extracted automatically from the image . moreover , due to the complexity of the tissue structure and subjectivity of the grading process , especially among the intermediate grades , there is no clear consensus as to which features or combination of features is to be used consistently . there is therefore a problem in identifying features explicitly , and moreover even when such features have been suggested by pathologists , the complexity and variability of the images and tissue structures in addition to variables relating to stain absorption can still obstruct the extraction of such features in a reliable manner that allows for automation . in general , there is a sensitive balance between overadapting to the complexity of the problem on the one hand and weakly accounting for it on the other hand such as the case when extracting global texture features without taking into account any knowledge of tissue architecture . in the approach that we propose , we avoid the explicit extraction of structure properties ( such as nuclei shape , glandular unit shape , and thickness of epithelium layer ) beyond a rough decomposition of images into a few classes based on the staining . yet , the method is still strongly founded on the architecture of glandular tissue ( such as breast or prostate ) and relies upon detecting glandular lumen and tissue components as a starting point . we use sequential region expansion to sample the space around lumen regions in the form of rings and preserve the statistics and component ratios within these rings in order to describe and represent these regions in an implicit manner . during the progression of cancer into advanced stages , when a glandular unit transforms into cribriform shape or splits into multiple lumen regions , such a phenomenon should be detected by the method due to the unusual presence of lumen and other structures in the outer sampling rings which reflects on the shape of the extracted profile curve and consequently on its classification and labeling . as opposed to most local neighborhood sampling or bag of features methods that either are patch - based or result in orderless , histogram - based features [ 10 , 11 ] , the method we propose does the sampling around a given ( lumen ) region as opposed to a pixel , while preserving the region 's boundary shape and encoding spatial distance from it . the contributions of our work can be stated as follows.we present a new approach to encode features in complex tissue images such as prostate and breast . the approach called statistical proximity sampling relies on a method of boundary expansion around lumen regions ; it uses rings or neighborhood strips around these regions while preserving the boundary shapes.the method is able to simultaneously encode the relative quantitative proportions of each tissue type around a lumen region as well as the spatial distribution of these proportions from the central lumen region , resulting in highly descriptive and discriminative features.combining this neighborhood - based feature description with multiple - instance learning , we are able to represent complex images in an efficient and information - preserving manner , which is more consequential than representing an entire image with a single feature vector . we present a new approach to encode features in complex tissue images such as prostate and breast . the approach called statistical proximity sampling relies on a method of boundary expansion around lumen regions ; it uses rings or neighborhood strips around these regions while preserving the boundary shapes . the method is able to simultaneously encode the relative quantitative proportions of each tissue type around a lumen region as well as the spatial distribution of these proportions from the central lumen region , resulting in highly descriptive and discriminative features . combining this neighborhood - based feature description with multiple - instance learning , we are able to represent complex images in an efficient and information - preserving manner , which is more consequential than representing an entire image with a single feature vector . analogously , gleason grading for prostate is based on five patterns , which are highly dependent on tissue architecture and the description of glandular units . in pattern 5 , thus , in conclusion , both gleason grading and the first component of elston grading are based on patterns that are defined by the amount and architecture of glandular units and tubules present in the tissue sample . while there is a lot of work on identifying nuclear pleomorphism and mitotic count , as most recently in , our contribution in this paper is to propose a new effective way of extracting information concerning glandular architecture , which is directly related to the first component in elston grading and is an essential part of gleason grading . in particular , what we present in this paper is a method that enables us to distinguish between images with tubular structures , denoted by c1 , and images lacking tubular structures , denoted by c0 , where these images are taken from both healthy and cancerous breast tissue , since we want to be able to identify tubules in both healthy and cancer tissue samples . the grading of cancerous tissue of glandular organs such as prostate and breast is to a large extent based on the tissue architecture around the glandular lumen regions . in the current work , we present a method that uses such types of maps as input for deriving a set of features based on statistically sampling the neighborhood of lumen regions . our method proceeds in the following manner.a tissue image is softly classified into a set of k probability maps using any method such as color decomposition ( see ) or a pattern analysis approach ( see ) ; an example is shown in figure 1 , where the k tissue types correspond to lumen , epithelium , nuclei , and stroma.starting from the lumen regions , each region is separately dilated by a square structuring element in sequential unit steps forming a set of rings or annuli around the original lumen space ( see figure 2 ) . the boundary shape is preserved within a reasonable number of sampling rings.within each ring , we compute the proportions of the different tissue types ( lumen , epithelium , nuclei , and stroma ) using the derived probability maps . thus , for each ring , we obtain a vector of length k. for instance , a vector such as [ 0.2 , 0.4 , 0.1 , 0.3 ] in a given ring indicates that the relative proportions of lumen , epithelium , nuclei , and stroma are 20% , 40% , 10% , and 30% , respectively.the vectors obtained in step ( 3 ) are stacked , forming a single vector of length r k , where r is the number of rings used , that is , neighborhood strips . thus each lumen region from step ( 1 ) will be represented by such a feature vector of length r k. an example of these vectors is shown in figure 4 , where there are 4 lumen regions and consequently 4 such feature vectors plotted using different colors.we present each image as a bag or collection of feature vectors corresponding to lumen regions in the image . thus , we use multiple - instance learning to represent an image using a collection of feature vectors and perform the classification of each image based on its contents . a tissue image is softly classified into a set of k probability maps using any method such as color decomposition ( see ) or a pattern analysis approach ( see ) ; an example is shown in figure 1 , where the k tissue types correspond to lumen , epithelium , nuclei , and stroma . starting from the lumen regions , each region is separately dilated by a square structuring element in sequential unit steps forming a set of rings or annuli around the original lumen space ( see figure 2 ) . these rings are regarded as neighborhood strips from which we will gather statistics on the quantity and location of surrounding tissue types . within each ring , we compute the proportions of the different tissue types ( lumen , epithelium , nuclei , and stroma ) using the derived probability maps . thus each lumen region from step ( 1 ) will be represented by such a feature vector of length r k. an example of these vectors is shown in figure 4 , where there are 4 lumen regions and consequently 4 such feature vectors plotted using different colors . we present each image as a bag or collection of feature vectors corresponding to lumen regions in the image . thus , we use multiple - instance learning to represent an image using a collection of feature vectors and perform the classification of each image based on its contents . in step ( 2 ) , it is possible to use dilations with larger steps or with a larger structuring element when deriving the rings . in what follows , we discuss in detail how the main feature vector of the statistical proximity sampling method is obtained . within each ring , the fraction of each class component , that is , lumen , epithelium , nuclei , and stroma , is computed as a ratio of the sum of class posterior probabilities within the ring to the total area of the ring . figure 3 shows lumen regions extracted from the corresponding posterior map and labeled according to their 4-connected neighborhood ; that is , pixels that are adjacent diagonally are not considered neighbors . figure 4 shows the profile curves obtained , one for each of the four lumen regions . the fourth part of the curve is particularly easy to notice since there is no stromal component close enough to the cyan luminal region . alternatively , in order to show how the different luminal regions compare to each other in terms of the spatial composition of their proximities , we replot the curves of figure 4 such that the proportions of the four different tissue types ( lumen , epithelium , nuclei , and stroma ) across the rings are shown in a relative frequency pie chart for each luminal region separately . this is illustrated in figure 5 , where each subplot represents a luminal region indicated by the color of the central rectangle , and we note here that each of these exhibits a different profile . we note that the previous example was based on the spectral decomposition of tissue that was specifically stained ( using sirius - hematoxylin ) in order to express the different relevant tissue components ( see ) . however , in order to illustrate that the proposed concept is robust and generally applicable , we have also applied it to images of breast cancer tissues from the human protein atlas database which are stained using hematoxylin - eosin + dab to visualize general background tissue structures and specific proteins . figure 7 shows four selected lumen regions from the posterior map of the lumen class in order to display their respective feature curves as based on the proximity sampling method described above . in a similar manner to the previous example , several detailed conclusions may be drawn from these figures ; however the most general one is that these feature curves capture the statistical distribution of the classes around each lumen region and may therefore be used to classify those regions . to test our method , we used a dataset consisting of images of breast tissue sections obtained from the human protein atlas project , where every image has been assigned a malignancy grade by an expert . the assigned class labels denoted by c0 and c1 are associated with the tubule - based elston grading , where the main factor is the absence ( c0 ) or presence ( c1 ) of milk ducts in the tissue . the dataset consists of tissue sections containing cells , glands , and luminal regions , and the proximity sampling method we have proposed applies in general also for images of tissue types that contain similar structures in living organisms . in the dataset , an image may contain several lumen regions . thus , there is an inherent relation between the formulation of this problem and multiple - instance learning . in the context of the latter , the feature vectors derived from the lumen regions in an image may be regarded as instances or objects and the image itself as a bag or compound object consisting of one or more instances . the instances themselves are not labeled , but rather only the bag carries a label , which in this case is the tubule - based grade assigned by the pathologists . some of the instances in a bag may be less important in contributing to the bag label , whereas one or more may be key instances , belonging to the so - called concept , that significantly define the bag label . for example , an image may contain one gland unit that characterizes a grade 3 cancer region , in addition to several noncontributing , background lumen regions . in such a case , one of the instances belongs to the concept that contributes to the grade 3 label . the multiple - instance learning approach is more flexible than standard classification approaches in that the representation allows us to encode more information from a single image by considering it as a collection of feature vectors rather than encoding the entire image by a single feature vector . the multiple - instance representation is highly informative in this situation since it encodes information from different regions in an image , each of which may contribute to the final grade or label of the image as a whole . therefore , in order not to add complexity to the construction of a classifier and preserve the flexibility of the task , we follow the bag dissimilarity approach described in , which does not attempt to locate a concept but rather uses a similarity measure across bags , which are seen as sets of instances . the dissimilarities computed between the bags become the new features , and this allows us to construct any classifier in this new feature space , thus decoupling the original multiple - instance problem from the classification task itself . moreover , the bag dissimilarity approach allows us to consider multiclass data , that is , data with several grades , whereas in the traditional multiple - instance learning problem only two classes , namely , a positive and a negative class , are considered at any given time , and a one - against - one or one - against - all approach is often used in the classification of multiclass situations . insofar , we have presented a new vectorial proximity - based feature for describing tissue architecture around glands , and we proceed in the next section to demonstrate its usefulness as a feature descriptor . however , in order to evaluate whether more conventional scalar features add any information to the new feature , we have implemented four classical , well - known scalar features that are simple to compute from each lumen region . the first is the size of the region , while the other measures relate to its shape and are the bending energy , area - to - perimeter ratio , and convexity ratio . bending energy is defined around the lumen perimeter based on the chain code sequence and is given by eb = p=1(p ) , where (p ) is a smoothed version of the curvature signal (p ) = tan((yc(p ) yc(p 1))/(xc(p ) xc(p 1 ) ) ) , where ( xc(p 1 ) , yc(p 1 ) ) and ( xc(p ) , yc(p ) ) are two consecutive points of the curvature . for each image in the dataset , we have used our proximity - based feature method to obtain a set of descriptive features . then we used multiple - instance learning to represent each image as a bag of instances and transform the feature space into a dissimilarity space by computing the distances among the different bags . note that ten sampling rings were used for the statistical proximity sampling method throughout all cases resulting in 40-element feature vectors for the case of the breast dataset since the number of classes was four using the hematoxylin - eosin + dab stain . we demonstrate in what follows how clustering may be applied to classify instances in the absence of bag labels . , image grade ) may then be obtained using a voting scheme over the cluster labels of the instances that belong to it . to study whether there may be an inherent number of clusters in the data possibly due to a certain fixed number of neighborhood descriptions that tend to recur around lumen regions we clustered the breast dataset using the gaussian mixture model ( gmm ) several times with a varying number of clusters k ranging from 1 to 10 , and we computed the bic values as defined by bic = 2ln(l ) + kln(n ) , where n is the number of objects in the data , l is the likelihood value of the mixture fit , and k is the number of clusters . note that the mixture model was initialized randomly 10 times for each value of k. as the number of clusters increases , we expect the log - likelihood to increase monotonically ; however the bic measure also includes the model parameters into the tradeoff , which in this case is the number of clusters k. the optimal mixture model would have a high log - likelihood yet at a lowest possible complexity k. a plot of the bic values versus the number of clusters is shown in figure 11 . we have presented a general and simple method for statistically describing the distribution of glandular structures around lumen regions . the method makes use of sampling based on an iterative region expansion procedure that preserves the shape of the lumen areas . one advantage of this approach is that , by analyzing the neighborhoods of lumen regions and preserving the spatial and statistical information in these proximities , we avoid the need to extract explicit features concerning the underlying tissue structures themselves . the result is a set of feature vectors containing spatial and statistical information that may be used to describe regions in tissue images for a large variety of purposes , among which is tubule - based grading , as we have demonstrated in this paper . the input required for the method can be either a set of probability or binary maps derived from soft or crisp classification regardless of the supervised or unsupervised method ( e.g. the method is also robust and its dependence on the quality of these maps is minimal since the approach does not attempt to derive any precise cellular or subcellular features , which would require accurate image segmentation . due to the natural complexity of biological tissue and the grading process , we have avoided the single feature vector based representation used in standard pattern recognition . automated grading was instead done using a bag dissimilarity approach while treating the problem in a similar manner to multiple - instance learning . classification results using cross - validation have shown that the statistical proximity sampling method presented is able to provide a set of discriminative features for tubule - based cancer grading . a possible drawback of the dissimilarity approach we have used in our classification is that although the classification task itself is accomplished and the diagnosis is automated , no single concept however , alternative multiple - instance learning methods that are based on the notion of finding a concept may be used for this purpose if needed . a comprehensive automated system that would be able to eventually assign high - level grading akin to that by pathologists would undoubtedly have to incorporate , in addition to the work described in this paper , methods that are designed to address nuclear pleomorphism and mitotic count ( as most recently in ) . as a first step towards that goal , we have in this paper addressed tubule - based grading , which contributes to one of the three components for malignancy grading under the ellis - elston system and which is also considered an important factor in gleason grading .

in recent years , the results after surgery for rectal cancer in norway , with a 5-year overall survival ( os ) rate of 60.1% , has surpassed that of colon cancer at 57.5% . this has been achieved because the surgical technique has been standardized according to total mesorectal excision ( tme ) with subsequent dramatic reductions of local recurrences . beginning in 2007 , all colon cancers were to be reported separately to the norwegian national cancer registry in an effort to systematically survey and hopefully improve results . nevertheless , a national strategy to standardize surgical treatment along the lines of radical surgery has neither been implemented in detail nor been generally accepted [ 2 , 3 ] . in this respect , the number of lymph nodes retrieved may act as a surrogate measure of radical surgery . the survival benefit of a large lymph node harvest has been shown in several reports [ 24 ] . it has been accepted nationally to offer patients with tumor node metastasis ( tnm ) stage iii below a certain age , usually 75 years , adjuvant chemotherapy . it has been decided that a rather arbitrary level of 12 retrieved nodes is enough to obtain adequate surgery and staging . pathologists may be a key factor for optimal lymph node harvest , and a conjoined effort between surgeon and pathologist would be ideal to improve results [ 26 ] . the aim of the study was to examine , after modest radical colon surgery removing mesocolic nodes and focus on lymph node yield , what would influence survival and where surgical improvement might be possible using data from a cohort of patients from three large norwegian teaching hospitals . patients from a national cohort were operated in 2000 , and follow - up was until december 2007 , a mean of 7.5 years later . three teaching community hospitals , haraldsplass deaconal hospital , stavanger university hospital , and akershus university hospital contributed patients . all three hospitals are teaching community hospitals , and the patients were operated with an open access by a large number of surgeons . at that time , extra radical surgery was unusual , and it is fair to assume that radical surgery usually constituted a moderate mesocolic resection . if metastases were diagnosed , patients and tumor conditions were assessed regarding feasibility for resection . patients usually went to the outpatient clinic every third month for the first 2 years and then every sixth month until 5 years had passed . blood tests with carcino - embryonic antigen measurement and ultrasonography of the liver and chest x - ray were carried out . elderly patients are stead - bound and even if a few of them were not followed up frequently , they could be tracked and life status ascertained through their identity number in the official national population registry . the specimen was examined and rinsed by the surgeons on the back table before being mounted on a board and placed in a box filled with enough formaldehyde for secure fixation . the specimen was examined by a junior pathologist ; after 4872 h , assisted by the consultant . tissue was paraffin - embedded , and hematoxylin eosin staining was used routinely before sections were examined microscopically . metastatic deposits were defined as lymph nodes if these structures resembled nodes but without containing visible lymphatic tissue . patients younger than 75 years of age that were classified as tnm stage iii were offered 12 courses of adjuvant treatment with 5-fluorouracil plus calsiumfolinate ( flv ) . the regional committee for medical and health research ethics of western norway and the data inspectorate for national registries approved the study . the chi - square test was used to compare groups with respect to categorical variables and analysis of variance for continuous variables . the following variables were analysed with respect to survival : hospital , age , gender , location , no of lymph nodes and lymph node ratio ( lnr ) , t stage , and tnm stage . survival curves were estimated by the kaplan meier method and compared using the log - rank test . multiple prognostic factors were analysed with the cox proportional hazards model using the spss 17 package . all three hospitals are teaching community hospitals , and the patients were operated with an open access by a large number of surgeons . at that time , extra radical surgery was unusual , and it is fair to assume that radical surgery usually constituted a moderate mesocolic resection . if metastases were diagnosed , patients and tumor conditions were assessed regarding feasibility for resection . patients usually went to the outpatient clinic every third month for the first 2 years and then every sixth month until 5 years had passed . blood tests with carcino - embryonic antigen measurement and ultrasonography of the liver and chest x - ray were carried out . elderly patients are stead - bound and even if a few of them were not followed up frequently , they could be tracked and life status ascertained through their identity number in the official national population registry . the specimen was examined and rinsed by the surgeons on the back table before being mounted on a board and placed in a box filled with enough formaldehyde for secure fixation . the specimen was examined by a junior pathologist ; after 4872 h , assisted by the consultant . tissue was paraffin - embedded , and hematoxylin eosin staining was used routinely before sections were examined microscopically . metastatic deposits were defined as lymph nodes if these structures resembled nodes but without containing visible lymphatic tissue . patients younger than 75 years of age that were classified as tnm stage iii were offered 12 courses of adjuvant treatment with 5-fluorouracil plus calsiumfolinate ( flv ) . the regional committee for medical and health research ethics of western norway and the data inspectorate for national registries approved the study . the chi - square test was used to compare groups with respect to categorical variables and analysis of variance for continuous variables . the following variables were analysed with respect to survival : hospital , age , gender , location , no of lymph nodes and lymph node ratio ( lnr ) , t stage , and tnm stage . survival curves were estimated by the kaplan meier method and compared using the log - rank test . multiple prognostic factors were analysed with the cox proportional hazards model using the spss 17 package . two hundred sixty - nine patients , 152 ( 56.5% ) women and 117 men , with a mean age of 71 years ( range , 2093 years ) were studied . one of the hospitals operated male patients that were younger , with a mean of 67 years . tumor locations were not different between the hospitals ( p = 0.059 ) . locoregional r0 resections for a single tumor location were done in 264 patients , while double resections ( n = 4 ) and a suspected locoregional r1 resection ( n = 1 ) were done in five patients . table 1tumor locations and pathology variables of patients resected for colon cancer at three teaching community hospitals in 2000variablesall patientschi - square testn = 269p valuelocation0.059 right hemicolon115 ( 42.8 ) transverse colon including flexures44 ( 16.4 ) descending colon9 ( 3.3 ) sigmoid colon and rectosigmoid96 ( 35.7 ) multiple or r125 ( 1.9)t category0.021 t111 ( 4.1 ) t230 ( 11.2 ) t3180 ( 66.9 ) t448 ( 17.8)tnm stage0.098 i34 ( 12.6 ) ii116 ( 43.1 ) iii93 ( 34.6 ) iv26 ( 9.7)resection of two tumors in separate locationsno radical loco - regional tumor resection tumor locations and pathology variables of patients resected for colon cancer at three teaching community hospitals in 2000 resection of two tumors in separate locations no radical loco - regional tumor resection there were no significant differences between hospitals relating to tnm stage distribution : 34 patients ( 12.6% ) were stage i , 116 patients ( 43.1% ) were stage ii , 93 patients ( 34.6% ) were stage iii , and 26 patients ( 9.6% ) were stage iv . the number of lymph nodes harvested for various stages were 8.7 ( stage i ) , 10.3 ( stage ii ) , 10.9 ( stage iii ) , and 10.3 ( stage iv ) . in 11 patients , the pathologist had classified the t category and tnm stage but omitted to specify the number of lymph nodes present . twelve or more lymph nodes were examined in 41.1% ( 106/258 ) of the resected specimens . significantly fewer lymph nodes ( p < 0.001 ) were harvested at one of the hospitals . otherwise , the three patient populations had similar characteristics . urgent surgery had a mortality of 12.5% ( 3/24 ) , whereas the elective group had a mortality of 4.5% ( 11/245 ) . did not differ between the hospitals ( log - rank p = 0.372 ; fig . 1kaplan meier survival curve for 269 patients resected for colon cancer in three norwegian hospitals in 2000 kaplan meier survival curve for 269 patients resected for colon cancer in three norwegian hospitals in 2000 whether categorizing number of lymph nodes harvested in three groups ( < 10 , 1019 , 20 or more ) or in two ( < 12 , 12 or more ) , no differences was found in os ( log - rank p = 0.423 and 0.270 , respectively ) . this was still the case after adjusting for hospital or tnm stage ( log - rank p = 0.449 ) . the uncategorized number of lymph nodes was not significant in a simple cox regression ( likelihood ratio p = 0.129 ) . however , in one hospital , better survival was found when the lymph node harvest was 12 compared to < 12 ( log - rank p = 0.037 ) as shown in fig . 2 . stage ii patients had os of 72.7% with 12 nodes harvested and 58.3% with < 12 nodes ( p = 0.124 ) , and stage iii patients had a 5-year os of 61.5% and 55.6% , respectively ( p = 0.508 ) . 2kaplan meier survival curve for 103 patients resected for colon cancer in one of three norwegian hospitals in 2000 . patients with a lymph node harvest of 12 or more showed significantly better overall survival ( log - rank test p = 0.037 ) kaplan meier survival curve for 103 patients resected for colon cancer in one of three norwegian hospitals in 2000 . patients with a lymph node harvest of 12 or more showed significantly better overall survival ( log - rank test p = 0.037 ) in stage iii patients , the lymph node ratio ( lnr ) was highly significant for patient survival , and os for lnr 1 ( < 0.25 ) was 83.3.5% , lnr 2 ( 0.250.50 ) 63.3% , lnr 3 ( 0.510.75 ) 18.8% , and lnr 4 ( 0.761 ) 18.2% ( log - rank test p < 0.001 ) . two of the hospitals did more resections of t4 tumors ( chi - square test p = 0.021 ) . adjusted for hospital , t4 tumors compared to t1t3 , was a significant adverse factor for survival in the log - rank test ( p = 0.049 ) . survival according to the different tnm stages is shown in table 2 . the 5-year os was 58.1% for stage iii and 63.8% for stage ii without differences between the hospitals in uni- and multivariate analyses . os for stage iv was significantly worse ( p < 0.001 ) . table 2five - year overall survival ( os ) according to the different tnm stages for 269 patients treated with resection for colon cancer in three norwegian teaching community hospitals in 2000tnm stagen ( % ) % osi34 ( 12.6)76.5ii116 ( 43.1)63.8iii93 ( 34.6)58.1iv26 ( 9.7)7.7all stages269 ( 100)58.0 five - year overall survival ( os ) according to the different tnm stages for 269 patients treated with resection for colon cancer in three norwegian teaching community hospitals in 2000 the results of univariate and multivariate cox - regression analyses are shown in table 3 . in the univariate cox regression old age , t category , high lnr , and tnm stage age as a variable was highly significant both as a continuous variable and with a cutoff value of 69 years . locations of the tumors with regard to the various resected segments were not significant ( table 3 ) . table 3univariate ( n = 269 patients ) and multivariate ( n = 258 ) cox regression models for analysis of overall survival for patients resected for colon cancer in three norwegian teaching community hospitals in 2000 univariatemultivariatelr testhr 95% cip valuehr 95% cip valueno . of sampled lymph nodes per increments of 100.79 ( 0.58 , 1.08)0.1290.82 ( 0.57 , 1.17)0.266 t category<0.0010.129 t1t21 ( reference)1 ( reference ) t31.69 ( 0.94 , 3.03)1.84 ( 0.55 , 6.15 ) t43.38 ( 1.79 , 6.40)2.64 ( 0.78 , 8.99)tumor location0.5680.716 right colon1 ( reference)1 ( reference ) transverse colon0.89 ( 0.55 , 1.46)0.79 ( 0.47 , 1.32 ) descending colon0.97 ( 0.39 , 2.42)1.44 ( 0.57 , 3.66 ) sigmoid colon0.70 ( 0.45 , 1.10)0.81 ( 0.50 , 1.31 ) rectosigmoid0.82 ( 0.44 , 1.53)0.62 ( 0.31 , 1.26 ) other / double0.96 ( 0.23 , 3.93)0.67 ( 0.15 , 2.91 ) r25.83 ( 0.79 , 42.81)0.61 ( 0.08 , 4.94)age per 10 years1.46 ( 1.23 , 1.74)0.0011.68 ( 1.38 , 2.03)<0.001gender0.1920.031 females1 ( reference)1 ( reference ) males1.26 ( 0.89 , 1.77)1.52 ( 1.04 , 2.23)tnm stage<0.001<0.001 i1 ( reference)1 ( reference ) ii1.61 ( 0.82 , 3.18)0.91 ( 0.23 , 3.69 ) iii2.07 ( 1.05 , 4.12)1.49 ( 0.39 , 5.66 ) iv10.50 ( 4.94 , 22.32)9.26 ( 2.15 , 39.85)hospital0.3900.169 ahus1 ( reference)1 ( reference ) sus1.06 ( 0.71 , 1.60)0.72 ( 0.46 , 1.14 ) hds1.34 ( 0.87 , 2.07)0.62 ( 0.37 , 1.03)hr hazard ratio ; ci confidence interval ; lr likelihood ratio ; sus stavanger university hospital ; hds haraldsplass deaconal hospital ; ahus akershus university hospitalanalyses based on 258 patients because of lack of specified number of lymph nodesfig . 3kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to hospital ( log - rank test p = 0.372 ) univariate ( n = 269 patients ) and multivariate ( n = 258 ) cox regression models for analysis of overall survival for patients resected for colon cancer in three norwegian teaching community hospitals in 2000 hr hazard ratio ; ci confidence interval ; lr likelihood ratio ; sus stavanger university hospital ; hds haraldsplass deaconal hospital ; ahus akershus university hospital analyses based on 258 patients because of lack of specified number of lymph nodes kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to hospital ( log - rank test p = 0.372 ) in the multivariate model age , male gender , high lnr , and tnm stage were adverse factors ( figs . 4 , 5 , and 6 ) . in the fully adjusted multivariate analysis , t category was not significant , but when removing tnm stage from the multivariate model , the t category again became significant ( p = 0.047 ) . these variables were not significant : hospital , tumor location , or number of harvested lymph nodes in these categories ( < 10 , 1019 , 20 ) . lnr was also highly significant for stage iii patients ( n = 93 ) when adjusted for all the variables in table 3 in a multivariate cox regression with hrs ( 95% cis ) of 1.72 ( 0.80 , 3.67 ) , 5.16 ( 2.48 , 10.74 ) , and 4.80 ( 1.92 , 11.99 ) for lnr 24 vs. lnr 1 , respectively ( p < 0.001 ) . 4kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to age ( log - rank test p < 0.001)fig . 5kaplan meier survival curves for 93 patients resected for colon cancer in three norwegian hospitals in 2000 according to lymph node ratio groups : < 0.25 ( group 1 ) , 0.250.5 ( group 2 ) , 0.510.75 ( group 3 ) , and > 0.75 ( group 4 ) ( log - rank test p < 0.001)fig . 6kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to tnm stage ( log - rank test p < 0.001 ) kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to age ( log - rank test p < 0.001 ) kaplan meier survival curves for 93 patients resected for colon cancer in three norwegian hospitals in 2000 according to lymph node ratio groups : < 0.25 ( group 1 ) , 0.250.5 ( group 2 ) , 0.510.75 ( group 3 ) , and > 0.75 ( group 4 ) ( log - rank test p < 0.001 ) kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to tnm stage ( log - rank test p < 0.001 ) the study may have been underpowered for detection of an assumed difference between patients with a lymph node harvest of < 12 lymph nodes and those with > 12 . with the number of patients included in our study , a power of at least 0.84 would be necessary to detect a difference in 5-year survival of 50% vs. 68% . an increase in sample size to detect a difference of 50% vs. 60% with a power of 0.80 in a one - tailed chi - square test tumor locations were not different between the hospitals ( p = 0.059 ) . locoregional r0 resections for a single tumor location were done in 264 patients , while double resections ( n = 4 ) and a suspected locoregional r1 resection ( n = 1 ) were done in five patients . table 1tumor locations and pathology variables of patients resected for colon cancer at three teaching community hospitals in 2000variablesall patientschi - square testn = 269p valuelocation0.059 right hemicolon115 ( 42.8 ) transverse colon including flexures44 ( 16.4 ) descending colon9 ( 3.3 ) sigmoid colon and rectosigmoid96 ( 35.7 ) multiple or r125 ( 1.9)t category0.021 t111 ( 4.1 ) t230 ( 11.2 ) t3180 ( 66.9 ) t448 ( 17.8)tnm stage0.098 i34 ( 12.6 ) ii116 ( 43.1 ) iii93 ( 34.6 ) iv26 ( 9.7)resection of two tumors in separate locationsno radical loco - regional tumor resection tumor locations and pathology variables of patients resected for colon cancer at three teaching community hospitals in 2000 resection of two tumors in separate locations no radical loco - regional tumor resection there were no significant differences between hospitals relating to tnm stage distribution : 34 patients ( 12.6% ) were stage i , 116 patients ( 43.1% ) were stage ii , 93 patients ( 34.6% ) were stage iii , and 26 patients ( 9.6% ) were stage iv . the number of lymph nodes harvested for various stages were 8.7 ( stage i ) , 10.3 ( stage ii ) , 10.9 ( stage iii ) , and 10.3 ( stage iv ) . in 11 patients , the pathologist had classified the t category and tnm stage but omitted to specify the number of lymph nodes present . twelve or more lymph nodes were examined in 41.1% ( 106/258 ) of the resected specimens . significantly fewer lymph nodes ( p < 0.001 ) were harvested at one of the hospitals . urgent surgery had a mortality of 12.5% ( 3/24 ) , whereas the elective group had a mortality of 4.5% ( 11/245 ) . the 5-year os was 58.0% ( fig . 1 ) and did not differ between the hospitals ( log - rank p = 0.372 ; fig . 1kaplan meier survival curve for 269 patients resected for colon cancer in three norwegian hospitals in 2000 kaplan meier survival curve for 269 patients resected for colon cancer in three norwegian hospitals in 2000 whether categorizing number of lymph nodes harvested in three groups ( < 10 , 1019 , 20 or more ) or in two ( < 12 , 12 or more ) , no differences was found in os ( log - rank p = 0.423 and 0.270 , respectively ) . this was still the case after adjusting for hospital or tnm stage ( log - rank p = 0.449 ) . the uncategorized number of lymph nodes was not significant in a simple cox regression ( likelihood ratio p = 0.129 ) . however , in one hospital , better survival was found when the lymph node harvest was 12 compared to < 12 ( log - rank p = 0.037 ) as shown in fig . 2 . stage ii patients had os of 72.7% with 12 nodes harvested and 58.3% with < 12 nodes ( p = 0.124 ) , and stage iii patients had a 5-year os of 61.5% and 55.6% , respectively ( p = 0.508 ) . 2kaplan meier survival curve for 103 patients resected for colon cancer in one of three norwegian hospitals in 2000 . patients with a lymph node harvest of 12 or more showed significantly better overall survival ( log - rank test p = 0.037 ) kaplan meier survival curve for 103 patients resected for colon cancer in one of three norwegian hospitals in 2000 . patients with a lymph node harvest of 12 or more showed significantly better overall survival ( log - rank test p = 0.037 ) in stage iii patients , the lymph node ratio ( lnr ) was highly significant for patient survival , and os for lnr 1 ( < 0.25 ) was 83.3.5% , lnr 2 ( 0.250.50 ) 63.3% , lnr 3 ( 0.510.75 ) 18.8% , and lnr 4 ( 0.761 ) 18.2% ( log - rank test p < 0.001 ) . two of the hospitals did more resections of t4 tumors ( chi - square test p = 0.021 ) . adjusted for hospital , t4 tumors compared to t1t3 , was a significant adverse factor for survival in the log - rank test ( p = 0.049 ) . the 5-year os was 58.1% for stage iii and 63.8% for stage ii without differences between the hospitals in uni- and multivariate analyses . os for stage iv was significantly worse ( p < 0.001 ) . table 2five - year overall survival ( os ) according to the different tnm stages for 269 patients treated with resection for colon cancer in three norwegian teaching community hospitals in 2000tnm stagen ( % ) % osi34 ( 12.6)76.5ii116 ( 43.1)63.8iii93 ( 34.6)58.1iv26 ( 9.7)7.7all stages269 ( 100)58.0 five - year overall survival ( os ) according to the different tnm stages for 269 patients treated with resection for colon cancer in three norwegian teaching community hospitals in 2000 the results of univariate and multivariate cox - regression analyses are shown in table 3 . in the univariate cox regression old age , t category , high lnr , and tnm stage were adverse factors for survival . age as a variable was highly significant both as a continuous variable and with a cutoff value of 69 years . locations of the tumors with regard to the various resected segments were not significant ( table 3 ) . table 3univariate ( n = 269 patients ) and multivariate ( n = 258 ) cox regression models for analysis of overall survival for patients resected for colon cancer in three norwegian teaching community hospitals in 2000 univariatemultivariatelr testhr 95% cip valuehr 95% cip valueno . of sampled lymph nodes per increments of 100.79 ( 0.58 , 1.08)0.1290.82 ( 0.57 , 1.17)0.266 t category<0.0010.129 t1t21 ( reference)1 ( reference ) t31.69 ( 0.94 , 3.03)1.84 ( 0.55 , 6.15 ) t43.38 ( 1.79 , 6.40)2.64 ( 0.78 , 8.99)tumor location0.5680.716 right colon1 ( reference)1 ( reference ) transverse colon0.89 ( 0.55 , 1.46)0.79 ( 0.47 , 1.32 ) descending colon0.97 ( 0.39 , 2.42)1.44 ( 0.57 , 3.66 ) sigmoid colon0.70 ( 0.45 , 1.10)0.81 ( 0.50 , 1.31 ) rectosigmoid0.82 ( 0.44 , 1.53)0.62 ( 0.31 , 1.26 ) other / double0.96 ( 0.23 , 3.93)0.67 ( 0.15 , 2.91 ) r25.83 ( 0.79 , 42.81)0.61 ( 0.08 , 4.94)age per 10 years1.46 ( 1.23 , 1.74)0.0011.68 ( 1.38 , 2.03)<0.001gender0.1920.031 females1 ( reference)1 ( reference ) males1.26 ( 0.89 , 1.77)1.52 ( 1.04 , 2.23)tnm stage<0.001<0.001 i1 ( reference)1 ( reference ) ii1.61 ( 0.82 , 3.18)0.91 ( 0.23 , 3.69 ) iii2.07 ( 1.05 , 4.12)1.49 ( 0.39 , 5.66 ) iv10.50 ( 4.94 , 22.32)9.26 ( 2.15 , 39.85)hospital0.3900.169 ahus1 ( reference)1 ( reference ) sus1.06 ( 0.71 , 1.60)0.72 ( 0.46 , 1.14 ) hds1.34 ( 0.87 , 2.07)0.62 ( 0.37 , 1.03)hr hazard ratio ; ci confidence interval ; lr likelihood ratio ; sus stavanger university hospital ; hds haraldsplass deaconal hospital ; ahus akershus university hospitalanalyses based on 258 patients because of lack of specified number of lymph nodesfig . 3kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to hospital ( log - rank test p = 0.372 ) univariate ( n = 269 patients ) and multivariate ( n = 258 ) cox regression models for analysis of overall survival for patients resected for colon cancer in three norwegian teaching community hospitals in 2000 hr hazard ratio ; ci confidence interval ; lr likelihood ratio ; sus stavanger university hospital ; hds haraldsplass deaconal hospital ; ahus akershus university hospital analyses based on 258 patients because of lack of specified number of lymph nodes kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to hospital ( log - rank test p = 0.372 ) in the multivariate model age , male gender , high lnr , and tnm stage were adverse factors ( figs . 4 , 5 , and 6 ) . in the fully adjusted multivariate analysis , t category was not significant , but when removing tnm stage from the multivariate model , the t category again became significant ( p = 0.047 ) . these variables were not significant : hospital , tumor location , or number of harvested lymph nodes in these categories ( < 10 , 1019 , 20 ) . lnr was also highly significant for stage iii patients ( n = 93 ) when adjusted for all the variables in table 3 in a multivariate cox regression with hrs ( 95% cis ) of 1.72 ( 0.80 , 3.67 ) , 5.16 ( 2.48 , 10.74 ) , and 4.80 ( 1.92 , 11.99 ) for lnr 24 vs. lnr 1 , respectively ( p < 0.001 ) . 4kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to age ( log - rank test p < 0.001)fig . 5kaplan meier survival curves for 93 patients resected for colon cancer in three norwegian hospitals in 2000 according to lymph node ratio groups : < 0.25 ( group 1 ) , 0.250.5 ( group 2 ) , 0.510.75 ( group 3 ) , and > 0.75 ( group 4 ) ( log - rank test p < 0.001)fig . 6kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to tnm stage ( log - rank test p < 0.001 ) kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to age ( log - rank test p < 0.001 ) kaplan meier survival curves for 93 patients resected for colon cancer in three norwegian hospitals in 2000 according to lymph node ratio groups : < 0.25 ( group 1 ) , 0.250.5 ( group 2 ) , 0.510.75 ( group 3 ) , and > 0.75 ( group 4 ) ( log - rank test p < 0.001 ) kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to tnm stage ( log - rank test p < 0.001 ) the study may have been underpowered for detection of an assumed difference between patients with a lymph node harvest of < 12 lymph nodes and those with > 12 . with the number of patients included in our study , a power of at least 0.84 would be necessary to detect a difference in 5-year survival of 50% vs. 68% . an increase in sample size to detect a difference of 50% vs. 60% with a power of 0.80 in a one - tailed chi - square test even though several groups have pioneered radical surgery for colon cancer ( complete mesocolic excision and a central tie ) with its potential benefits similar to ( tme ) of rectal cancer surgery , such an approach has not been widely adopted in our country . therefore , we wanted to examine our background results based on a less standardized approach and potential areas for improvement before embarking on more radical procedures as championed by others . advanced tnm stage had an adverse influence on outcome , as the results for stage iv versus i iii and stage iii versus i ii were significant in the multivariate analysis . for stage iii patients , the 5-year os was 58.1% . in one hospital , the 5-year os for stage iii was worse with 47.8% . in the multivariate analysis , however , when adjusted for hospital , there was no significant survival difference . a recently published international multicenter study of a colorectal population reported an identical os of 58.8% for patients treated in 20002002 . others have found a 5-year os of 90.796.3% for stage ii and 64.671.7% for stage iii using radical surgical procedures . thus , we consider our figures to show room for improvement , although where improvement may be best realised is debatable . an advanced stage may theoretically be better treated with more radical surgery even though the presence of skip lesions in rare instances may hardly explain this . radical or complete mesocolic excision has been shown to increase both the absolute lymph node harvest and prognosis for the patients [ 2 , 3 ] . however , it has been contested that patients will benefit from more radical surgery in advanced cases . in a swedish population - based retrospective study , a median number of six lymph nodes were detected in the specimens from 1,856 patients operated between 1996 and 2000 . there were a very low number of nodes in the specimens from the left colon , whereas in the right flexure tumors , the lymph node count was not inferior . in both instances , the feeding vessel areas were not divided completely , i.e. , the inferior mesenteric artery proximally and the root of the medial colic artery . in a population - based study from the netherlands by kelder and co - workers , the median lymph node harvest was six and in only 21% of the specimens were 12 or more nodes examined . thus , we consider that the principle of radical colon surgery may have been violated in these studies when tumors were in those locations . more radical surgery for colon cancer has been recommended in reports from the usa , europe , and japan [ 2 , 15 , 16 ] . a retrospective national report based on a small number with supposedly radical colon surgery found that this approach significantly increased survival compared with a much larger and older group subject to a standard the number of lymph nodes may be seen only as a surrogate marker for the extent of surgery without a proper oncologic explanation for the importance of a large harvest [ 2 , 3 ] . the node count has been correlated to survival both for stage ii and iii disease [ 2 , 18 ] . the ontario cancer registry study showed better survival with a lymph node harvest of more than ten lymph nodes . increasing in contrast , there is good evidence reported by others that increasing the total number of lymph nodes increases survival significantly [ 2 , 4 ] . in one study , a mean harvest of more than 28 lymph nodes data from the seer database demonstrated that a cutoff value of 15 was useful , and perhaps , there is an inherent limit to the number of nodes necessary to achieve this effect on survival . nevertheless , the hypothesis that a larger ( negative ) lymph node yield is beneficial is contradicted even from highly rated institutions . in the univariate analysis , one of the hospitals achieved a significantly better survival with a harvest of more than 12 lymph nodes . however , no significant survival benefit was found with harvest of more than 12 lymph nodes in the multivariate analysis . many pathologists were involved in this trial even though the specimen handling was supposed to be equal . in a recent study , the pathologist was found to be the dependent factor in lymph node harvest in multivariate analysis , not the operating surgeon . the overall poor lymph node harvest may also indicate that more extensive surgery is one way to improve outcome as found in one of the hospitals . it may be speculated that the poorer outcome in the other two hospitals may have levelled out any benefit by an improved lymph node harvest , i.e. , more radical surgery . patients below 75 years were given flv chemotherapy , and staging was considered important for that reason . for proper staging of colon cancer , the minimum number of lymph nodes needed has been somewhat arbitrarily suggested as 12 by a national cancer institute expert panel . although some authors [ 14 , 18 ] have concluded with stage migration as an explanation for an increased survival benefit , other studies have shown that detection of positive nodes beyond six or seven lymph nodes examined had no effect on staging [ 19 , 23 ] . doubtless , a standardized surgical approach in cooperation with a dedicated pathology service is necessary if a minimum number of nodes shall be found and consequently help in outcome assessment . studies have shown that the lnr is an independent and better marker than pn+ for survival . in our study , lnr was highly significant for os when adjusted for hospitals in the multivariate analysis . five - year os varied from 83.5% in lnr group 1 , 63.3% in lnr 2 , to 18.8% in lnr group 3 . according to wang et al . [ 26 , 27 ] , the prognostic effect of lnr did not depend on the total number of lymph nodes nor on the number of positive nodes . in contrast , berger et al . found that lnr was not a significant prognostic indicator with less than ten examined nodes but became highly significant for os and disease - free survival with more than ten lymph nodes harvested . a mayo clinic study found a positive correlation between the number of positive lymph nodes and survival but did not analyze the effect of lnr . fifty - eight percent of our stage iii patients had less than 12 lymph nodes examined . still , we found a significant survival difference of 44.5% between lnr group 2 and 3 and 65.3% between group 1 and 4 . a study from new zealand found that both the absolute number of lymph nodes retrieved up to 16 , as well as the lnr , were important for prognosis . the share of stage iii compared to stage ii cancers in their patients amounted to 54.9% , higher than in most other reports . it seems obvious that a large negative lymph node yield will down - regulate lnr , but the importance of this with regard to outcome may need to be examined in more detail . another national report of colon cancer from a single institution found that an emergency operation , some colon locations , blood transfusion of more than two units , old age , and tnm stage were negative predictive factors . we did not study the effect of blood transfusion , and emergency operations were not separated from the rest partly because they were few , i.e. , 15% reported by sjo et al . , and the definition may be debatable . the different colon locations found to be statistically significant ( transverse , left flexure , and descending colon ) were not so in our multivariate analysis . we suspect that this is partly because they were too few , 20% , to make an impact . even though the number of urgently operated patients was small in this series , as expected , this small group had a higher mortality of 12.5% compared to 4.5% in the elective patients . severe co - morbidities in old age may have contributed to these figures as has been reported by others . however , it should be possible to set an optimal target of < 3% in elective cases . the average age of our study population was 71 years and that may be older than in series of selected patients . the average age of men and women in our country for those that have reached the age of 62 ( earliest oap retirement age ) is 81 and 85 years . to circumvent the analytic problem of age , sjo et al thus , their survival figures improved from 62% actuarial survival to 74% for women and 79% for men . the method is cumbersome , as it necessitates life tables to calculate this for every patient year . three - year disease - free survival has been shown to parallel overall 5-year survival . however , it requires close follow - up with ct instead of the conventional ultrasonography and chest x - ray examinations . close follow - up may even result in better treatment of metastases according to japanese results . only 41% had a lymph node harvest of 12 or more lymph nodes , and although this influenced os in one hospital , it did not overall . more radical surgery may increase the lymph node yield , but if this has the potential to increase overall survival in our patients remains to be seen in the prospective registry . mortality should be kept low with adequate assessment and treatment of co - morbidities as well as meticulous surgery to avoid complications .

backgrounda national surveillance program of colon cancer treatment was introduced in 2007 . we examined prognostic factors for colon cancer operated in 2000 with an aim of improving survival in the new program and a special focus on the merit of lymph node yield.methodsa cohort of 269 patients , 152 women ( 56.5% ) , with a mean age of 71 years , was operated for colon cancer in 2000 at three teaching hospitals and followed up for 7 years.resultsoverall 5-year survival was 58.0% , and overall hospital mortality was 5.2% , with 4.5% in elective cases and 12.5% after urgent surgery . in only 41.1% of the specimens were 12 or more lymph nodes retrieved , but this did not affect survival in the combined cohort , although one of the hospitals achieved a significantly better result with a harvest of 12 or more lymph nodes . in a multivariate analysis , old age , gender , a high lymph node ratio ( lnr ) at stage iii , and tumor node metastasis stage were adverse factors for survival.conclusionsthe operative mortality was high and should be reassessed . the lymph node count did not have a significant impact on outcome overall , whereas the lnr proved significant for stage iii . a prospective protocol using overall lymph node yield as a surrogate measure for more radical surgery , nevertheless , seems warranted to improve the lymph node harvest according to international recommendations .

Introduction Material and methods Surgery Follow-up Pathology Oncology Ethics Statistical analysis Results Surgery Pathology Survival analysis Statistical analysis Discussion Conclusion

in recent years , the results after surgery for rectal cancer in norway , with a 5-year overall survival ( os ) rate of 60.1% , has surpassed that of colon cancer at 57.5% . in this respect , the number of lymph nodes retrieved may act as a surrogate measure of radical surgery . the aim of the study was to examine , after modest radical colon surgery removing mesocolic nodes and focus on lymph node yield , what would influence survival and where surgical improvement might be possible using data from a cohort of patients from three large norwegian teaching hospitals . the following variables were analysed with respect to survival : hospital , age , gender , location , no of lymph nodes and lymph node ratio ( lnr ) , t stage , and tnm stage . the following variables were analysed with respect to survival : hospital , age , gender , location , no of lymph nodes and lymph node ratio ( lnr ) , t stage , and tnm stage . two hundred sixty - nine patients , 152 ( 56.5% ) women and 117 men , with a mean age of 71 years ( range , 2093 years ) were studied . one of the hospitals operated male patients that were younger , with a mean of 67 years . table 1tumor locations and pathology variables of patients resected for colon cancer at three teaching community hospitals in 2000variablesall patientschi - square testn = 269p valuelocation0.059 right hemicolon115 ( 42.8 ) transverse colon including flexures44 ( 16.4 ) descending colon9 ( 3.3 ) sigmoid colon and rectosigmoid96 ( 35.7 ) multiple or r125 ( 1.9)t category0.021 t111 ( 4.1 ) t230 ( 11.2 ) t3180 ( 66.9 ) t448 ( 17.8)tnm stage0.098 i34 ( 12.6 ) ii116 ( 43.1 ) iii93 ( 34.6 ) iv26 ( 9.7)resection of two tumors in separate locationsno radical loco - regional tumor resection tumor locations and pathology variables of patients resected for colon cancer at three teaching community hospitals in 2000 resection of two tumors in separate locations no radical loco - regional tumor resection there were no significant differences between hospitals relating to tnm stage distribution : 34 patients ( 12.6% ) were stage i , 116 patients ( 43.1% ) were stage ii , 93 patients ( 34.6% ) were stage iii , and 26 patients ( 9.6% ) were stage iv . the number of lymph nodes harvested for various stages were 8.7 ( stage i ) , 10.3 ( stage ii ) , 10.9 ( stage iii ) , and 10.3 ( stage iv ) . urgent surgery had a mortality of 12.5% ( 3/24 ) , whereas the elective group had a mortality of 4.5% ( 11/245 ) . 1kaplan meier survival curve for 269 patients resected for colon cancer in three norwegian hospitals in 2000 kaplan meier survival curve for 269 patients resected for colon cancer in three norwegian hospitals in 2000 whether categorizing number of lymph nodes harvested in three groups ( < 10 , 1019 , 20 or more ) or in two ( < 12 , 12 or more ) , no differences was found in os ( log - rank p = 0.423 and 0.270 , respectively ) . 2kaplan meier survival curve for 103 patients resected for colon cancer in one of three norwegian hospitals in 2000 . patients with a lymph node harvest of 12 or more showed significantly better overall survival ( log - rank test p = 0.037 ) kaplan meier survival curve for 103 patients resected for colon cancer in one of three norwegian hospitals in 2000 . patients with a lymph node harvest of 12 or more showed significantly better overall survival ( log - rank test p = 0.037 ) in stage iii patients , the lymph node ratio ( lnr ) was highly significant for patient survival , and os for lnr 1 ( < 0.25 ) was 83.3.5% , lnr 2 ( 0.250.50 ) 63.3% , lnr 3 ( 0.510.75 ) 18.8% , and lnr 4 ( 0.761 ) 18.2% ( log - rank test p < 0.001 ) . adjusted for hospital , t4 tumors compared to t1t3 , was a significant adverse factor for survival in the log - rank test ( p = 0.049 ) . table 2five - year overall survival ( os ) according to the different tnm stages for 269 patients treated with resection for colon cancer in three norwegian teaching community hospitals in 2000tnm stagen ( % ) % osi34 ( 12.6)76.5ii116 ( 43.1)63.8iii93 ( 34.6)58.1iv26 ( 9.7)7.7all stages269 ( 100)58.0 five - year overall survival ( os ) according to the different tnm stages for 269 patients treated with resection for colon cancer in three norwegian teaching community hospitals in 2000 the results of univariate and multivariate cox - regression analyses are shown in table 3 . in the univariate cox regression old age , t category , high lnr , and tnm stage age as a variable was highly significant both as a continuous variable and with a cutoff value of 69 years . table 3univariate ( n = 269 patients ) and multivariate ( n = 258 ) cox regression models for analysis of overall survival for patients resected for colon cancer in three norwegian teaching community hospitals in 2000 univariatemultivariatelr testhr 95% cip valuehr 95% cip valueno . 3kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to hospital ( log - rank test p = 0.372 ) univariate ( n = 269 patients ) and multivariate ( n = 258 ) cox regression models for analysis of overall survival for patients resected for colon cancer in three norwegian teaching community hospitals in 2000 hr hazard ratio ; ci confidence interval ; lr likelihood ratio ; sus stavanger university hospital ; hds haraldsplass deaconal hospital ; ahus akershus university hospital analyses based on 258 patients because of lack of specified number of lymph nodes kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to hospital ( log - rank test p = 0.372 ) in the multivariate model age , male gender , high lnr , and tnm stage were adverse factors ( figs . lnr was also highly significant for stage iii patients ( n = 93 ) when adjusted for all the variables in table 3 in a multivariate cox regression with hrs ( 95% cis ) of 1.72 ( 0.80 , 3.67 ) , 5.16 ( 2.48 , 10.74 ) , and 4.80 ( 1.92 , 11.99 ) for lnr 24 vs. lnr 1 , respectively ( p < 0.001 ) . 4kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to age ( log - rank test p < 0.001)fig . 5kaplan meier survival curves for 93 patients resected for colon cancer in three norwegian hospitals in 2000 according to lymph node ratio groups : < 0.25 ( group 1 ) , 0.250.5 ( group 2 ) , 0.510.75 ( group 3 ) , and > 0.75 ( group 4 ) ( log - rank test p < 0.001)fig . 6kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to tnm stage ( log - rank test p < 0.001 ) kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to age ( log - rank test p < 0.001 ) kaplan meier survival curves for 93 patients resected for colon cancer in three norwegian hospitals in 2000 according to lymph node ratio groups : < 0.25 ( group 1 ) , 0.250.5 ( group 2 ) , 0.510.75 ( group 3 ) , and > 0.75 ( group 4 ) ( log - rank test p < 0.001 ) kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to tnm stage ( log - rank test p < 0.001 ) the study may have been underpowered for detection of an assumed difference between patients with a lymph node harvest of < 12 lymph nodes and those with > 12 . table 1tumor locations and pathology variables of patients resected for colon cancer at three teaching community hospitals in 2000variablesall patientschi - square testn = 269p valuelocation0.059 right hemicolon115 ( 42.8 ) transverse colon including flexures44 ( 16.4 ) descending colon9 ( 3.3 ) sigmoid colon and rectosigmoid96 ( 35.7 ) multiple or r125 ( 1.9)t category0.021 t111 ( 4.1 ) t230 ( 11.2 ) t3180 ( 66.9 ) t448 ( 17.8)tnm stage0.098 i34 ( 12.6 ) ii116 ( 43.1 ) iii93 ( 34.6 ) iv26 ( 9.7)resection of two tumors in separate locationsno radical loco - regional tumor resection tumor locations and pathology variables of patients resected for colon cancer at three teaching community hospitals in 2000 resection of two tumors in separate locations no radical loco - regional tumor resection there were no significant differences between hospitals relating to tnm stage distribution : 34 patients ( 12.6% ) were stage i , 116 patients ( 43.1% ) were stage ii , 93 patients ( 34.6% ) were stage iii , and 26 patients ( 9.6% ) were stage iv . the number of lymph nodes harvested for various stages were 8.7 ( stage i ) , 10.3 ( stage ii ) , 10.9 ( stage iii ) , and 10.3 ( stage iv ) . twelve or more lymph nodes were examined in 41.1% ( 106/258 ) of the resected specimens . urgent surgery had a mortality of 12.5% ( 3/24 ) , whereas the elective group had a mortality of 4.5% ( 11/245 ) . 1kaplan meier survival curve for 269 patients resected for colon cancer in three norwegian hospitals in 2000 kaplan meier survival curve for 269 patients resected for colon cancer in three norwegian hospitals in 2000 whether categorizing number of lymph nodes harvested in three groups ( < 10 , 1019 , 20 or more ) or in two ( < 12 , 12 or more ) , no differences was found in os ( log - rank p = 0.423 and 0.270 , respectively ) . patients with a lymph node harvest of 12 or more showed significantly better overall survival ( log - rank test p = 0.037 ) kaplan meier survival curve for 103 patients resected for colon cancer in one of three norwegian hospitals in 2000 . patients with a lymph node harvest of 12 or more showed significantly better overall survival ( log - rank test p = 0.037 ) in stage iii patients , the lymph node ratio ( lnr ) was highly significant for patient survival , and os for lnr 1 ( < 0.25 ) was 83.3.5% , lnr 2 ( 0.250.50 ) 63.3% , lnr 3 ( 0.510.75 ) 18.8% , and lnr 4 ( 0.761 ) 18.2% ( log - rank test p < 0.001 ) . adjusted for hospital , t4 tumors compared to t1t3 , was a significant adverse factor for survival in the log - rank test ( p = 0.049 ) . table 2five - year overall survival ( os ) according to the different tnm stages for 269 patients treated with resection for colon cancer in three norwegian teaching community hospitals in 2000tnm stagen ( % ) % osi34 ( 12.6)76.5ii116 ( 43.1)63.8iii93 ( 34.6)58.1iv26 ( 9.7)7.7all stages269 ( 100)58.0 five - year overall survival ( os ) according to the different tnm stages for 269 patients treated with resection for colon cancer in three norwegian teaching community hospitals in 2000 the results of univariate and multivariate cox - regression analyses are shown in table 3 . in the univariate cox regression old age , t category , high lnr , and tnm stage were adverse factors for survival . table 3univariate ( n = 269 patients ) and multivariate ( n = 258 ) cox regression models for analysis of overall survival for patients resected for colon cancer in three norwegian teaching community hospitals in 2000 univariatemultivariatelr testhr 95% cip valuehr 95% cip valueno . 3kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to hospital ( log - rank test p = 0.372 ) univariate ( n = 269 patients ) and multivariate ( n = 258 ) cox regression models for analysis of overall survival for patients resected for colon cancer in three norwegian teaching community hospitals in 2000 hr hazard ratio ; ci confidence interval ; lr likelihood ratio ; sus stavanger university hospital ; hds haraldsplass deaconal hospital ; ahus akershus university hospital analyses based on 258 patients because of lack of specified number of lymph nodes kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to hospital ( log - rank test p = 0.372 ) in the multivariate model age , male gender , high lnr , and tnm stage were adverse factors ( figs . lnr was also highly significant for stage iii patients ( n = 93 ) when adjusted for all the variables in table 3 in a multivariate cox regression with hrs ( 95% cis ) of 1.72 ( 0.80 , 3.67 ) , 5.16 ( 2.48 , 10.74 ) , and 4.80 ( 1.92 , 11.99 ) for lnr 24 vs. lnr 1 , respectively ( p < 0.001 ) . 4kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to age ( log - rank test p < 0.001)fig . 5kaplan meier survival curves for 93 patients resected for colon cancer in three norwegian hospitals in 2000 according to lymph node ratio groups : < 0.25 ( group 1 ) , 0.250.5 ( group 2 ) , 0.510.75 ( group 3 ) , and > 0.75 ( group 4 ) ( log - rank test p < 0.001)fig . 6kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to tnm stage ( log - rank test p < 0.001 ) kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to age ( log - rank test p < 0.001 ) kaplan meier survival curves for 93 patients resected for colon cancer in three norwegian hospitals in 2000 according to lymph node ratio groups : < 0.25 ( group 1 ) , 0.250.5 ( group 2 ) , 0.510.75 ( group 3 ) , and > 0.75 ( group 4 ) ( log - rank test p < 0.001 ) kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to tnm stage ( log - rank test p < 0.001 ) the study may have been underpowered for detection of an assumed difference between patients with a lymph node harvest of < 12 lymph nodes and those with > 12 . an increase in sample size to detect a difference of 50% vs. 60% with a power of 0.80 in a one - tailed chi - square test even though several groups have pioneered radical surgery for colon cancer ( complete mesocolic excision and a central tie ) with its potential benefits similar to ( tme ) of rectal cancer surgery , such an approach has not been widely adopted in our country . in a swedish population - based retrospective study , a median number of six lymph nodes were detected in the specimens from 1,856 patients operated between 1996 and 2000 . there were a very low number of nodes in the specimens from the left colon , whereas in the right flexure tumors , the lymph node count was not inferior . in a population - based study from the netherlands by kelder and co - workers , the median lymph node harvest was six and in only 21% of the specimens were 12 or more nodes examined . more radical surgery for colon cancer has been recommended in reports from the usa , europe , and japan [ 2 , 15 , 16 ] . a retrospective national report based on a small number with supposedly radical colon surgery found that this approach significantly increased survival compared with a much larger and older group subject to a standard the number of lymph nodes may be seen only as a surrogate marker for the extent of surgery without a proper oncologic explanation for the importance of a large harvest [ 2 , 3 ] . the ontario cancer registry study showed better survival with a lymph node harvest of more than ten lymph nodes . in one study , a mean harvest of more than 28 lymph nodes data from the seer database demonstrated that a cutoff value of 15 was useful , and perhaps , there is an inherent limit to the number of nodes necessary to achieve this effect on survival . in the univariate analysis , one of the hospitals achieved a significantly better survival with a harvest of more than 12 lymph nodes . however , no significant survival benefit was found with harvest of more than 12 lymph nodes in the multivariate analysis . the overall poor lymph node harvest may also indicate that more extensive surgery is one way to improve outcome as found in one of the hospitals . another national report of colon cancer from a single institution found that an emergency operation , some colon locations , blood transfusion of more than two units , old age , and tnm stage were negative predictive factors . only 41% had a lymph node harvest of 12 or more lymph nodes , and although this influenced os in one hospital , it did not overall . more radical surgery may increase the lymph node yield , but if this has the potential to increase overall survival in our patients remains to be seen in the prospective registry .

glomerulonephritides ( gns ) comprise a group of complex and heterogeneous disease entities , caused by many different underlying conditions . these include primary forms , for example , iga nephropathy or membranous gn , as well as secondary forms developing as a consequence of systemic diseases as lupus nephritis and anca - associated vasculitides . however , regardless of their etiology , gns have in common the fact that they are the result of misdirected immune responses . therefore , in most forms of gn , a pronounced renal glomerular and often also tubulointerstitial inflammatory cell infiltrate is found . numerous studies from the past have shown that cd4 t cells are crucial mediators of most forms of gn . it has been shown that especially cells of the th1 and th17 responses are highly nephritogenic [ 38 ] . dysregulated systemic th1 and th17 immunity in addition , both t helper cell lineages are important mediators of local renal tissue injury as well [ 911 ] . in contrast to pathogenic th1 and th17 responses , regulatory t cells ( tregs ) were proven to be potent anti - inflammatory players in gn . several studies of the past have highlighted their protective effects [ 7 , 1214 ] . given the central roles of t helper effector and t regulatory cells in gn , interestingly , increasing evidence suggests that systemic expansion and renal infiltration of different cd4 t cell subtypes follow a concerted time course . after rapid renal and systemic expansion , their percentages , however , steadily decrease over time and often decline to reach baseline levels . cells of th1 polarization , in contrast , occur later during renal inflammation . they expand at a somewhat slower rate than th17 cells , but their numbers seem to persist at high levels . finally , tregs were shown to steadily expand in a continuous process until a stable equilibrium with their proinflammatory th1 and th17 counterparts is established [ 12 , 17 ] . this defined time course of renal infiltration , which is initially dominated by th17 cells , followed by th1 cells and tregs at later stages , has led to speculations of possible transdifferentiation of th17 cells into another cell type . one possibility , which has been suggested , is reprogramming of th17 cells to acquire a th1 phenotype [ 18 , 19 ] . alternatively , th17 cells could lose their pathogenic properties and might be reprogrammed to become foxp3 tregs . the change in characteristics of single cd4 t cells , that is , plasticity , has been addressed using different methods over the past decade in human and mouse . in vitro approaches as well as transfer experiments using highly purified populations of cd4 t cell subsets have established the view that cd4 t cells can change their polarity under certain conditions [ 18 , 2026 ] . to follow the fate of single cd4 t cells , lineage - tracing systems using cre - recombinase expression under the control of key cytokines or transcription factors and subsequent permanent fluorochrome expression have been established [ 19 , 2729 ] . mice overcome technical limitations in single cell tracing , which were present in transfer experiments using highly purified or even bulk populations of in vitro polarized t cell subsets . in very elegant studies with il-17a - cre fate reporter mice , hirota et al . have established the concept that encephalitogenic th17 cells have a high degree of plasticity into the th1 phenotype in experimental autoimmune encephalomyelitis ( eae ) , the mouse model for multiple sclerosis . furthermore , studies in these mice have revealed that , in specialized environments , namely , intestinal peyer 's patches , th17 cells potentially develop into t follicular helper cells ( tfh ) and drive antigen - specific iga responses in germinal center b cells . moreover , regulatory type 1 cells ( tr1 ) , an intriguing t cell subtype with potent immunosuppressive properties , have only recently been recognized as important players in intestinal inflammation . accumulating evidence suggests that , upon the right stimuli , th17 cells can transdifferentiate to acquire the ability of il-10 secretion and become cells with a tr1 phenotype . a high degree of heterogeneity within certain t cell subsets was also apparent in studies that performed single cell sequencing of th17 cells from eae and from in vitro culture [ 32 , 33 ] . plasticity of human cd4 t cells , on the other hand , can be addressed by using t cell receptors ( tcr ) as an endogenous barcoding system . sequencing of tcr revealed a great diversity in the phenotype of cells that presumably descend from a single cd4 t , cell indicating potential transdifferentiation [ 34 , 35 ] . studies that focus on plasticity of human cd4 t cells have been reviewed recently in detail by dupage and bluestone . in summary , increasing data suggest instability or plasticity , especially , of th17 cells . however , to complicate things , numerous studies have also postulated a diametrically opposite concept ; namely , th17 cells might derive from transdifferentiation of foxp3 tregs [ 29 , 3740 ] . the following paragraphs will summarize our current knowledge of cd4 t cell plasticity with a particular focus on glomerulonephritis . given the high nephritogenic potential of th17 cells [ 6 , 41 ] , their plasticity in renal autoimmune disease is of great clinical interest . two opposing fates have been proposed : transdifferentiation into th1 cells or alternatively into anti - inflammatory tr1 cells . thus , the question clearly arises , if therapeutic interventions targeting th17 t cells might be of dual benefit , since these could also hamper development of th1 responses . on the other hand , blockade of th17 cell development might also interfere with generation of regulatory t cell subsets and thus impede resolution of tissue injury . however , until now only limited data have been published on the potential plasticity of th17 cells in glomerulonephritis . in a previous study , we have transferred in vitro polarized th1 and th17 cells into t cell deficient rag1 knockout mice and analyzed the pathogenicity of these cell types in a planted - antigen model of gn . analysis of systemic immune responses revealed that only ifn but no il-17 or il-4 was produced by splenocytes after the transfer of th1 cells . in contrast , some ifn was also produced by spleen cells after the transfer of th17 cells , indicating that some th17 cells might have adopted a th1 phenotype . it is , however , important to note that t cell pathogenicity rather than plasticity was the primary focus of this study . as a result , in particular , the in vitro polarized th17 cells contained a relevant fraction of ifn producing th1 cells even before transfer , which clearly limits analysis . furthermore , only systemic but not organ specific t cell responses in the kidney were addressed . in summary , this study indicates stability of splenic th1 cells , without significant th1 to th17 or th2 plasticity but suggests some degree of th17 cell transdifferentiation into cells of the th1 type during gn . traced the fate of in vitro th17 polarized cells in another planted - antigen model of gn . these authors evaluated cytokine expression of splenic and renal t cells after transfer and found somewhat lower th17 cell frequencies than expected . these findings therefore indicate partial loss of the th17 effector phenotype but do not support significant th17 to th1 transdifferentiation . in a recent study after transfer of highly purified in vitro polarized th17 cells from fluorescence reporter mice and subsequent induction of crescentic glomerulonephritis , reanalysis of t cells from the kidney displayed a relatively high degree of stability . moreover , using il-17a fate reporter mice , these findings were confirmed in immunocompetent mice in two models of experimental glomerulonephritis . importantly , in these studies , no relevant transdifferentiation into th1 or th2 cells was detected among ex - th17 cells , leaving their fate unknown . in this context , it is important to note that th17 cells have been reported to have a high rate of instability and conversion into th1 cells in nonrenal models of autoimmune diseases . this indicates that the kidney provides a unique environment that supports the stability of th17 cells . since th17 cells are pathogenic in crescentic glomerulonephritis [ 6 , 41 ] , we aimed at actively interfering with their stability . we thus treated mice with a monoclonal anti - cd3 antibody , which resulted in induction of a tolerogenic phenotype , characterized by il-10 coexpression , in otherwise stable renal th17 cells . the great therapeutic potential of this finding for the treatment of renal autoimmune diseases clearly warrants further exploration . since renal th17 cells do not seem to acquire either th1 or th2 phenotypes , an alternative scenario would be th17 transdifferentiation into anti - inflammatory foxp3 tregs . however , available data from renal disease do not support this notion either . in a recent study , we have transferred treg depleted spleen cells into rag1-deficient mice and induced a model of accelerated crescentic glomerulonephritis . interestingly , flow cytometry of splenocytes revealed persistent absence of foxp3 cells at the end of the experiment . this finding indicates that there is no transdifferentiation of non - treg t cell subsets , including th17 cells , into foxp3 tregs in this model . furthermore , in another study , we transferred highly purified th17 cells into lymphocyte intact mice and traced their fate during gn , using a congenic marker system . importantly , though , none of these ex - th17 cells had upregulated foxp3 in kidneys or spleens , which excludes transdifferentiation into tregs . in summary , increasing evidence supports some degree of th17 instability during gn , which , however , does not lead to generation of th1 cells or tregs . on possible scenario would thus be that th17 cells adopt a tr1 phenotype as has recently been suggested . another much discussed aspect of cd4 t cell plasticity is the hypothesis that foxp3 tregs might possess the capacity to transdifferentiate into t effector cells . this notion was supported by early experiments , in which tregs were highly purified from foxp3 fluorescence reporter mouse strains and transferred into different t cell deficient recipients . surprisingly , around half of the transferred tregs had lost foxp3 expression , as well as other treg hallmark molecules as cd25 and ctla-4 . assessment of their function revealed loss of suppressive capacity , and even more suggestive of transdifferentiation , these ex - tregs had started to produce inflammatory cytokines as il-17 and ifn. further rounds of experiments , however , made clear that this observed high degree of instability was overestimated due to the lymphopenic environment in the recipient mice . when tregs were cotransferred with t helper effector cells or transferred into lymphocyte intact recipients , only few tregs lost their phenotype [ 37 , 40 , 47 ] . this new and refined view on treg plasticity was confirmed by later elegant studies using foxp3 fate reporter mice . these studies from different groups consistently reported a high degree of treg stability with , however , minor populations of between 5 and 10% showing loss of foxp3 [ 29 , 4850 ] . the fate of these ex - foxp3 cells is currently unsolved and remains a highly discussed topic . while most tregs seem to stably maintain foxp3 expression , substantial fractions were found to coexpress master transcription factors and key cytokines of t helper effector cell lineages [ 5254 ] . four possible scenarios regarding these t cells with a hybrid phenotype are currently under debate . firstly , effector t helper cells could transiently coexpress foxp3 during early stages of activation [ 49 , 55 , 56 ] . secondly alternatively , tregs might be upregulating classical t helper effector cell transcription factors to gain certain functional characteristics and , last but not least , they could be independent and previously unrecognized t cell lineages . to date , not many published data support the first and second concepts . in contrast , an increasing body of evidence , including data from human and experimental glomerulonephritis , points towards the third hypothesis [ 5760 ] . in analogy to lineage diversity among t helper effector cells , different corresponding treg subtypes might exist . lineage specificity of these th1- , th2- , or th17-type tregs seems to be achieved by sharing some transcription factors with their respective t helper effector counterpart [ 14 , 6163 ] . th1 specific treg1 cells coexpress the th1 master transcription factor t - bet [ 53 , 60 ] and treg17 cells , targeting th17 responses , rely on coactivation of stat3 [ 52 , 57 , 58 ] . finally , very recent data suggest existence of a third and independent t cell lineage , different from t helper effector cells and conventional tregs , which coexpresses the unusual combination of the treg master transcription factor foxp3 with the th17 characteristic rort [ 17 , 54 , 64 , 65 ] . the following paragraphs will summarize available data on treg diversification and their subphenotypes and stability in gn . this prompted the question whether stat3 and foxp3 double positive cells might be tregs transdifferentiating into th17 cells or even bifunctional treg / th17 hybrids . if either of these concepts was true , preventing stat3 activation in tregs should result in reduced th17 immunity . however , this was not the case . in contrast , specific deletion of stat3 in tregs surprisingly resulted in spontaneous exacerbation of th17 immunity and development of fatal colitis . this indicated control of th17 responses , specifically by a subset of tregs , which coexpresses stat3 . furthermore , this finding supported previously unrecognized treg heterogeneity , rather than instability or transdifferentiation . since nothing was known about the role of these th17 specific stat3 dependent treg17 cells in inflammatory diseases , our group studied their function in experimental gn . in line with the data by chaudhry et al . , we found that loss of stat3 in tregs significantly enhanced type-17 immunity and aggravated renal disease in models of acute crescentic gn and chronically developing systemic lupus erythematosus . importantly , the enhanced levels of il-17 were not a result of cytokine production by tregs but rather by foxp3 negative th17 cells . furthermore , treg numbers and percentages in blood and spleens were not reduced , indicating preserved treg stability . interestingly , however , we found significantly impaired treg accumulation in the inflamed kidneys . as an underlying mechanism this trafficking receptor is characteristically found on th17 cells and mediates their infiltration into inflamed kidneys . stat3 mediated expression of ccr6 on tregs therefore enables their trafficking into areas of th17 inflammation and facilitates close cell contacts which optimizes direct immunosuppression . studies in humans confirmed close colocalization of ccr6 positive tregs with ccr6 positive th17 cells in kidneys of patients with anca - associated gn . furthermore , analyses of blood from patients with hyper - ige syndrome ( hies ) , caused by dominant negative stat3 mutations , showed normal treg percentages but significant reduction of treg expressed ccr6 . collectively , these observations indicate that stat3 activation , both in mice and in humans , does not reflect treg reprogramming or instability but rather specialization for counterregulating th17 immunity . similar to activation of the th17 characteristic transcription factor stat3 , pioneering studies by koch et al . have noted coexpression of foxp3 with the th1 master inducer t - bet . again , the question arose whether this might represent transdifferentiation of tregs into th1 cells . this hypothesis was supported by the observation that t - bet tregs express large amounts of the th1-type cytokine ifn. however , the opposite was the case . elaborate transfer studies , using tregs from t - bet pan knockout mice , showed that t - bet confers tregs with the capacity to effectively downregulate type-1 immunity . absence of treg1 cells resulted in overshooting th1 responses , underlining their regulatory rather than proinflammatory function . in analogy to the shared expression of ccr6 by stat3 positive th17 and treg17 cells , t - bet positive th1 and treg1 cells were shown to share the chemokine receptor cxcr3 , which facilitates their colocalization . importantly , treg1 cells arose from t - bet negative uncommitted tregs , which activated t - bet during inflammation and not from transient promiscuous upregulation of foxp3 in th1 cells . taken together , t - bet expressing tregs do not seem to be transdifferentiating intermediates on their way to a th1 phenotype but rather constitute a th1 specialized suppressor population . not much is known regarding treg1 cells in renal disease . our group therefore addressed this question and induced experimental crescentic gn in mice with a treg selective defect in t - bet activation . if t - bet expression in tregs resulted in transdifferentiation towards th1 cells , treg numbers in these mice should increase , while th1 responses should be diminished . however , treg homeostasis was not impaired and systemic treg frequencies were normal . furthermore , instead of reduced numbers of th1 cells , mice with a treg specific defect of t - bet activation developed significantly overshooting th1 immunity and showed worsened glomerular disease . these observations clearly refute the concept of treg / th1 transdifferentiation and support the view of t - bet tregs as effector treg population , specialized for the control of pathogenic th1-type inflammation . taken together the available data from studies of gn suggest treg lineage heterogeneity and specialization during organ inflammation rather than instability or plasticity . the observation that tregs can coactivate certain transcription factors of t helper effector cell lineages has led to the discovery of cells , expressing foxp3 together with the th17 master transcription factor rort . these rort tregs were found to be present not only in mice but also in healthy humans as well as in many inflammatory conditions [ 6668 ] . several authors had suggested before that some treg subpopulations might transdifferentiate into th17 cells , making cells that coexpress foxp3 with rort likely candidates [ 29 , 3740 , 48 ] . our group therefore decided to address this open question and studied origin , function , and plasticity of rortfoxp3 t cells . during different models of gn , we found that rort tregs massively expanded systemically and also in the nephritic kidneys , early during the course of inflammation . interestingly , analysis of the rort tregs cytokine profile revealed production of both , treg characteristic cytokines tgf- , il-35/ebi-3 , and il-10 and the th17 hallmark cytokine il-17a . since this observation indeed suggested that rort tregs are cells undergoing transdifferentiation , either from th17 into tregs or the other way around , we performed transfer studies . transfer of highly purified rortfoxp3 th17 cells or rort foxp3 conventional tregs into congenic lymphocyte intact recipients revealed that none of the single transcription factor positive cells upregulated the other transcription factor during the course of experimental gn . this observation surprisingly indicated that rort tregs did not derive from treg / th17 transdifferentiation but rather represent an independent cell lineage . however , a second hypothesis , explaining the origin of rort tregs , might be transient and promiscuous upregulation of foxp3 during early activation of th17 cells [ 49 , 55 , 56 ] . in order to test this possibility , we generated fate reporter mice , in which cells are permanently marked , once they have upregulated foxp3 at any state of development . antigen challenge of these mice , however , showed that virtually all cells , which had activated foxp3 , indeed remained foxp3 positive during the following activation period of 7 days . we could thus exclude transient foxp3 upregulation in rort th17 cells , as event leading to generation of rortfoxp3 tregs . a third possible scenario , underlying the origin of rort tregs , however , remained . since rort is a downstream target of stat3 , we wanted to evaluate whether rort tregs might possibly resemble treg17 cells , which we had previously shown to be induced by activation of stat3 [ 57 , 58 ] . analysis of mice with a treg specific deficiency of stat3 activation , however , showed unaltered rort expression in tregs in both spleens and nephritic kidneys , excluding this possibility and further underlining the independent nature of rort tregs . next , we decided to generate mice with selectively impaired rort activation in foxp3 tregs . if rort tregs were treg to th17 transdifferentiating cells , this transdifferentiation would be impaired in these mice and they should progressively accumulate tregs , while th17 cell numbers should decline over time . in case that bi - tregs resembled treg17 cells , th17 responses should be overshooting in mice with deficient rort activation in tregs . analyses at different time points in different models of gn , however , revealed unchanged treg and th17 cell homeostasis . again , this observation indicated an independent nature of rort tregs , with no signs of th17 transdifferentiation or treg17 specialization . finally , we wanted to study the direct fate of rort tregs . for this purpose , they were highly purified by flow cytometric sorting and subsequently transferred into congenic recipient mice . 10 days after induction of gn , the transferred cells were reanalyzed in spleens and nephritic kidneys . our results showed that the transferred rort tregs had massively expanded in both organs during inflammation . much to our surprise , the vast majority had lost both transcription factors , rort and foxp3 , to become double negative ( ex - rort tregs ) . importantly , however , we did not observe relevant transdifferentiation of rort tregs into either rortfoxp3 th17 cells or conventional rortfoxp3 tregs , leaving the fate of ex - rort tregs unknown . taken together , our studies thus support the following concepts : ( 1 ) rort tregs are not tregs transdifferentiating into th17 cells , ( 2 ) rort tregs are not th17 cells on their way to become tregs , ( 3 ) rort tregs are not th17 cells transiently expressing foxp3 during activation , and ( 4 ) rort tregs do not resemble th17 specific treg17 cells . rather , they represent an independent effector treg lineage , which rapidly expands during inflammation and subsequently retracts by as to yet unknown mechanisms . based on currently available data , we thus propose the concept outlined in figure 3 . the independent nature of rort tregs has recently been confirmed by three highly ranked studies [ 64 , 65 , 69 ] . after transfer into lymphopenic rag mice , rort tregs showed a high degree of stability with no relevant transdifferentiation into th17 cells or conventional tregs . with respect to the function of rort tregs , concepts are , however , still evolving . our studies have shown that exogenously transferred rort tregs were highly protective in a model of acute gn . however , endogenous rort tregs had additional proinflammatory functions . selective inhibition of rort activation in tregs resulted in complete abrogation of their il-17 production and ameliorated renal tissue damage in acute gn . in addition , the recent landmark report by ohnmacht et al . surprisingly showed that rort expression in tregs is crucial for suppression of th2 immunity . our own previous work in a model of acute crescentic gn , and unpublished data using the pristane model of murine sle , strongly supports this observation . rort tregs might thus be potent regulators of th2 responses and could be important to protect from allergies . in summary , foxp3 and rort coexpressing tregs are not treg / th17 transdifferentiating or th17 specialized treg17 cells but represent a unique , stable , and independent t cell lineage with both regulatory and proinflammatory functions . a thorough understanding of cd4 t cell plasticity is of high clinical relevance in the light of newly emerging t helper cell directed therapies . while th1 cells display a relatively high degree of stability in gn , th17 cells may have different fates . in several experimental models of nonrenal inflammatory disease , most th17 cells transdifferentiate into a th1 phenotype . in gn , however , a significant proportion of th17 cells seems to be stable and maintains the differentiation status . another fraction , in contrast , appears to undergo functional changes and loses th17 characteristics . nevertheless , currently available data do not support th17 conversion into th1 , th2 , or treg cells during the course of gn , leaving the nature of ex - th17 cells unknown . interestingly , however , th17 cells can be pushed into a tolerogenic phenotype by anti - cd3 treatment , which represents a promising therapeutic strategy . in the case of tregs , data from gn underline a very high degree of stability and show no hint for transdifferentiation . rather , a new paradigm is emerging , suggesting activation and diversification of naive tregs into different effector treg lineages as rort tregs , t - bet treg1 , and stat3 treg17 cells .

multiple studies have identified cd4 + t cells as central players of glomerulonephritis ( gn ) . cells of the th1 and th17 responses cause renal tissue damage , while tregs mediate protection . recently , a high degree of plasticity among these t cell lineages was proposed . during inflammation , th17 cells were shown to have the potential of transdifferentiation into th1 , th2 , or alternatively anti - inflammatory tr1 cells . currently available data from studies in gn , however , do not indicate relevant th17 to th1 or th2 conversion , leaving the th17 cell fate enigmatic . tregs , on the other hand , were speculated to transdifferentiate into th17 cells . again , data from gn do not support this concept . rather , it seems that previously unrecognized subspecialized effector treg lineages exist . these include th1 specific treg1 as well as th17 directed treg17 cells . furthermore , a bifunctional treg subpopulation was recently identified in gn , which secrets il-17 and coexpresses foxp3 together with the th17 characteristic transcription factor rort . similarities between these different and highly specialized effector treg subpopulations with the corresponding t helper effector cell lineages might have resulted in previous misinterpretation as treg transdifferentiation . in summary , th17 cells have a relatively stable phenotype during gn , while , in the case of tregs , currently available data suggest lineage heterogeneity rather than plasticity .

1. Introduction 2. Concept of CD4 3. The Fate of Th17 Cells in Glomerulonephritis 4. Treg Stability: A Matter of Debate 5. Lineage Specific Tregs: One Size Does Not Fit All 6. ROR 7. Conclusions

these include primary forms , for example , iga nephropathy or membranous gn , as well as secondary forms developing as a consequence of systemic diseases as lupus nephritis and anca - associated vasculitides . therefore , in most forms of gn , a pronounced renal glomerular and often also tubulointerstitial inflammatory cell infiltrate is found . numerous studies from the past have shown that cd4 t cells are crucial mediators of most forms of gn . it has been shown that especially cells of the th1 and th17 responses are highly nephritogenic [ 38 ] . dysregulated systemic th1 and th17 immunity in addition , both t helper cell lineages are important mediators of local renal tissue injury as well [ 911 ] . in contrast to pathogenic th1 and th17 responses , regulatory t cells ( tregs ) were proven to be potent anti - inflammatory players in gn . several studies of the past have highlighted their protective effects [ 7 , 1214 ] . given the central roles of t helper effector and t regulatory cells in gn , interestingly , increasing evidence suggests that systemic expansion and renal infiltration of different cd4 t cell subtypes follow a concerted time course . after rapid renal and systemic expansion , their percentages , however , steadily decrease over time and often decline to reach baseline levels . cells of th1 polarization , in contrast , occur later during renal inflammation . finally , tregs were shown to steadily expand in a continuous process until a stable equilibrium with their proinflammatory th1 and th17 counterparts is established [ 12 , 17 ] . this defined time course of renal infiltration , which is initially dominated by th17 cells , followed by th1 cells and tregs at later stages , has led to speculations of possible transdifferentiation of th17 cells into another cell type . one possibility , which has been suggested , is reprogramming of th17 cells to acquire a th1 phenotype [ 18 , 19 ] . alternatively , th17 cells could lose their pathogenic properties and might be reprogrammed to become foxp3 tregs . in vitro approaches as well as transfer experiments using highly purified populations of cd4 t cell subsets have established the view that cd4 t cells can change their polarity under certain conditions [ 18 , 2026 ] . to follow the fate of single cd4 t cells , lineage - tracing systems using cre - recombinase expression under the control of key cytokines or transcription factors and subsequent permanent fluorochrome expression have been established [ 19 , 2729 ] . mice overcome technical limitations in single cell tracing , which were present in transfer experiments using highly purified or even bulk populations of in vitro polarized t cell subsets . have established the concept that encephalitogenic th17 cells have a high degree of plasticity into the th1 phenotype in experimental autoimmune encephalomyelitis ( eae ) , the mouse model for multiple sclerosis . furthermore , studies in these mice have revealed that , in specialized environments , namely , intestinal peyer 's patches , th17 cells potentially develop into t follicular helper cells ( tfh ) and drive antigen - specific iga responses in germinal center b cells . moreover , regulatory type 1 cells ( tr1 ) , an intriguing t cell subtype with potent immunosuppressive properties , have only recently been recognized as important players in intestinal inflammation . accumulating evidence suggests that , upon the right stimuli , th17 cells can transdifferentiate to acquire the ability of il-10 secretion and become cells with a tr1 phenotype . a high degree of heterogeneity within certain t cell subsets was also apparent in studies that performed single cell sequencing of th17 cells from eae and from in vitro culture [ 32 , 33 ] . plasticity of human cd4 t cells , on the other hand , can be addressed by using t cell receptors ( tcr ) as an endogenous barcoding system . studies that focus on plasticity of human cd4 t cells have been reviewed recently in detail by dupage and bluestone . in summary , increasing data suggest instability or plasticity , especially , of th17 cells . however , to complicate things , numerous studies have also postulated a diametrically opposite concept ; namely , th17 cells might derive from transdifferentiation of foxp3 tregs [ 29 , 3740 ] . given the high nephritogenic potential of th17 cells [ 6 , 41 ] , their plasticity in renal autoimmune disease is of great clinical interest . two opposing fates have been proposed : transdifferentiation into th1 cells or alternatively into anti - inflammatory tr1 cells . on the other hand , blockade of th17 cell development might also interfere with generation of regulatory t cell subsets and thus impede resolution of tissue injury . however , until now only limited data have been published on the potential plasticity of th17 cells in glomerulonephritis . in a previous study , we have transferred in vitro polarized th1 and th17 cells into t cell deficient rag1 knockout mice and analyzed the pathogenicity of these cell types in a planted - antigen model of gn . in contrast , some ifn was also produced by spleen cells after the transfer of th17 cells , indicating that some th17 cells might have adopted a th1 phenotype . it is , however , important to note that t cell pathogenicity rather than plasticity was the primary focus of this study . as a result , in particular , the in vitro polarized th17 cells contained a relevant fraction of ifn producing th1 cells even before transfer , which clearly limits analysis . furthermore , only systemic but not organ specific t cell responses in the kidney were addressed . in summary , this study indicates stability of splenic th1 cells , without significant th1 to th17 or th2 plasticity but suggests some degree of th17 cell transdifferentiation into cells of the th1 type during gn . these authors evaluated cytokine expression of splenic and renal t cells after transfer and found somewhat lower th17 cell frequencies than expected . these findings therefore indicate partial loss of the th17 effector phenotype but do not support significant th17 to th1 transdifferentiation . in a recent study after transfer of highly purified in vitro polarized th17 cells from fluorescence reporter mice and subsequent induction of crescentic glomerulonephritis , reanalysis of t cells from the kidney displayed a relatively high degree of stability . importantly , in these studies , no relevant transdifferentiation into th1 or th2 cells was detected among ex - th17 cells , leaving their fate unknown . in this context , it is important to note that th17 cells have been reported to have a high rate of instability and conversion into th1 cells in nonrenal models of autoimmune diseases . we thus treated mice with a monoclonal anti - cd3 antibody , which resulted in induction of a tolerogenic phenotype , characterized by il-10 coexpression , in otherwise stable renal th17 cells . the great therapeutic potential of this finding for the treatment of renal autoimmune diseases clearly warrants further exploration . since renal th17 cells do not seem to acquire either th1 or th2 phenotypes , an alternative scenario would be th17 transdifferentiation into anti - inflammatory foxp3 tregs . however , available data from renal disease do not support this notion either . interestingly , flow cytometry of splenocytes revealed persistent absence of foxp3 cells at the end of the experiment . this finding indicates that there is no transdifferentiation of non - treg t cell subsets , including th17 cells , into foxp3 tregs in this model . furthermore , in another study , we transferred highly purified th17 cells into lymphocyte intact mice and traced their fate during gn , using a congenic marker system . importantly , though , none of these ex - th17 cells had upregulated foxp3 in kidneys or spleens , which excludes transdifferentiation into tregs . in summary , increasing evidence supports some degree of th17 instability during gn , which , however , does not lead to generation of th1 cells or tregs . on possible scenario would thus be that th17 cells adopt a tr1 phenotype as has recently been suggested . another much discussed aspect of cd4 t cell plasticity is the hypothesis that foxp3 tregs might possess the capacity to transdifferentiate into t effector cells . this notion was supported by early experiments , in which tregs were highly purified from foxp3 fluorescence reporter mouse strains and transferred into different t cell deficient recipients . surprisingly , around half of the transferred tregs had lost foxp3 expression , as well as other treg hallmark molecules as cd25 and ctla-4 . assessment of their function revealed loss of suppressive capacity , and even more suggestive of transdifferentiation , these ex - tregs had started to produce inflammatory cytokines as il-17 and ifn. further rounds of experiments , however , made clear that this observed high degree of instability was overestimated due to the lymphopenic environment in the recipient mice . when tregs were cotransferred with t helper effector cells or transferred into lymphocyte intact recipients , only few tregs lost their phenotype [ 37 , 40 , 47 ] . these studies from different groups consistently reported a high degree of treg stability with , however , minor populations of between 5 and 10% showing loss of foxp3 [ 29 , 4850 ] . while most tregs seem to stably maintain foxp3 expression , substantial fractions were found to coexpress master transcription factors and key cytokines of t helper effector cell lineages [ 5254 ] . four possible scenarios regarding these t cells with a hybrid phenotype are currently under debate . secondly alternatively , tregs might be upregulating classical t helper effector cell transcription factors to gain certain functional characteristics and , last but not least , they could be independent and previously unrecognized t cell lineages . in analogy to lineage diversity among t helper effector cells , different corresponding treg subtypes might exist . lineage specificity of these th1- , th2- , or th17-type tregs seems to be achieved by sharing some transcription factors with their respective t helper effector counterpart [ 14 , 6163 ] . th1 specific treg1 cells coexpress the th1 master transcription factor t - bet [ 53 , 60 ] and treg17 cells , targeting th17 responses , rely on coactivation of stat3 [ 52 , 57 , 58 ] . finally , very recent data suggest existence of a third and independent t cell lineage , different from t helper effector cells and conventional tregs , which coexpresses the unusual combination of the treg master transcription factor foxp3 with the th17 characteristic rort [ 17 , 54 , 64 , 65 ] . the following paragraphs will summarize available data on treg diversification and their subphenotypes and stability in gn . this prompted the question whether stat3 and foxp3 double positive cells might be tregs transdifferentiating into th17 cells or even bifunctional treg / th17 hybrids . however , this was not the case . in contrast , specific deletion of stat3 in tregs surprisingly resulted in spontaneous exacerbation of th17 immunity and development of fatal colitis . this indicated control of th17 responses , specifically by a subset of tregs , which coexpresses stat3 . furthermore , this finding supported previously unrecognized treg heterogeneity , rather than instability or transdifferentiation . in line with the data by chaudhry et al . importantly , the enhanced levels of il-17 were not a result of cytokine production by tregs but rather by foxp3 negative th17 cells . interestingly , however , we found significantly impaired treg accumulation in the inflamed kidneys . as an underlying mechanism this trafficking receptor is characteristically found on th17 cells and mediates their infiltration into inflamed kidneys . studies in humans confirmed close colocalization of ccr6 positive tregs with ccr6 positive th17 cells in kidneys of patients with anca - associated gn . similar to activation of the th17 characteristic transcription factor stat3 , pioneering studies by koch et al . have noted coexpression of foxp3 with the th1 master inducer t - bet . again , the question arose whether this might represent transdifferentiation of tregs into th1 cells . however , the opposite was the case . elaborate transfer studies , using tregs from t - bet pan knockout mice , showed that t - bet confers tregs with the capacity to effectively downregulate type-1 immunity . absence of treg1 cells resulted in overshooting th1 responses , underlining their regulatory rather than proinflammatory function . in analogy to the shared expression of ccr6 by stat3 positive th17 and treg17 cells , t - bet positive th1 and treg1 cells were shown to share the chemokine receptor cxcr3 , which facilitates their colocalization . importantly , treg1 cells arose from t - bet negative uncommitted tregs , which activated t - bet during inflammation and not from transient promiscuous upregulation of foxp3 in th1 cells . taken together , t - bet expressing tregs do not seem to be transdifferentiating intermediates on their way to a th1 phenotype but rather constitute a th1 specialized suppressor population . if t - bet expression in tregs resulted in transdifferentiation towards th1 cells , treg numbers in these mice should increase , while th1 responses should be diminished . furthermore , instead of reduced numbers of th1 cells , mice with a treg specific defect of t - bet activation developed significantly overshooting th1 immunity and showed worsened glomerular disease . taken together the available data from studies of gn suggest treg lineage heterogeneity and specialization during organ inflammation rather than instability or plasticity . the observation that tregs can coactivate certain transcription factors of t helper effector cell lineages has led to the discovery of cells , expressing foxp3 together with the th17 master transcription factor rort . these rort tregs were found to be present not only in mice but also in healthy humans as well as in many inflammatory conditions [ 6668 ] . several authors had suggested before that some treg subpopulations might transdifferentiate into th17 cells , making cells that coexpress foxp3 with rort likely candidates [ 29 , 3740 , 48 ] . our group therefore decided to address this open question and studied origin , function , and plasticity of rortfoxp3 t cells . during different models of gn , we found that rort tregs massively expanded systemically and also in the nephritic kidneys , early during the course of inflammation . interestingly , analysis of the rort tregs cytokine profile revealed production of both , treg characteristic cytokines tgf- , il-35/ebi-3 , and il-10 and the th17 hallmark cytokine il-17a . since this observation indeed suggested that rort tregs are cells undergoing transdifferentiation , either from th17 into tregs or the other way around , we performed transfer studies . transfer of highly purified rortfoxp3 th17 cells or rort foxp3 conventional tregs into congenic lymphocyte intact recipients revealed that none of the single transcription factor positive cells upregulated the other transcription factor during the course of experimental gn . however , a second hypothesis , explaining the origin of rort tregs , might be transient and promiscuous upregulation of foxp3 during early activation of th17 cells [ 49 , 55 , 56 ] . antigen challenge of these mice , however , showed that virtually all cells , which had activated foxp3 , indeed remained foxp3 positive during the following activation period of 7 days . a third possible scenario , underlying the origin of rort tregs , however , remained . since rort is a downstream target of stat3 , we wanted to evaluate whether rort tregs might possibly resemble treg17 cells , which we had previously shown to be induced by activation of stat3 [ 57 , 58 ] . analysis of mice with a treg specific deficiency of stat3 activation , however , showed unaltered rort expression in tregs in both spleens and nephritic kidneys , excluding this possibility and further underlining the independent nature of rort tregs . if rort tregs were treg to th17 transdifferentiating cells , this transdifferentiation would be impaired in these mice and they should progressively accumulate tregs , while th17 cell numbers should decline over time . in case that bi - tregs resembled treg17 cells , th17 responses should be overshooting in mice with deficient rort activation in tregs . analyses at different time points in different models of gn , however , revealed unchanged treg and th17 cell homeostasis . again , this observation indicated an independent nature of rort tregs , with no signs of th17 transdifferentiation or treg17 specialization . 10 days after induction of gn , the transferred cells were reanalyzed in spleens and nephritic kidneys . importantly , however , we did not observe relevant transdifferentiation of rort tregs into either rortfoxp3 th17 cells or conventional rortfoxp3 tregs , leaving the fate of ex - rort tregs unknown . taken together , our studies thus support the following concepts : ( 1 ) rort tregs are not tregs transdifferentiating into th17 cells , ( 2 ) rort tregs are not th17 cells on their way to become tregs , ( 3 ) rort tregs are not th17 cells transiently expressing foxp3 during activation , and ( 4 ) rort tregs do not resemble th17 specific treg17 cells . rather , they represent an independent effector treg lineage , which rapidly expands during inflammation and subsequently retracts by as to yet unknown mechanisms . based on currently available data , we thus propose the concept outlined in figure 3 . after transfer into lymphopenic rag mice , rort tregs showed a high degree of stability with no relevant transdifferentiation into th17 cells or conventional tregs . with respect to the function of rort tregs , concepts are , however , still evolving . selective inhibition of rort activation in tregs resulted in complete abrogation of their il-17 production and ameliorated renal tissue damage in acute gn . in summary , foxp3 and rort coexpressing tregs are not treg / th17 transdifferentiating or th17 specialized treg17 cells but represent a unique , stable , and independent t cell lineage with both regulatory and proinflammatory functions . a thorough understanding of cd4 t cell plasticity is of high clinical relevance in the light of newly emerging t helper cell directed therapies . while th1 cells display a relatively high degree of stability in gn , th17 cells may have different fates . in several experimental models of nonrenal inflammatory disease , most th17 cells transdifferentiate into a th1 phenotype . in gn , however , a significant proportion of th17 cells seems to be stable and maintains the differentiation status . nevertheless , currently available data do not support th17 conversion into th1 , th2 , or treg cells during the course of gn , leaving the nature of ex - th17 cells unknown . interestingly , however , th17 cells can be pushed into a tolerogenic phenotype by anti - cd3 treatment , which represents a promising therapeutic strategy . in the case of tregs , data from gn underline a very high degree of stability and show no hint for transdifferentiation . rather , a new paradigm is emerging , suggesting activation and diversification of naive tregs into different effector treg lineages as rort tregs , t - bet treg1 , and stat3 treg17 cells .

decompressive craniectomy is very effective method of reducing intracranial pressure . in a recent prospective clinical trial , decompressive craniectomy was confirmed to have reduced the mortality by 50% in the patients with cerebral edema caused by cerebral infarction17,23,42,43 ) . in addition , it was effective for cerebral edema caused by traumatic brain damage2,19 ) , subarachnoid hemorrhage10,16 ) , intracerebral hemorrhage29 ) , and cranial venous and sinus thromboses7 ) . cranioplasty is performed after craniectomy when intracranial pressure is under control for functional and aesthetic restorations . during the cranioplasty procedure , autologous bone flaps are preferably used due to their advantages in storage , viability , cost , prevention of disease transmission , and aesthetics . however , cranioplasty that uses the autologous bone flap has a risk of developing complications such as infection , intracranial hemorrhage , seizure , and hydrocephalus . long - term complications , such as bone flap resorption and bone non - union , have incidence rates that reached 2 - 17%18,21,33 ) . according to the studies on human cranioplasty , the timing of performing cranioplasty is one of the critical factors that may develop autologous bone flap resorption and bone non - union15 ) . although generally accepted concept about timing of cranioplasty using autologous bone is that early cranioplasty has more risk of infection and delayed cranioplasty has risk of non - union or resoprtion of bone flap4,6,25,44 ) , the debates are still remained . another report showed non - union and/or resorption of bone flap occurred more frequently on early cranioplasty patients who had surgery - related infections35 ) . and the other said there were no relation of bone non - union to timing of cranioplasty32 ) . in this study , cranioplasty was performed on rabbits using frozen autologous bone flaps , while the levels of new bone formation and bone fusion after craniectomy were observed , according to the timing of cranioplasty to evaluate its efficacy . experiments were conducted in compliance with the animal experimentation ethics upon approval from the institutional animal care and use committee of our institute . eight adult ( 16 weeks or older ) male new zealand white rabbits with the body weights ranging from 3.2 kg to 4.2 kg were used in this study . four out of eight subjects were assigned to the delayed cranioplasty group ( dg ) , and the remaining four subjects were assigned to the early cranioplasty group ( eg ) . at day 0 , craniectomy was performed at the right frontoparietal bone of the dg rabbit , and the obtained bone flap was stored in a freezer at a temperature of -80. at day 28 or week 4 , bone defect was made in the right frontoparietal bone of the eg rabbit , and then , the bone flap was stored at the same condition . at day 56 or week 8 , the frozen bone flap was fixed on the bone defect area of the dg and eg rabbits . in order to obtain control data , the same procedures were made on the left cranial bone of the rabbits , and the bone flap was immediately fixed ( control group , cg ) . at day 112 or week 16 , the eg and dg rabbits were sacrificed to conduct radiological and histological tests on their cranial bones ( fig . the time span between craniectomy and cranioplasty was the healing time of bone defect and defined as " the primary healing time " , while the time between the cranioplasty and rabbit sacrifice was the bone formation time of the gap between bone flap and cranium and defined as " the secondary healing time " . in order to induce anesthesia on the rabbits , 0.5 ml / kg of ketamin hcl ( ketalar , yuhan corporation , seoul , korea ) and 0.25 mg / kg of xylazine hcl ( rompun , bayer , pittsburgh , pa , usa ) were mixed and injected intramuscularly . for local anesthesia and hemorrhage control , 2% lidocaine hcl ( yuhan corporation , seoul , korea ) containing 1 : 100000 epinephrine was injected subcutaneously . after a 4-cm long incision was made along the midline of the cranial skin , the muscles and periosteum were incised layer by layer to expose the cranial bone ( fig . in order to make the bone flap , a bone flap margin was formed 3 mm lateral to the sagittal suture , which was located at the right side for the experimental groups and the left side for the control group , using a trephine drill with a 10-mm outer diameter and 9-mm inner diameter . to avoid damaging the dura mater , using a 1-mm round burr , the space between the round bone - flap margin and the cranial bone was expanded out of the bone flap in order to create a 12-mm bone defect diameter ( fig . the reason why the size of the bone defect was decided at 12 mm was that the critical bone defect , which did not cause a spontaneous bone healing in rabbits , was reported to be 12 mm or larger37 ) . 2e ) , while the 9-mm bone flap was lifted in order to form a bone defect ( fig . the blood and soft tissues of the bone flap were removed , and the flap was wrapped up with gauze soaked sterilized saline . each bone flap was put in a sterilized and labeled plastic container before they were kept in a freezer ( cln-540u , nihon freezer , tokyo , japan ) at a temperature of -80. layer - to - layer suture was performed using absorbable sutures ( 3 - 0 vycryl ) and unabsorbable sutures ( 3 - 0 nylon ) . after anesthesia and skin incision that mentioned above , the fibrous tissues , which were formed during the primary healing time , were removed using a curette in order to expose the bone defect area ( fig . afterwards , the autologous bone flap , which has been kept in the freezer , was applied to each labeled rabbit in order to perform cranioplasty . the cranial bone and the bone flap were fixed using a titanium - alloy miniplate and 3-mm screws ( synthes inc . , , the cranial bone of the sacrificed rabbit was scanned by the micro - computed tomography ( micro - ct ) ( skyscan 1173 , skyscan , kontich , belgium ) . 4a , b ) and the three dimensional reconstructed images ( nrecon reconstruction program , skyscan , kontich , belgium ) ( fig . 4c , d ) were obtained . after confirming the round bone flap through the micro - ct images and 3d remodeling images , we defined the region of interest ( roi ) as the region within a 14-mm diameter from center point of bone flap . we measured the bone volume ( volroi ) and the bone surface ( surroi ) of roi ( fig . 5a ) . from volroi and surroi , the bone volume ( volbone flap ) and the bone surface ( surbone flap ) of the bone flap , and the volume afterwards , the ct scan images ( fig . 5b ) and the 3d reconstruction images ( fig . 5c ) of the new bone volume ( volnew ) and the new bone surface ( surnew ) were quantified and measured . volnew = volroi-(volbone flap+volmetal ) surnew = surroi-(surbone flap+surmetal ) after dehydration processing was completed , the graft was put in a methyl methacrylate solution in order to maintain a vacuum state . after the methyl methacrylate solution was permeated , the tissues were put in a 5 ml of methyl methacrylate solution and 50 l of jb-4 embedding kit component b was added in order to maintain a vacuum state for 10 minutes . nitrogen gas was injected to be activated at room temperature ( 24 - 28 ) for 24 hours and to prepare a plastic block . the tissues were consecutively cut in a 10-m thickness using a hard tissue cutter ( jung polycuts , leica biosystems , nussloch , germany ) . the sliced tissues were stained through the use of hematoxylin and eosin ( h - e ) , while goldner 's modified masson trichrome methods were used for optical microscopic observations . chicago , il , usa ) was used for statistical analyses . as a non - parametric test , kruskal - wallis test was used for the comparison among the dg , eg , and cg . as a post hoc test experiments were conducted in compliance with the animal experimentation ethics upon approval from the institutional animal care and use committee of our institute . eight adult ( 16 weeks or older ) male new zealand white rabbits with the body weights ranging from 3.2 kg to 4.2 kg were used in this study . four out of eight subjects were assigned to the delayed cranioplasty group ( dg ) , and the remaining four subjects were assigned to the early cranioplasty group ( eg ) . at day 0 , craniectomy was performed at the right frontoparietal bone of the dg rabbit , and the obtained bone flap was stored in a freezer at a temperature of -80. at day 28 or week 4 , bone defect was made in the right frontoparietal bone of the eg rabbit , and then , the bone flap was stored at the same condition . at day 56 or week 8 , the frozen bone flap was fixed on the bone defect area of the dg and eg rabbits . in order to obtain control data , the same procedures were made on the left cranial bone of the rabbits , and the bone flap was immediately fixed ( control group , cg ) . at day 112 or week 16 , the eg and dg rabbits were sacrificed to conduct radiological and histological tests on their cranial bones ( fig . the time span between craniectomy and cranioplasty was the healing time of bone defect and defined as " the primary healing time " , while the time between the cranioplasty and rabbit sacrifice was the bone formation time of the gap between bone flap and cranium and defined as " the secondary healing time " . in order to induce anesthesia on the rabbits , 0.5 ml / kg of ketamin hcl ( ketalar , yuhan corporation , seoul , korea ) and 0.25 mg / kg of xylazine hcl ( rompun , bayer , pittsburgh , pa , usa ) were mixed and injected intramuscularly . for local anesthesia and hemorrhage control , 2% lidocaine hcl ( yuhan corporation , seoul , korea ) containing 1 : 100000 epinephrine was injected subcutaneously . after a 4-cm long incision was made along the midline of the cranial skin , the muscles and periosteum were incised layer by layer to expose the cranial bone ( fig . in order to make the bone flap , a bone flap margin was formed 3 mm lateral to the sagittal suture , which was located at the right side for the experimental groups and the left side for the control group , using a trephine drill with a 10-mm outer diameter and 9-mm inner diameter . to avoid damaging the dura mater , the space between the round bone - flap margin and the cranial bone was expanded out of the bone flap in order to create a 12-mm bone defect diameter ( fig . 2d ) . the reason why the size of the bone defect was decided at 12 mm was that the critical bone defect , which did not cause a spontaneous bone healing in rabbits , was reported to be 12 mm or larger37 ) . 2e ) , while the 9-mm bone flap was lifted in order to form a bone defect ( fig . the blood and soft tissues of the bone flap were removed , and the flap was wrapped up with gauze soaked sterilized saline . each bone flap was put in a sterilized and labeled plastic container before they were kept in a freezer ( cln-540u , nihon freezer , tokyo , japan ) at a temperature of -80. layer - to - layer suture was performed using absorbable sutures ( 3 - 0 vycryl ) and unabsorbable sutures ( 3 - 0 nylon ) . after anesthesia and skin incision that mentioned above , the fibrous tissues , which were formed during the primary healing time , were removed using a curette in order to expose the bone defect area ( fig . afterwards , the autologous bone flap , which has been kept in the freezer , was applied to each labeled rabbit in order to perform cranioplasty . the cranial bone and the bone flap were fixed using a titanium - alloy miniplate and 3-mm screws ( synthes inc . , in order to induce anesthesia on the rabbits , 0.5 ml / kg of ketamin hcl ( ketalar , yuhan corporation , seoul , korea ) and 0.25 mg / kg of xylazine hcl ( rompun , bayer , pittsburgh , pa , usa ) were mixed and injected intramuscularly . for local anesthesia and hemorrhage control , 2% lidocaine hcl ( yuhan corporation , seoul , korea ) containing 1 : 100000 epinephrine was injected subcutaneously . after a 4-cm long incision was made along the midline of the cranial skin , the muscles and periosteum were incised layer by layer to expose the cranial bone ( fig . in order to make the bone flap , a bone flap margin was formed 3 mm lateral to the sagittal suture , which was located at the right side for the experimental groups and the left side for the control group , using a trephine drill with a 10-mm outer diameter and 9-mm inner diameter . to avoid damaging the dura mater , the space between the round bone - flap margin and the cranial bone was expanded out of the bone flap in order to create a 12-mm bone defect diameter ( fig . 2d ) . the reason why the size of the bone defect was decided at 12 mm was that the critical bone defect , which did not cause a spontaneous bone healing in rabbits , was reported to be 12 mm or larger37 ) . 2e ) , while the 9-mm bone flap was lifted in order to form a bone defect ( fig . the blood and soft tissues of the bone flap were removed , and the flap was wrapped up with gauze soaked sterilized saline . each bone flap was put in a sterilized and labeled plastic container before they were kept in a freezer ( cln-540u , nihon freezer , tokyo , japan ) at a temperature of -80. layer - to - layer suture was performed using absorbable sutures ( 3 - 0 vycryl ) and unabsorbable sutures ( 3 - 0 nylon ) . after anesthesia and skin incision that mentioned above , the fibrous tissues , which were formed during the primary healing time , were removed using a curette in order to expose the bone defect area ( fig . afterwards , the autologous bone flap , which has been kept in the freezer , was applied to each labeled rabbit in order to perform cranioplasty . the cranial bone and the bone flap were fixed using a titanium - alloy miniplate and 3-mm screws ( synthes inc . , west chester , pa , usa ) ( fig . at week 16 , the cranial bone of the sacrificed rabbit was scanned by the micro - computed tomography ( micro - ct ) ( skyscan 1173 , skyscan , kontich , belgium ) . 4a , b ) and the three dimensional reconstructed images ( nrecon reconstruction program , skyscan , kontich , belgium ) ( fig . 4c , d ) were obtained . after confirming the round bone flap through the micro - ct images and 3d remodeling images , we defined the region of interest ( roi ) as the region within a 14-mm diameter from center point of bone flap . we measured the bone volume ( volroi ) and the bone surface ( surroi ) of roi ( fig . 5a ) . from volroi and surroi , the bone volume ( volbone flap ) and the bone surface ( surbone flap ) of the bone flap , and the volume afterwards , the ct scan images ( fig . 5b ) and the 3d reconstruction images ( fig . 5c ) of the new bone volume ( volnew ) and the new bone surface ( surnew ) were quantified and measured . after dehydration processing was completed , the graft was put in a methyl methacrylate solution in order to maintain a vacuum state . the tissues were put in a 5 ml of methyl methacrylate solution and 50 l of jb-4 embedding kit component b was added in order to maintain a vacuum state for 10 minutes . nitrogen gas was injected to be activated at room temperature ( 24 - 28 ) for 24 hours and to prepare a plastic block . the tissues were consecutively cut in a 10-m thickness using a hard tissue cutter ( jung polycuts , leica biosystems , nussloch , germany ) . the sliced tissues were stained through the use of hematoxylin and eosin ( h - e ) , while goldner 's modified masson trichrome methods were used for optical microscopic observations . chicago , il , usa ) was used for statistical analyses . as a non - parametric test , kruskal - wallis test was used for the comparison among the dg , eg , and cg . as a post hoc test no subject died or experienced complications such as infection , and none of them dropped out of the experiments conducted in this study . the new bone formation observed in the micro - ct images was represented as bone volume ( volnew ) and bone surface ( surnew ) ( table 1 ) . the mean new bone volume of the dg was 53.7810.86 mm , while the mean bone surface was 726.60170.99 mm . the mean new bone volume and mean bone surface of the eg were 50.137.18 mm and 706.2377.26 mm , respectively . the mean new bone volume of the cg was 31.5112.84 mm , while the mean bone surface was 436.65132.24 mm . statistically significant differences were observed in the mean new bone volume ( p=0.024 ) and bone surface ( p=0.007 ) among the three groups ( table 2 ) . in the post hoc test , no statistically significant difference in bone volume ( p=0.886 ) and bone surface ( p=0.886 ) was observed between the dg and eg . when the cg was compared with the eg and dg , significant differences in bone volume ( p=0.028 ) and bone surface ( p=0.008 ) similar to the findings in the micro - ct images , new bone formation was observed in the microscopic examination of the h - e stain and goldner 's stain in all of the rabbit tissues . the new bone formation was observed as a form of 1 ) a bony islet between the bone flap and the edge of the cranial bone ( fig . 6a ) , 2 ) a bony islet at the lower part of the bone flap ( fig . 6b ) , 3 ) a new bone formation at the edge of the cranial bone ( fig . 6c ) , and 4 ) a bone flap incorporation between the bone flap and the edge of the cranial bone ( fig . cranioplasty , which is performed after craniectomy , has aesthetic and functional advantages , and it is used in a wide variety of diseases . however , various complications such as infection , intracranial hemorrhage , bone flap resorption , depressed bone flap , cerebral vasospasm , and hydrocephalus can develop after the cranioplasty procedure , and their incidence rate has been reported to be 15 - 36.5%4,5,8,14,20,27,32,36 ) . the materials that are most frequently used in cranioplasty include autologous bone flap , poly - methyl - methacrylate ( pmma ) , and hydroxy apatite . the pmma is known to show a similar frequency of complication development to that of the autologous bone flap , but its osteogenesis capability is much less than that of the autologous bone flap22,24 ) . hydroxy apatite can supplement the low osteogenesis capability of pmma ; however , it is expensive38 ) . the autologous bone flap is the most widely used cranioplasty material due to its superiority in the viability , cost , prevention of disease tranmission , and aesthetics to artificial bones . nevertheless , long - term complications such as bone non - union and resorption , which develop after the cranioplasty procedure using the autologous bone flap , are the major causes that require re - operation . in a cranioplasty using autologous bone flap , bone flap incorporation occurs in the process of revascularization , osteoconduction , resorption , and osteogenesis12 ) . in a bone union process , bone remodeling is completed through the repetition of bone resorption and new bone formation . bone resorption is known to happen when problems arise during the bone reunion process39 ) . the patient - side risk factors of bone resorption are known to include young age , traumatic cranial damage , multiple cranial fractures , and the size of the cranial defect . meanwhile , iatrogenic factors , which include the timing of cranioplasty , have been reported13,15,21,40 ) . according to previous studies conducted on the timing of cranioplasty , its early performance resulted in an increase in the incidence of infection , while delayed performance resulted in an increase in the resorption of the autologous bone flap4,6,25,35,44 ) . however , piedra et al.32 ) reported that there is no difference in the frequency of the development of complications , such as bone non - union and resorption , and in the infection between the early and delayed cranioplasty performance groups . in the case of cranioplasty performed within two months after craniectomy , schuss et al.35 ) reported a high frequency of infection , and resultant bone non - union and resorption . in this study , the timing of cranioplasty was set at 4 and 8 weeks with the eg and dg , and no significant difference in new bone formation was observed between the groups . furthermore , when the frozen autologous bone flap was used on rabbits within 8 weeks , an active new bone formation was radiologically and histologically confirmed , and no significant difference in new bone formation was observed according to the freezing period . there are several methods of storing the autologous cranial bone during the period between the procedures of craniectomy and cranioplasty . the method of freezing the autologous bone flap was introduced in the 1950s1,11,30 ) , and since then , it has been the most widely used method . the most important factor to consider for a successful bone flap incorporation after cranioplasty is the viability of the bone flap9,28,34 ) . furthermore , the frozen autologous bone flap is known to have a comparatively favorable viability . when the frozen autologous bone flap was histologically analyzed at temperatures between -17 and -80 , the harvesian system of the bone tissues and structural proteins were confirmed to have remained the same regardless of the freezing period3,31,34 ) , and the osteocytes were observed even until the 35th month after freezing34 ) . moreover , the freezing storage method is superior to the other methods in terms of preserving the cellular architecture30,34 ) . an example of this is the cytoplasm of the lacunar cells , which was well preserved41 ) . in the frozen cranial bone , new blood vessels are remodeled , and they play the role of an architectural frame that assists the growth of the osteoprogenitor cells . in addition , revascularization and infiltration of the osteoblasts occur from the edge of the cranial bone toward the bone flap34 ) . a new bone formation was observed at the edge of the cranial bone close to the bone flaps of all the dg and eg rabbits not only in the micro - ct , but also in the histological results . a bony islet formation was also observed . according to sultan et al.41 ) , the bony islet formation contributed to the promotion of bone flap incorporation and the reduction of bone flap resorption . when cranioplasty is performed immediately after craniectomy , the best bone flap viability and a favorable new bone formation are usually observed41 ) . in this study , however , the cg 's new bone formation was significantly the worst when compared with the eg and dg . this may be due to the difference in the total healing time - no primary healing time in the cg , 8 weeks in the dg and 4 weeks in the eg - even though the three groups had the same secondary healing time of 8 weeks . according to sohn et al.37 ) , osteogenesis is a consistent process , but the time of initiating bone marrow maturity and bony islet formation in rabbits after the craniectomy procedure was 8 weeks after the surgery . this means that it takes 8 to 12 weeks to clearly observe and assess bone flap incorporation , bone remodeling , and osteogenesis . in this study , early bone flap incorporation was assessed through the early assessment on the healing process of the defect area . the primary healing time of the eg and dg was 4 weeks and 8 weeks , respectively , and their total healing time was 12 weeks and 16 weeks , respectively , while the total healing time of the cg was 8 weeks . this implies that the time of initiating new bone formation and bone flap incorporation is affected not only by the healing time after cranioplasty ( secondary healing time ) , but also by the total period after craniectomy ( primary and secondary healing time ) . accordingly , the primary healing time given prior to bone transplantation may positively affect the bone healing process . the bone remodeling period after fracture on rabbits is widely known as 6 - 8 weeks of period-1 week of resoprtion , 0.5 - 1 week of reversal and 4.5 - 6 weeks of bone formation ( on human , total 17 weeks-2 weeks of resorption , 2 weeks of reversal and 13 weeks of bone formation)26 ) . at this point of view , the cranioplasty performing before end of bone remodeling period would have some advantages to obtain bone fusion . thus , we think that cranioplasty performed within 8 weeks of primary healing time could result in good bone fusion . small sample size and the lack of power analysis for estimating appropriate sample are major concerns of this study . despite of small sample size , the experimental result using animal models that the period between craniectomy and cranioplasty could affect the bone fusion is a meaningful result of this study . another limitation is the different periods of total healing time and primary healing time among groups , because the duration of healing time after craniectomy has been known as one of important factors of bone fusion and union . we suggested more precisely designed study that focused on variations of healing duration among the groups should be mandatory . considering the preliminary characteristic of this study , these limitations are expected to be surmounted on well - designed large study on future . in this study , the cranioplasty procedure conducted on rabbits using the autologous bone flap frozen for less than 8 weeks resulted in favorable new bone formation and bone flap incorporation . furthermore , not only the post - cranioplasty healing time ( secondary healing time ) , but also the primary healing time , which was the time between the procedures of craniectomy and cranioplasty , were considered to positively affect the new bone formation and bone flap incorporation processes . based on the confirmation in this study regarding the positive healing process of rabbits even during the delayed period , the delayed period between the craniectomy and cranioplasty procedures in humans may positively affect the bone flap incorporation process .

objectivethe timing of cranioplasty and method of bone flap storage are known risk factors of non - union and resorption of bone flaps . in this animal experimental study , we evaluated the efficacy of cranioplasty using frozen autologous bone flap , and examined whether the timing of cranioplasty after craniectomy affects bone fusion and new bone formation.methodstotal 8 rabbits ( male , older than 16 weeks ) were divided into two groups of early cranioplasty group ( eg , 4 rabbits ) and delayed cranioplasty group ( dg , 4 rabbits ) . the rabbits of each group were performed cranioplasty via frozen autologous bone flaps 4 weeks ( eg ) and 8 weeks ( dg ) after craniectomy . in order to obtain control data , the cranioplasty immediate after craniectomy were made on the contralateral cranial bone of the rabbits ( control group , cg).the bone fusion and new bone formation were evaluated by micro - ct scan and histological examination 8 weeks after cranioplasty on both groups.resultsin the micro - ct scans , the mean values of the volume and the surface of new bone were 50.137.18 mm3 and 706.2377.26 mm2 in eg , 53.7810.86 mm3 and 726.60170.99 mm2 in dg , and 31.5112.84 mm3 and 436.65132.24 mm2 in cg . in the statistical results , significant differences were shown between eg and cg and between dg and cg ( volume : p=0.028 and surface : p=0.008 ) . the histological results confirmed new bone formation in all rabbits.conclusionwe observed new bone formation on all the frozen autologous bone flaps that was stored within 8 weeks . the timing of cranioplasty may showed no difference of degree of new bone formation . not only the healing period after cranioplasty but the time interval from craniectomy to cranioplasty could affect the new bone formation .

INTRODUCTION MATERIALS AND METHODS Experimental animals and groups Preparation of the experimental model Formation of bone defect and storage of bone flap Cranioplasty Radiologic observation Tissue processing and histological observation Statistics RESULTS DISCUSSION CONCLUSION

during the cranioplasty procedure , autologous bone flaps are preferably used due to their advantages in storage , viability , cost , prevention of disease transmission , and aesthetics . according to the studies on human cranioplasty , the timing of performing cranioplasty is one of the critical factors that may develop autologous bone flap resorption and bone non - union15 ) . although generally accepted concept about timing of cranioplasty using autologous bone is that early cranioplasty has more risk of infection and delayed cranioplasty has risk of non - union or resoprtion of bone flap4,6,25,44 ) , the debates are still remained . another report showed non - union and/or resorption of bone flap occurred more frequently on early cranioplasty patients who had surgery - related infections35 ) . and the other said there were no relation of bone non - union to timing of cranioplasty32 ) . in this study , cranioplasty was performed on rabbits using frozen autologous bone flaps , while the levels of new bone formation and bone fusion after craniectomy were observed , according to the timing of cranioplasty to evaluate its efficacy . four out of eight subjects were assigned to the delayed cranioplasty group ( dg ) , and the remaining four subjects were assigned to the early cranioplasty group ( eg ) . at day 0 , craniectomy was performed at the right frontoparietal bone of the dg rabbit , and the obtained bone flap was stored in a freezer at a temperature of -80. at day 28 or week 4 , bone defect was made in the right frontoparietal bone of the eg rabbit , and then , the bone flap was stored at the same condition . at day 56 or week 8 , the frozen bone flap was fixed on the bone defect area of the dg and eg rabbits . in order to obtain control data , the same procedures were made on the left cranial bone of the rabbits , and the bone flap was immediately fixed ( control group , cg ) . the time span between craniectomy and cranioplasty was the healing time of bone defect and defined as " the primary healing time " , while the time between the cranioplasty and rabbit sacrifice was the bone formation time of the gap between bone flap and cranium and defined as " the secondary healing time " . in order to induce anesthesia on the rabbits , 0.5 ml / kg of ketamin hcl ( ketalar , yuhan corporation , seoul , korea ) and 0.25 mg / kg of xylazine hcl ( rompun , bayer , pittsburgh , pa , usa ) were mixed and injected intramuscularly . after a 4-cm long incision was made along the midline of the cranial skin , the muscles and periosteum were incised layer by layer to expose the cranial bone ( fig . in order to make the bone flap , a bone flap margin was formed 3 mm lateral to the sagittal suture , which was located at the right side for the experimental groups and the left side for the control group , using a trephine drill with a 10-mm outer diameter and 9-mm inner diameter . to avoid damaging the dura mater , using a 1-mm round burr , the space between the round bone - flap margin and the cranial bone was expanded out of the bone flap in order to create a 12-mm bone defect diameter ( fig . the blood and soft tissues of the bone flap were removed , and the flap was wrapped up with gauze soaked sterilized saline . after anesthesia and skin incision that mentioned above , the fibrous tissues , which were formed during the primary healing time , were removed using a curette in order to expose the bone defect area ( fig . afterwards , the autologous bone flap , which has been kept in the freezer , was applied to each labeled rabbit in order to perform cranioplasty . the cranial bone and the bone flap were fixed using a titanium - alloy miniplate and 3-mm screws ( synthes inc . , , the cranial bone of the sacrificed rabbit was scanned by the micro - computed tomography ( micro - ct ) ( skyscan 1173 , skyscan , kontich , belgium ) . after confirming the round bone flap through the micro - ct images and 3d remodeling images , we defined the region of interest ( roi ) as the region within a 14-mm diameter from center point of bone flap . we measured the bone volume ( volroi ) and the bone surface ( surroi ) of roi ( fig . from volroi and surroi , the bone volume ( volbone flap ) and the bone surface ( surbone flap ) of the bone flap , and the volume afterwards , the ct scan images ( fig . 5c ) of the new bone volume ( volnew ) and the new bone surface ( surnew ) were quantified and measured . volnew = volroi-(volbone flap+volmetal ) surnew = surroi-(surbone flap+surmetal ) after dehydration processing was completed , the graft was put in a methyl methacrylate solution in order to maintain a vacuum state . as a non - parametric test , kruskal - wallis test was used for the comparison among the dg , eg , and cg . four out of eight subjects were assigned to the delayed cranioplasty group ( dg ) , and the remaining four subjects were assigned to the early cranioplasty group ( eg ) . at day 0 , craniectomy was performed at the right frontoparietal bone of the dg rabbit , and the obtained bone flap was stored in a freezer at a temperature of -80. at day 28 or week 4 , bone defect was made in the right frontoparietal bone of the eg rabbit , and then , the bone flap was stored at the same condition . at day 56 or week 8 , the frozen bone flap was fixed on the bone defect area of the dg and eg rabbits . in order to obtain control data , the same procedures were made on the left cranial bone of the rabbits , and the bone flap was immediately fixed ( control group , cg ) . the time span between craniectomy and cranioplasty was the healing time of bone defect and defined as " the primary healing time " , while the time between the cranioplasty and rabbit sacrifice was the bone formation time of the gap between bone flap and cranium and defined as " the secondary healing time " . in order to induce anesthesia on the rabbits , 0.5 ml / kg of ketamin hcl ( ketalar , yuhan corporation , seoul , korea ) and 0.25 mg / kg of xylazine hcl ( rompun , bayer , pittsburgh , pa , usa ) were mixed and injected intramuscularly . after a 4-cm long incision was made along the midline of the cranial skin , the muscles and periosteum were incised layer by layer to expose the cranial bone ( fig . in order to make the bone flap , a bone flap margin was formed 3 mm lateral to the sagittal suture , which was located at the right side for the experimental groups and the left side for the control group , using a trephine drill with a 10-mm outer diameter and 9-mm inner diameter . to avoid damaging the dura mater , the space between the round bone - flap margin and the cranial bone was expanded out of the bone flap in order to create a 12-mm bone defect diameter ( fig . the blood and soft tissues of the bone flap were removed , and the flap was wrapped up with gauze soaked sterilized saline . after anesthesia and skin incision that mentioned above , the fibrous tissues , which were formed during the primary healing time , were removed using a curette in order to expose the bone defect area ( fig . afterwards , the autologous bone flap , which has been kept in the freezer , was applied to each labeled rabbit in order to perform cranioplasty . the cranial bone and the bone flap were fixed using a titanium - alloy miniplate and 3-mm screws ( synthes inc . , in order to induce anesthesia on the rabbits , 0.5 ml / kg of ketamin hcl ( ketalar , yuhan corporation , seoul , korea ) and 0.25 mg / kg of xylazine hcl ( rompun , bayer , pittsburgh , pa , usa ) were mixed and injected intramuscularly . after a 4-cm long incision was made along the midline of the cranial skin , the muscles and periosteum were incised layer by layer to expose the cranial bone ( fig . in order to make the bone flap , a bone flap margin was formed 3 mm lateral to the sagittal suture , which was located at the right side for the experimental groups and the left side for the control group , using a trephine drill with a 10-mm outer diameter and 9-mm inner diameter . to avoid damaging the dura mater , the space between the round bone - flap margin and the cranial bone was expanded out of the bone flap in order to create a 12-mm bone defect diameter ( fig . the blood and soft tissues of the bone flap were removed , and the flap was wrapped up with gauze soaked sterilized saline . after anesthesia and skin incision that mentioned above , the fibrous tissues , which were formed during the primary healing time , were removed using a curette in order to expose the bone defect area ( fig . afterwards , the autologous bone flap , which has been kept in the freezer , was applied to each labeled rabbit in order to perform cranioplasty . the cranial bone and the bone flap were fixed using a titanium - alloy miniplate and 3-mm screws ( synthes inc . at week 16 , the cranial bone of the sacrificed rabbit was scanned by the micro - computed tomography ( micro - ct ) ( skyscan 1173 , skyscan , kontich , belgium ) . after confirming the round bone flap through the micro - ct images and 3d remodeling images , we defined the region of interest ( roi ) as the region within a 14-mm diameter from center point of bone flap . from volroi and surroi , the bone volume ( volbone flap ) and the bone surface ( surbone flap ) of the bone flap , and the volume afterwards , the ct scan images ( fig . 5c ) of the new bone volume ( volnew ) and the new bone surface ( surnew ) were quantified and measured . as a non - parametric test , kruskal - wallis test was used for the comparison among the dg , eg , and cg . the new bone formation observed in the micro - ct images was represented as bone volume ( volnew ) and bone surface ( surnew ) ( table 1 ) . the mean new bone volume of the dg was 53.7810.86 mm , while the mean bone surface was 726.60170.99 mm . the mean new bone volume and mean bone surface of the eg were 50.137.18 mm and 706.2377.26 mm , respectively . the mean new bone volume of the cg was 31.5112.84 mm , while the mean bone surface was 436.65132.24 mm . statistically significant differences were observed in the mean new bone volume ( p=0.024 ) and bone surface ( p=0.007 ) among the three groups ( table 2 ) . when the cg was compared with the eg and dg , significant differences in bone volume ( p=0.028 ) and bone surface ( p=0.008 ) similar to the findings in the micro - ct images , new bone formation was observed in the microscopic examination of the h - e stain and goldner 's stain in all of the rabbit tissues . the new bone formation was observed as a form of 1 ) a bony islet between the bone flap and the edge of the cranial bone ( fig . 6b ) , 3 ) a new bone formation at the edge of the cranial bone ( fig . 6c ) , and 4 ) a bone flap incorporation between the bone flap and the edge of the cranial bone ( fig . however , various complications such as infection , intracranial hemorrhage , bone flap resorption , depressed bone flap , cerebral vasospasm , and hydrocephalus can develop after the cranioplasty procedure , and their incidence rate has been reported to be 15 - 36.5%4,5,8,14,20,27,32,36 ) . the materials that are most frequently used in cranioplasty include autologous bone flap , poly - methyl - methacrylate ( pmma ) , and hydroxy apatite . the pmma is known to show a similar frequency of complication development to that of the autologous bone flap , but its osteogenesis capability is much less than that of the autologous bone flap22,24 ) . the autologous bone flap is the most widely used cranioplasty material due to its superiority in the viability , cost , prevention of disease tranmission , and aesthetics to artificial bones . nevertheless , long - term complications such as bone non - union and resorption , which develop after the cranioplasty procedure using the autologous bone flap , are the major causes that require re - operation . in a cranioplasty using autologous bone flap , bone flap incorporation occurs in the process of revascularization , osteoconduction , resorption , and osteogenesis12 ) . in a bone union process , bone remodeling is completed through the repetition of bone resorption and new bone formation . the patient - side risk factors of bone resorption are known to include young age , traumatic cranial damage , multiple cranial fractures , and the size of the cranial defect . according to previous studies conducted on the timing of cranioplasty , its early performance resulted in an increase in the incidence of infection , while delayed performance resulted in an increase in the resorption of the autologous bone flap4,6,25,35,44 ) . however , piedra et al.32 ) reported that there is no difference in the frequency of the development of complications , such as bone non - union and resorption , and in the infection between the early and delayed cranioplasty performance groups . in the case of cranioplasty performed within two months after craniectomy , schuss et al.35 ) reported a high frequency of infection , and resultant bone non - union and resorption . in this study , the timing of cranioplasty was set at 4 and 8 weeks with the eg and dg , and no significant difference in new bone formation was observed between the groups . furthermore , when the frozen autologous bone flap was used on rabbits within 8 weeks , an active new bone formation was radiologically and histologically confirmed , and no significant difference in new bone formation was observed according to the freezing period . the method of freezing the autologous bone flap was introduced in the 1950s1,11,30 ) , and since then , it has been the most widely used method . the most important factor to consider for a successful bone flap incorporation after cranioplasty is the viability of the bone flap9,28,34 ) . furthermore , the frozen autologous bone flap is known to have a comparatively favorable viability . when the frozen autologous bone flap was histologically analyzed at temperatures between -17 and -80 , the harvesian system of the bone tissues and structural proteins were confirmed to have remained the same regardless of the freezing period3,31,34 ) , and the osteocytes were observed even until the 35th month after freezing34 ) . in the frozen cranial bone , new blood vessels are remodeled , and they play the role of an architectural frame that assists the growth of the osteoprogenitor cells . a new bone formation was observed at the edge of the cranial bone close to the bone flaps of all the dg and eg rabbits not only in the micro - ct , but also in the histological results . according to sultan et al.41 ) , the bony islet formation contributed to the promotion of bone flap incorporation and the reduction of bone flap resorption . when cranioplasty is performed immediately after craniectomy , the best bone flap viability and a favorable new bone formation are usually observed41 ) . in this study , however , the cg 's new bone formation was significantly the worst when compared with the eg and dg . this may be due to the difference in the total healing time - no primary healing time in the cg , 8 weeks in the dg and 4 weeks in the eg - even though the three groups had the same secondary healing time of 8 weeks . according to sohn et al.37 ) , osteogenesis is a consistent process , but the time of initiating bone marrow maturity and bony islet formation in rabbits after the craniectomy procedure was 8 weeks after the surgery . in this study , early bone flap incorporation was assessed through the early assessment on the healing process of the defect area . the primary healing time of the eg and dg was 4 weeks and 8 weeks , respectively , and their total healing time was 12 weeks and 16 weeks , respectively , while the total healing time of the cg was 8 weeks . this implies that the time of initiating new bone formation and bone flap incorporation is affected not only by the healing time after cranioplasty ( secondary healing time ) , but also by the total period after craniectomy ( primary and secondary healing time ) . the bone remodeling period after fracture on rabbits is widely known as 6 - 8 weeks of period-1 week of resoprtion , 0.5 - 1 week of reversal and 4.5 - 6 weeks of bone formation ( on human , total 17 weeks-2 weeks of resorption , 2 weeks of reversal and 13 weeks of bone formation)26 ) . at this point of view , the cranioplasty performing before end of bone remodeling period would have some advantages to obtain bone fusion . thus , we think that cranioplasty performed within 8 weeks of primary healing time could result in good bone fusion . despite of small sample size , the experimental result using animal models that the period between craniectomy and cranioplasty could affect the bone fusion is a meaningful result of this study . another limitation is the different periods of total healing time and primary healing time among groups , because the duration of healing time after craniectomy has been known as one of important factors of bone fusion and union . in this study , the cranioplasty procedure conducted on rabbits using the autologous bone flap frozen for less than 8 weeks resulted in favorable new bone formation and bone flap incorporation . furthermore , not only the post - cranioplasty healing time ( secondary healing time ) , but also the primary healing time , which was the time between the procedures of craniectomy and cranioplasty , were considered to positively affect the new bone formation and bone flap incorporation processes . based on the confirmation in this study regarding the positive healing process of rabbits even during the delayed period , the delayed period between the craniectomy and cranioplasty procedures in humans may positively affect the bone flap incorporation process .

in the last decades and almost all around the world , the disease burden profile has changed from communicable diseases in childhood to non - communicable diseases ( ncds ) in adulthood ( 1 , 2 ) . meanwhile , the worldwide healthy adjusted life expectancy ( hale ) has grown slower than life expectancy ( 0.8 years for each 1 year ) and in a few countries hale even has been reduced ( 3 , 4 ) . in addition , non - fatal health outcomes have resulted in increasing years lived with disability ( yld ) , worldwide ( 5 ) . the global burden of diseases ( gbd ) study on 2010 identified high blood pressure ( bp ) as the top global burden risk factor . in most parts of asia , middle east , north africa , and central europe , it had worldwide attributable deaths of about 9.4 million and disability adjusted life years ( dalys ) of about 173.6 million and comprised 53 percent of ischemic heart disease ( ihd ) dalys ( 1 ) . moreover , this study defined ihd , lower respiratory infections , and stroke as the three major causes for years of life lost ( yll ) due to premature death ( 6 ) , with high bp as one of the most important risk factors for all of them . in 2001 , high bp resulted in 7.6 million premature deaths and 92 million dalys ; it also caused 54% of strokes and 47% of ihds worldwide ( 7 ) . in 2002 , ihd and stroke were the two principal causes of global death ( 8) . the world health organization ( who ) ranked high bp ( 13% of global death ) ( 9 ) and , cardiovascular diseases ( cvds ) ( 45% of global deaths ) ( 10 ) as the leading risk factors for global death in 2009 and 2011 , respectively . it is well documented that elevated bp during adulthood roots in childhood ( 11 - 13 ) . given the importance of tracking bp from childhood , the fourth report on the diagnosis , evaluation , and treatment of high bp in children and adolescents highlighted that all children aged above 3 years , who are seen in a medical setting the growing pattern of ncds and the pattern of the gbd are of special concern in low- and middle - income countries ( 1 ) . as a low - middle income country located in the middle east and north africa ( mena ) region , iran is facing rapid epidemiological transition and change in the disease pattern ( 15 ) . therefore , increasing our knowledge on the risk factors of ncds , would be helpful for designing and implementing timely preventive health programs . it seems necessary to cumulate their findings and estimate the overall burden of elevated bp for the country . furthermore , comparing data at the local and sub - national levels might yield useful results , which could be used by health policy makers and health workers . even the gbd study has drawn an overview of the situation at global level and does not provide each country s specific situation . therefore in the current review , first we looked for the studies conducted on bp among iranian pediatric population . thereafter , we tried to deliberate the relevant trends and burdens in the country . due to the scarcity of accessible information in many areas of the country , we need to use advanced statistical modeling methods in order to impute the missed data ( 16 , 17 ) . the present systematic review is a sub - component of cardio - metabolic risk factors burden study in iranian children and adolescents which itself is a part of the national and sub - national burden of diseases , injuries and risk factors ( nasbod ) study in iran . we have described detailed designs and protocols elsewhere ( 18 , 19 ) , and here we explain it in brief . the major outcome in the current review was elevated bp . according to the us national heart , lung , and blood institute ( nhlbi ) , bp is considered normal when the systolic and diastolic values are less than the 90 percentile for the child s age , sex , and height . prehypertension , is diagnosed when a child s average bp is above the 90th percentile but below the 95th . every adolescent with a bp greater than 120.80 mmhg is also diagnosed to have prehypertension , even if the bp would be below the 90 percentile . stage i hypertension is diagnosed if a child s bp is greater than the 95th percentile but less than or equal to the 99th percentile plus 5 mmhg . stage ii hypertension is diagnosed if a child s bp is greater than the 99th percentile plus 5 mmhg ( 14 ) . in the current review , the majority of the retrieved articles had used these cut points or earlier definitions proposed by nhlbi ( 20 , 21 ) . measures consisted of the values / mean ( standard deviation , sd ) values of sbp and/or dbp , or the frequency rates of high bp . in all studies selected for this review , bp value was measured by sphygmomanometer through brachial artery with appropriate cuff size and was reported in mmhg or cmhg . in most of them , bp was measured two to three times in a single session and the mean value was recorded . in some others , two to three measurements were made on separate occasions and the last one was included in their analysis . we searched international databases including pubmed / medline , isi web of science , and scopus , as well as the iranian scientific databases ( iranmedex , scientific information database ( sid ) , irandoc ) from january 1990 to january 2014 . the search terms were as follows : hypertension , blood pressure , high blood pressure , systolic pressure , diastolic pressure , arterial pressure in any possible combination with school or student or girl or boy or child * or adolescen * or pediatr * or paediatr * and iran for searching in the international databases ; the farsi equivalent of these terms were used for searching the national databases . a definite search strategy for each database we also looked at the reference list of relevant retrieved articles to find more publications . to the best of our knowledge the only nationwide study on cardio - metabolic risk factors in iranian pediatric population with the age specific definitions are the surveys of a national surveillance program entitled : childhood and adolescence surveillance and prevention of adult non - communicable diseases ( caspian ) study ( 24 , 25 ) . it has been conducted in four different surveys from 2003 to 2012 ( 25 - 29 ) . we included all cross sectional studies on bp values and prevalence of high bp in 6 - 18 year - old iranian population . we also included cohort and case - control studies if they had reported their baseline data . we limited the search to human studies conducted from january 1990 to january 2014 in iran ( iran , i.r.iran , islamic republic of iran ) or with iran in the affiliation of the authors with no restriction in the language . we excluded the studies that reported population normal values or percentiles exclusively or reported findings in more extended age groups than our study age range , or did not use pediatric specific definitions for bp classification . in the case of finding multiple publications from one study , we selected the more comprehensive one . the selected documents were saved in computer files with back - ups . in the first stage , the retrieved titles were screened to find relevant articles , at the second stage the abstracts were screened , and at the third stage full texts of relevant papers were screened ( appendix 2 ) . for quality assessment , we considered our study eligibility criteria , study design , sample size , sampling method , response rates , measurement tools and their calibration , as well as measurement methods and estimates . two independent reviewers ( zf and rk ) qualified the articles and the poor rated ones were excluded . the data extraction process is explained in detail elsewhere ( 19 ) . for most papers , confidence intervals ( ci ) , we tried to make contact with main authors but unfortunately the response rate was not favorable . the major outcome in the current review was elevated bp . according to the us national heart , lung , and blood institute ( nhlbi ) , bp is considered normal when the systolic and diastolic values are less than the 90 percentile for the child s age , sex , and height . prehypertension , is diagnosed when a child s average bp is above the 90th percentile but below the 95th . every adolescent with a bp greater than 120.80 mmhg is also diagnosed to have prehypertension , even if the bp would be below the 90 percentile . stage i hypertension is diagnosed if a child s bp is greater than the 95th percentile but less than or equal to the 99th percentile plus 5 mmhg . stage ii hypertension is diagnosed if a child s bp is greater than the 99th percentile plus 5 mmhg ( 14 ) . in the current review , the majority of the retrieved articles had used these cut points or earlier definitions proposed by nhlbi ( 20 , 21 ) . measures consisted of the values / mean ( standard deviation , sd ) values of sbp and/or dbp , or the frequency rates of high bp . in all studies selected for this review , bp value was measured by sphygmomanometer through brachial artery with appropriate cuff size and was reported in mmhg or cmhg . in most of them , bp was measured two to three times in a single session and the mean value was recorded . in some others , two to three measurements were made on separate occasions and the last one was included in their analysis . we searched international databases including pubmed / medline , isi web of science , and scopus , as well as the iranian scientific databases ( iranmedex , scientific information database ( sid ) , irandoc ) from january 1990 to january 2014 . the search terms were as follows : hypertension , blood pressure , high blood pressure , systolic pressure , diastolic pressure , arterial pressure in any possible combination with school or student or girl or boy or child * or adolescen * or pediatr * or paediatr * and iran for searching in the international databases ; the farsi equivalent of these terms were used for searching the national databases . a definite search strategy for each database we also looked at the reference list of relevant retrieved articles to find more publications . to the best of our knowledge the only nationwide study on cardio - metabolic risk factors in iranian pediatric population with the age specific definitions are the surveys of a national surveillance program entitled : childhood and adolescence surveillance and prevention of adult non - communicable diseases ( caspian ) study ( 24 , 25 ) . it has been conducted in four different surveys from 2003 to 2012 ( 25 - 29 ) . to the best of our knowledge the only nationwide study on cardio - metabolic risk factors in iranian pediatric population with the age specific definitions are the surveys of a national surveillance program entitled : childhood and adolescence surveillance and prevention of adult non - communicable diseases ( caspian ) study ( 24 , 25 ) . it has been conducted in four different surveys from 2003 to 2012 ( 25 - 29 ) . we included all cross sectional studies on bp values and prevalence of high bp in 6 - 18 year - old iranian population . we also included cohort and case - control studies if they had reported their baseline data . we limited the search to human studies conducted from january 1990 to january 2014 in iran ( iran , i.r.iran , islamic republic of iran ) or with iran in the affiliation of the authors with no restriction in the language . we excluded the studies that reported population normal values or percentiles exclusively or reported findings in more extended age groups than our study age range , or did not use pediatric specific definitions for bp classification . in the case of finding multiple publications from one study , we selected the more comprehensive one . all retrieved publications underwent selection and qualification processes . the search was repeated throughout the study period to add any newly published articles . in the first stage , the retrieved titles were screened to find relevant articles , at the second stage the abstracts were screened , and at the third stage full texts of relevant papers were screened ( appendix 2 ) . for quality assessment , we considered our study eligibility criteria , study design , sample size , sampling method , response rates , measurement tools and their calibration , as well as measurement methods and estimates . two independent reviewers ( zf and rk ) qualified the articles and the poor rated ones were excluded . the data extraction process is explained in detail elsewhere ( 19 ) . for most papers , confidence intervals ( ci ) for which complementary data were needed , we tried to make contact with main authors but unfortunately the response rate was not favorable . the search algorithm including the number of initial search results and included studies are shown in appendix 2 . at first step after three steps of selection and then qualifying processes , we included 36 articles ( 30 - 68 ) ( tables 1 and 2 ) in our systematic review . 2 . sbp more than 95th percentile , defined as hypertension , dbp more than 95th percentile , defined as hypertension or bp under 90th percentile for age and sex : normal , between 90th to 95th : high normal , above 95th : high blood pressure . prehypertension was defined as an average systolic or diastolic blood pressure between the 90th and 95th percentiles for gender , age , and height . 4 . for children aged 6 - 9 years , significant hypertension and severe hypertension were defined as systolic blood pressure greater than 122 mmhg and 130 mmhg and diastolic blood pressure greater than 78 mmhg and 86 mmhg respectively . significant and severe hypertension in children aged10 - 12 years was defined as systolic blood pressure greater than 126 mmhg and 134mmhg and diastolic blood pressure greater than 82 mmhg and 90 mmhg respectively(second task force ) ( 20 - 21 ) . report of the second task force on blood pressure control in children1987.task force on blood pressure control in children.national heart , lung , and blood institute , bethesda , maryland.pediatrics . according to the harriet lane handbook : a manual for pediatric house officers ( 23 ) . according to the fourth report on the diagnosis , evaluation , and treatment of high blood pressure in children and adolescents(between 90 and 95 percentile : prehtn , between 95th percentile and 99th percentile+5 mmhg : stage 1 htn and over 99th percentile+5 mmhg : stage 2 htn ( 14 ) . according to the update on the 1987 task force report on high blood pressure in children and adolescents : a working group report from the national high blood pressure education program ( between 90th and 95th percentiles : significant htn and over 99th percentile severe htn ) ( 21 ) . 11 . bp above the 95th percentile for that age and sex after adjusting for weight and height = htn ( 19 - 21 ) . 12 . bp in the 90th percentile for their age , sex , and height = htn ( 19 - 21 ) . 13 . pre htn = bp equal or greater than the age- and gender - specific 90th percentile after adjusting for weight and height or bp equal or more than 120/80 mm hg when bp was equal or over the age- and gender - specific 95th percentile value , it was considered as htn ( 14 ) . 1 . hypertension ( systolic : 120 + , and diastolic : 100 + ) . 2 . sbp more than 95th percentile , defined as hypertension , dbp more than 95th percentile , defined as hypertension or bp under 90th percentile for age and sex : normal , between 90th to 95th : high normal , above 95th : high blood pressure . prehypertension was defined as an average systolic or diastolic blood pressure between the 90th and 95th percentiles for gender , age , and height . , significant hypertension and severe hypertension were defined as systolic blood pressure greater than 122 mmhg and 130 mmhg and diastolic blood pressure greater than 78 mmhg and 86 mmhg respectively . significant and severe hypertension in children aged 10 - 12 years was defined as systolic blood pressure greater than 126 mmhg and 134 mmhg and diastolic blood pressure greater than 82 mmhg and 90 mmhg respectively ( second task force ) ( 20 - 21 ) . report of the second task force on blood pressure control in children - 1987.task force on blood pressure control in children.national heart , lung , and blood institute , bethesda , maryland.pediatrics . according to the harriet lane handbook : a manual for pediatric house officers ( 23 ) . according to the fourth report on the diagnosis , evaluation , and treatment of high blood pressure in children and adolescents(between 90 and 95 percentile : prehtn , between 95th percentile and 99th percentile+5 mmhg : stage 1 htn and over 99th percentile+5 mmhg : stage 2 htn ( 14 ) . according to the update on the 1987 task force report on high blood pressure in children and adolescents : a working group report from the national high blood pressure education program ( between 90th and 95th percentiles : significant htn and over 99th percentile severe htn ) ( 21 ) . altogether , this review included 1,096,263 total population ( 515,098 girls , 47% ) and 277 data - points . they were from four national , one provincial , 29 district and two community study levels . the least value of mean sbp ( 90.10 4.00 mmhg ( 95% ci 89.25 , 90.94 ) ) and mean dbp ( 50.7 11.40 ( 50.01 , 51.38 ) mmhg ) are both reported from tehran city in the same study ( 54 ) . the highest mean values reported are 120.2 12.30 ( 118.98 , 121.41 ) mmhg for mean sbp from kerman city ( 59 ) and 79.20 12.30 ( 77.95 , 80.44 ) mmhg for mean dbp from isfahan city ( 53 ) . the study of kerman city also had the second rank of highest dbp , i.e. 76 11.4 ( 4.87 , 77.12 ) mmhg ( 59 ) . as presented in tables 1 and 2 , almost all articles reported mean bp values . from 36 published articles , fifteen reported prevalence rates of isolated high sbp and high dbp ( table 1 ) . fifteen articles reported the prevalence of high sbp and/or high dbp ( table 2 ) . table 1 also presents the national data of caspian studies ( 66 - 68 ) . the caspian - i study was a nationwide survey conducted in 2003 - 2004 in 23 provinces , and included 21,111 students , aged 6 - 18 years . elevated bp was defined as values equal or greater than 95th percentile , and the prevalence of high sbp , dbp as well as sbp and/or dbp in total population was 4.2% ( 3.96 , 4.51 ) , 5.4% ( 5.05 , 5.67 ) and 7.7 % ( 7.38 , 8.11 ) , respectively ( 66 ) . the caspian - iii study was conducted in 2009 - 2010 among 5,738 students aged 10 - 18 years living in 27 provinces . it reported prevalence rates for high bp ( values equal or greater than 90th percentile ) as follows : 3.8% ( 3.31 , 4.32 ) , 3.3% ( 2.86 , 3.8 ) , and 6% ( 5.41 , 6.65 ) ( 66 ) . the corresponding figures reported by caspian - iv study , conducted in 2011 - 2012 on 13,486 students from 30 provinces , were 4.17% ( 3.84 , 4.52 ) , 4.33% ( 3.99 , 4.68 ) and 6.88% ( 6.45 , 7.32 ) , respectively ( 68 ) . as it is obvious from these two tables , the reported range of high bp in iranian pediatric population in sub - national studies in the two past decades varies widely , with rates as low as 0.40% ( 95% ci 0.009 , 1.98 ) for high sbp in tehran province for 15 - 19-year - old boys ( 39 - 41 ) and as high as 24.10% ( 20.80 , 27.67 ) for high dbp in khorasan - razavi province ( 46 ) . moreover , in national screening of 6-year - old children at school entry , the prevalence of high bp was 0.10% ( 0.09 , 0.109 ) ( 31 ) . based on the use of 95th percentile to define hypertension , the prevalence of hypertension would be expected to be around 5% and in fact 1 - 3% ( 69 ) . our findings showed that the number of sub - national studies in iranian pediatric population that reported high bp rates above 5% ( 30 , 45 , 46 , 55 , 58 , 61 , 62 , 65 ) was equal to studies that reported rates below 5% ( 31 , 34 , 36 , 42 , 44 , 48 , 49 , 64 ) . in eight papers , the rates from both ranges ( below and above 5% ) for different age or sex categories are reported ( 37 - 41 , 43 , 50 , 57 , 63 ) . seven studies reported at least one prevalence rate of elevated bp of more than 10 percent ( 30 , 37 - 39 , 41 , 43 , 50 , 62 , 63 ) . to the best of our knowledge , this study is the first systematic review on studies related to bp in the pediatric population , not only in iran , but also , in the mena region . as expected , we found that the prevalence of elevated bp was not negligible in healthy children and adolescents in iran , and varied in different regions . it should be considered that in addition to detecting prehypertension and hypertension at early stages , bp screening programs would help in monitoring the mean sbp and dbp over time . though a decrease from 41% in 1990 to 25% in 2010 has occurred in the contribution of pediatric mortality and morbidity to global dalys ( 3 ) , considering that many adult ncds actually start from earlier stages of life , the importance of this side of pediatric health and its significant sequels in the adult life should not be neglected . even because of the high susceptibility of fetal life to environmental and nutritional disorders , preventive measures should focus on this stage of life ( 70 ) . the childhood obesity epidemic has resulted in left ventricular hypertrophy and evidence for premature development of atherosclerosis , therefore it makes the issue of tracking bp and screening for elevated bp of special concern in the pediatric age group ( 71 ) . children and adolescents with pre - hypertension and hypertension tend to maintain this situation over time ( 1 , 72 - 76 ) . many risky behaviors as smoking , alcohol use , physical inactivity , and unhealthy dietary habits are developed during adolescence period . changing such lifestyle habits would be difficult after establishment , and would have lifelong consequences including ncds and their risk factors as high bp ( 77 ) . a longitudinal study with 17 years of follow up of a pediatric population showed a 6.88 fold increase in the prevalence of hypertension in those who had high bp at baseline ( 78 ) . national studies in american children and adolescents found that that the mean bp levels ( 79 ) , as well as the prevalence of high bp ( 80 ) are gradually rising over time . the mean age of iranian population is estimated to be increasing by 6.5 years in the two coming decades . presuming the persistent prevalence of risk and protective factors and only because of that increase in the age , the country s yll from cvds will be duplicated in 2025 compared to 2005 , without difference in terms of gender ( 81 ) . a study in 2003 showed that in iran 58 percent of dalys resulted from ncds and the country health burden is changing from communicable diseases to ncds and road accidents ( 82 ) . who has reported an age - standardized mortality rate of 420.8 ( males ) and 348.0 ( females ) per 100000 for cvd and diabetes in iran for 2008 , and estimated a prevalence rate of around 33.7% for hypertension ( 7 ) . a national survey ( surfncd ) in 2007 showed a remarkable prevalence of ncds and their risk factors among iranians aged 15 65 ( 83 ) . another national study showed that high sbp is responsible for most deaths in all regions of iran . it is expected that by optimizing sbp , the years of life expectancy would increase by 3.2 ( 2.6 , 3.9 95% uncertainty interval ) years in men and by 4.1 ( 3.2 , 4.9 ui ) years in women , therefore it is concluded that prevention and control of high bp should be considered as a health priority for iran ( 84 ) . many studies documented high rates of cardio - metabolic predisposing factors in iranian pediatric population ( 25 - 29 , 85 - 90 ) . the caspian - i study ( 25 , 26 ) , showed that the percentiles of sbp and dbp of iranian children and adolescents were in close agreement with reference values ( 14 ) . as mentioned earlier , the study has continued to the 4th survey till now and different rates of elevated bp are reported . overall , these surveys showed that in recent years , the prevalence of elevated bp has increased slightly . the variation between surveys could be attributed to differences between definitions of elevated bp ( the first phase was conducted before the introduction of pediatric pre - htn ) and differences in study populations , i.e. different proportion of age and gender subgroups , different provinces , different total populations and changes in the prevalence of associated conditions as obesity , unhealthy dietary habits , inactivity , air and noise pollution , urbanization , etc . given the association of bp level with excess weight as well as with environmental factors as air pollution , noise pollution , and passive smoking ( 91 ) , and the high sodium intake of iranian children ( 33 ) , it is proposed that in the near future , the mean bp and the prevalence of high bp will be escalating in iranian children and adolescents . the high prevalence of ncd risk factors in various age groups of iranians , underscores the necessity of conducting comprehensive research and preventive programs to achieve a multidisciplinary intervention plan involving the whole health system of iran ( 92 ) . both in the 53rd session of eastern mediterranean region ( emro ) ( 93 ) and september 2011 session of the united nations ( un ) in new york , all members committed to develop national strategies as well as preventive and controlling action plans , to involve all stakeholders and to organize financial resources targeting ncds . it seems that still no effective public and community interventions have been done in this regard . the gbd study 2010 revealed that some parts of underachievement of the who 4th millennium goal is because of the lack of relevant information from the pediatric age group ( 94 ) . presenting the information regarding pediatric studies to policymakers in a systematic and concluding manner would be of help for planning action - oriented programs . moreover , there is increasing need to know where and how the complementary research and action plans must be implemented . the current study summarized the information on the mean bp and the prevalence of high bp in the iranian pediatric population . the wide heterogeneity of the retrieved papers and their point estimates led the researchers not to consider a meta - analysis . it is planned to draw the trend of bp changes across these years . as mentioned before after that , ylds , ylls , dalys and burden of the hbp in pediatric population of iran will be estimated . the importance of bp tracking and the pediatric hypertension per se and as a cvd risk factor becomes more obvious , making it important for including bp measurement in screening programs and conducting further comprehensive and cumulative studies on its time trends , primary prevention and early diagnosis . health policies on prevention and early control of high bp can be effective in reducing the prevalence and the adverse consequences of high bp in adulthood . therefore , the findings of the current systematic review and succeeding works on bp of iranian children and adolescents would be useful for future health policies and research activities aimed to reduce the burden of high bp at individual and public health levels . follow up surveillance programs and comparison of bp trends of children and adolescents over time are recommended .

context : blood pressure ( bp ) tracks from childhood to adulthood , and has ethnic variations . therefore , it is important to assess the situation of pediatric bp in different populations . this study aims to systematically review the studies conducted on bp in iranian children and adolescents.evidence acquisition : we conducted a systematic review on published and national data about pediatric bp in iran , our search was conducted in pub med , medline , isi , and scopus , as well as in national databases including scientific information database ( sid ) , iranmedex and irandoc from 1990 to 2014.results:we found 1373 records in the primary search including 840 from international and 533 from national databases . after selection and quality assessment phases , data were extracted from 36 papers and four national data sources . mean systolic bp ( sbp ) varied from 90.1 14 mmhg ( 95% ci 89.25 , 90.94 ) to 120.2 12.3 ( 118.98 , 121.41 ) mmhg , and for diastolic bp ( dbp ) from 50.7 11.4 ( 50.01 , 51.38 ) to 79.2 12.3 ( 77.95 , 80.44 ) mmhg . the frequency of elevated bp had large variation in sub - national studies with rates as low as 0.4% ( 0.009 , 1.98 ) for high sbp and as high as 24.1% ( 20.8 , 27.67 ) for high dbp . at national level , three surveys reported slightly raised rates of elevated bp from 2009 to 2012.conclusions:the findings provide practical information on bp levels in iranian pediatric population . although differences exist on the findings of various studies , this review underscores the necessity of tracking bp from childhood , and implementing interventions for primordial prevention of hypertension .

1. Context 2. Evidence Acquisition 2.1. Outcome Definition 2.2. Measures Definition 2.3. Search Strategy 2.3.1. National Data 2.4. Inclusion and Exclusion Criteria 2.5. Data Management 2.6. Selection Process 2.7. Quality Assessment 2.8. Data Extraction 3. Results 4. Conclusions

in the last decades and almost all around the world , the disease burden profile has changed from communicable diseases in childhood to non - communicable diseases ( ncds ) in adulthood ( 1 , 2 ) . the global burden of diseases ( gbd ) study on 2010 identified high blood pressure ( bp ) as the top global burden risk factor . in most parts of asia , middle east , north africa , and central europe , it had worldwide attributable deaths of about 9.4 million and disability adjusted life years ( dalys ) of about 173.6 million and comprised 53 percent of ischemic heart disease ( ihd ) dalys ( 1 ) . moreover , this study defined ihd , lower respiratory infections , and stroke as the three major causes for years of life lost ( yll ) due to premature death ( 6 ) , with high bp as one of the most important risk factors for all of them . it is well documented that elevated bp during adulthood roots in childhood ( 11 - 13 ) . given the importance of tracking bp from childhood , the fourth report on the diagnosis , evaluation , and treatment of high bp in children and adolescents highlighted that all children aged above 3 years , who are seen in a medical setting the growing pattern of ncds and the pattern of the gbd are of special concern in low- and middle - income countries ( 1 ) . as a low - middle income country located in the middle east and north africa ( mena ) region , iran is facing rapid epidemiological transition and change in the disease pattern ( 15 ) . therefore , increasing our knowledge on the risk factors of ncds , would be helpful for designing and implementing timely preventive health programs . it seems necessary to cumulate their findings and estimate the overall burden of elevated bp for the country . furthermore , comparing data at the local and sub - national levels might yield useful results , which could be used by health policy makers and health workers . therefore in the current review , first we looked for the studies conducted on bp among iranian pediatric population . the present systematic review is a sub - component of cardio - metabolic risk factors burden study in iranian children and adolescents which itself is a part of the national and sub - national burden of diseases , injuries and risk factors ( nasbod ) study in iran . we have described detailed designs and protocols elsewhere ( 18 , 19 ) , and here we explain it in brief . the major outcome in the current review was elevated bp . according to the us national heart , lung , and blood institute ( nhlbi ) , bp is considered normal when the systolic and diastolic values are less than the 90 percentile for the child s age , sex , and height . in the current review , the majority of the retrieved articles had used these cut points or earlier definitions proposed by nhlbi ( 20 , 21 ) . measures consisted of the values / mean ( standard deviation , sd ) values of sbp and/or dbp , or the frequency rates of high bp . we searched international databases including pubmed / medline , isi web of science , and scopus , as well as the iranian scientific databases ( iranmedex , scientific information database ( sid ) , irandoc ) from january 1990 to january 2014 . the search terms were as follows : hypertension , blood pressure , high blood pressure , systolic pressure , diastolic pressure , arterial pressure in any possible combination with school or student or girl or boy or child * or adolescen * or pediatr * or paediatr * and iran for searching in the international databases ; the farsi equivalent of these terms were used for searching the national databases . to the best of our knowledge the only nationwide study on cardio - metabolic risk factors in iranian pediatric population with the age specific definitions are the surveys of a national surveillance program entitled : childhood and adolescence surveillance and prevention of adult non - communicable diseases ( caspian ) study ( 24 , 25 ) . it has been conducted in four different surveys from 2003 to 2012 ( 25 - 29 ) . we included all cross sectional studies on bp values and prevalence of high bp in 6 - 18 year - old iranian population . we limited the search to human studies conducted from january 1990 to january 2014 in iran ( iran , i.r.iran , islamic republic of iran ) or with iran in the affiliation of the authors with no restriction in the language . in the first stage , the retrieved titles were screened to find relevant articles , at the second stage the abstracts were screened , and at the third stage full texts of relevant papers were screened ( appendix 2 ) . for quality assessment , we considered our study eligibility criteria , study design , sample size , sampling method , response rates , measurement tools and their calibration , as well as measurement methods and estimates . the major outcome in the current review was elevated bp . according to the us national heart , lung , and blood institute ( nhlbi ) , bp is considered normal when the systolic and diastolic values are less than the 90 percentile for the child s age , sex , and height . in the current review , the majority of the retrieved articles had used these cut points or earlier definitions proposed by nhlbi ( 20 , 21 ) . measures consisted of the values / mean ( standard deviation , sd ) values of sbp and/or dbp , or the frequency rates of high bp . we searched international databases including pubmed / medline , isi web of science , and scopus , as well as the iranian scientific databases ( iranmedex , scientific information database ( sid ) , irandoc ) from january 1990 to january 2014 . the search terms were as follows : hypertension , blood pressure , high blood pressure , systolic pressure , diastolic pressure , arterial pressure in any possible combination with school or student or girl or boy or child * or adolescen * or pediatr * or paediatr * and iran for searching in the international databases ; the farsi equivalent of these terms were used for searching the national databases . to the best of our knowledge the only nationwide study on cardio - metabolic risk factors in iranian pediatric population with the age specific definitions are the surveys of a national surveillance program entitled : childhood and adolescence surveillance and prevention of adult non - communicable diseases ( caspian ) study ( 24 , 25 ) . it has been conducted in four different surveys from 2003 to 2012 ( 25 - 29 ) . to the best of our knowledge the only nationwide study on cardio - metabolic risk factors in iranian pediatric population with the age specific definitions are the surveys of a national surveillance program entitled : childhood and adolescence surveillance and prevention of adult non - communicable diseases ( caspian ) study ( 24 , 25 ) . it has been conducted in four different surveys from 2003 to 2012 ( 25 - 29 ) . we included all cross sectional studies on bp values and prevalence of high bp in 6 - 18 year - old iranian population . we limited the search to human studies conducted from january 1990 to january 2014 in iran ( iran , i.r.iran , islamic republic of iran ) or with iran in the affiliation of the authors with no restriction in the language . we excluded the studies that reported population normal values or percentiles exclusively or reported findings in more extended age groups than our study age range , or did not use pediatric specific definitions for bp classification . in the first stage , the retrieved titles were screened to find relevant articles , at the second stage the abstracts were screened , and at the third stage full texts of relevant papers were screened ( appendix 2 ) . for quality assessment , we considered our study eligibility criteria , study design , sample size , sampling method , response rates , measurement tools and their calibration , as well as measurement methods and estimates . for most papers , confidence intervals ( ci ) for which complementary data were needed , we tried to make contact with main authors but unfortunately the response rate was not favorable . at first step after three steps of selection and then qualifying processes , we included 36 articles ( 30 - 68 ) ( tables 1 and 2 ) in our systematic review . sbp more than 95th percentile , defined as hypertension , dbp more than 95th percentile , defined as hypertension or bp under 90th percentile for age and sex : normal , between 90th to 95th : high normal , above 95th : high blood pressure . prehypertension was defined as an average systolic or diastolic blood pressure between the 90th and 95th percentiles for gender , age , and height . significant and severe hypertension in children aged10 - 12 years was defined as systolic blood pressure greater than 126 mmhg and 134mmhg and diastolic blood pressure greater than 82 mmhg and 90 mmhg respectively(second task force ) ( 20 - 21 ) . report of the second task force on blood pressure control in children1987.task force on blood pressure control in children.national heart , lung , and blood institute , bethesda , maryland.pediatrics . according to the fourth report on the diagnosis , evaluation , and treatment of high blood pressure in children and adolescents(between 90 and 95 percentile : prehtn , between 95th percentile and 99th percentile+5 mmhg : stage 1 htn and over 99th percentile+5 mmhg : stage 2 htn ( 14 ) . according to the update on the 1987 task force report on high blood pressure in children and adolescents : a working group report from the national high blood pressure education program ( between 90th and 95th percentiles : significant htn and over 99th percentile severe htn ) ( 21 ) . bp in the 90th percentile for their age , sex , and height = htn ( 19 - 21 ) . prehypertension was defined as an average systolic or diastolic blood pressure between the 90th and 95th percentiles for gender , age , and height . significant and severe hypertension in children aged 10 - 12 years was defined as systolic blood pressure greater than 126 mmhg and 134 mmhg and diastolic blood pressure greater than 82 mmhg and 90 mmhg respectively ( second task force ) ( 20 - 21 ) . report of the second task force on blood pressure control in children - 1987.task force on blood pressure control in children.national heart , lung , and blood institute , bethesda , maryland.pediatrics . according to the fourth report on the diagnosis , evaluation , and treatment of high blood pressure in children and adolescents(between 90 and 95 percentile : prehtn , between 95th percentile and 99th percentile+5 mmhg : stage 1 htn and over 99th percentile+5 mmhg : stage 2 htn ( 14 ) . according to the update on the 1987 task force report on high blood pressure in children and adolescents : a working group report from the national high blood pressure education program ( between 90th and 95th percentiles : significant htn and over 99th percentile severe htn ) ( 21 ) . altogether , this review included 1,096,263 total population ( 515,098 girls , 47% ) and 277 data - points . the least value of mean sbp ( 90.10 4.00 mmhg ( 95% ci 89.25 , 90.94 ) ) and mean dbp ( 50.7 11.40 ( 50.01 , 51.38 ) mmhg ) are both reported from tehran city in the same study ( 54 ) . the highest mean values reported are 120.2 12.30 ( 118.98 , 121.41 ) mmhg for mean sbp from kerman city ( 59 ) and 79.20 12.30 ( 77.95 , 80.44 ) mmhg for mean dbp from isfahan city ( 53 ) . 76 11.4 ( 4.87 , 77.12 ) mmhg ( 59 ) . from 36 published articles , fifteen reported prevalence rates of isolated high sbp and high dbp ( table 1 ) . fifteen articles reported the prevalence of high sbp and/or high dbp ( table 2 ) . the caspian - i study was a nationwide survey conducted in 2003 - 2004 in 23 provinces , and included 21,111 students , aged 6 - 18 years . elevated bp was defined as values equal or greater than 95th percentile , and the prevalence of high sbp , dbp as well as sbp and/or dbp in total population was 4.2% ( 3.96 , 4.51 ) , 5.4% ( 5.05 , 5.67 ) and 7.7 % ( 7.38 , 8.11 ) , respectively ( 66 ) . the caspian - iii study was conducted in 2009 - 2010 among 5,738 students aged 10 - 18 years living in 27 provinces . it reported prevalence rates for high bp ( values equal or greater than 90th percentile ) as follows : 3.8% ( 3.31 , 4.32 ) , 3.3% ( 2.86 , 3.8 ) , and 6% ( 5.41 , 6.65 ) ( 66 ) . the corresponding figures reported by caspian - iv study , conducted in 2011 - 2012 on 13,486 students from 30 provinces , were 4.17% ( 3.84 , 4.52 ) , 4.33% ( 3.99 , 4.68 ) and 6.88% ( 6.45 , 7.32 ) , respectively ( 68 ) . as it is obvious from these two tables , the reported range of high bp in iranian pediatric population in sub - national studies in the two past decades varies widely , with rates as low as 0.40% ( 95% ci 0.009 , 1.98 ) for high sbp in tehran province for 15 - 19-year - old boys ( 39 - 41 ) and as high as 24.10% ( 20.80 , 27.67 ) for high dbp in khorasan - razavi province ( 46 ) . moreover , in national screening of 6-year - old children at school entry , the prevalence of high bp was 0.10% ( 0.09 , 0.109 ) ( 31 ) . based on the use of 95th percentile to define hypertension , the prevalence of hypertension would be expected to be around 5% and in fact 1 - 3% ( 69 ) . our findings showed that the number of sub - national studies in iranian pediatric population that reported high bp rates above 5% ( 30 , 45 , 46 , 55 , 58 , 61 , 62 , 65 ) was equal to studies that reported rates below 5% ( 31 , 34 , 36 , 42 , 44 , 48 , 49 , 64 ) . seven studies reported at least one prevalence rate of elevated bp of more than 10 percent ( 30 , 37 - 39 , 41 , 43 , 50 , 62 , 63 ) . to the best of our knowledge , this study is the first systematic review on studies related to bp in the pediatric population , not only in iran , but also , in the mena region . as expected , we found that the prevalence of elevated bp was not negligible in healthy children and adolescents in iran , and varied in different regions . it should be considered that in addition to detecting prehypertension and hypertension at early stages , bp screening programs would help in monitoring the mean sbp and dbp over time . though a decrease from 41% in 1990 to 25% in 2010 has occurred in the contribution of pediatric mortality and morbidity to global dalys ( 3 ) , considering that many adult ncds actually start from earlier stages of life , the importance of this side of pediatric health and its significant sequels in the adult life should not be neglected . the childhood obesity epidemic has resulted in left ventricular hypertrophy and evidence for premature development of atherosclerosis , therefore it makes the issue of tracking bp and screening for elevated bp of special concern in the pediatric age group ( 71 ) . children and adolescents with pre - hypertension and hypertension tend to maintain this situation over time ( 1 , 72 - 76 ) . changing such lifestyle habits would be difficult after establishment , and would have lifelong consequences including ncds and their risk factors as high bp ( 77 ) . a longitudinal study with 17 years of follow up of a pediatric population showed a 6.88 fold increase in the prevalence of hypertension in those who had high bp at baseline ( 78 ) . national studies in american children and adolescents found that that the mean bp levels ( 79 ) , as well as the prevalence of high bp ( 80 ) are gradually rising over time . the mean age of iranian population is estimated to be increasing by 6.5 years in the two coming decades . presuming the persistent prevalence of risk and protective factors and only because of that increase in the age , the country s yll from cvds will be duplicated in 2025 compared to 2005 , without difference in terms of gender ( 81 ) . who has reported an age - standardized mortality rate of 420.8 ( males ) and 348.0 ( females ) per 100000 for cvd and diabetes in iran for 2008 , and estimated a prevalence rate of around 33.7% for hypertension ( 7 ) . many studies documented high rates of cardio - metabolic predisposing factors in iranian pediatric population ( 25 - 29 , 85 - 90 ) . the caspian - i study ( 25 , 26 ) , showed that the percentiles of sbp and dbp of iranian children and adolescents were in close agreement with reference values ( 14 ) . as mentioned earlier , the study has continued to the 4th survey till now and different rates of elevated bp are reported . overall , these surveys showed that in recent years , the prevalence of elevated bp has increased slightly . the variation between surveys could be attributed to differences between definitions of elevated bp ( the first phase was conducted before the introduction of pediatric pre - htn ) and differences in study populations , i.e. different proportion of age and gender subgroups , different provinces , different total populations and changes in the prevalence of associated conditions as obesity , unhealthy dietary habits , inactivity , air and noise pollution , urbanization , etc . given the association of bp level with excess weight as well as with environmental factors as air pollution , noise pollution , and passive smoking ( 91 ) , and the high sodium intake of iranian children ( 33 ) , it is proposed that in the near future , the mean bp and the prevalence of high bp will be escalating in iranian children and adolescents . the high prevalence of ncd risk factors in various age groups of iranians , underscores the necessity of conducting comprehensive research and preventive programs to achieve a multidisciplinary intervention plan involving the whole health system of iran ( 92 ) . both in the 53rd session of eastern mediterranean region ( emro ) ( 93 ) and september 2011 session of the united nations ( un ) in new york , all members committed to develop national strategies as well as preventive and controlling action plans , to involve all stakeholders and to organize financial resources targeting ncds . the current study summarized the information on the mean bp and the prevalence of high bp in the iranian pediatric population . health policies on prevention and early control of high bp can be effective in reducing the prevalence and the adverse consequences of high bp in adulthood . therefore , the findings of the current systematic review and succeeding works on bp of iranian children and adolescents would be useful for future health policies and research activities aimed to reduce the burden of high bp at individual and public health levels . follow up surveillance programs and comparison of bp trends of children and adolescents over time are recommended .

oral mucocutaneous conditions are a group of disorders which are observed in the dental practice . oral mucosal manifestations may be the initial feature , or the only sign of such diseases . in other cases , lesions occur in both the skin and mucosae , with severe clinical manifestations involving the tissues . correct diagnosis is critical , since proper treatment and follow - up will depend on which disease is involved . however , vesiculo - bullous lesions frequently present diagnostic problems because the lesions often resemble each other clinically and routine histological examination sometimes can not differentiate between them . thus immunohistology particularly immunofluorescence is increasingly being used with routine histology to accurately diagnose vesiculo - bullous lesions . these conditions are caused by a genetic mutation or are due to an autoimmune response . this study was carried out in the department of oral pathology , sri ramachandra dental college & hospital , chennai . the study population consisted of 26 patients with oral mucocutaneous diseases who were selected randomly . of the 26 patients,6 patients were under pulse therapy who were clinically and histopathologically diagnosed as pemphigus . these patients were free of active lesions and their disease activity was assessed by indirect immunofluorescence ( iif ) who were grouped separately . based on the clinical and provisional diagnosis , the remaining 20 patients who had active lesions were subjected to both direct immunofluorscence ( dif ) and iif and were divided into four groups . we carried out histopathology study with subsequent immunofluorescent technique to study the pattern of immunofluorescence in each group . they were immediately washed in normal saline and placed in michel 's medium and transported to the laboratory within 24 hours . whole blood was allowed to clot , centrifuged , and the serum was separated and transported to the laboratory . the study consisted of 26 patients of pemphigus , pemphigoid , lichen planus , and lupus erythematosus . they were grouped as follows : group i pemphigus ( seven cases , patients with active lesions ) group ii pemphigus ( six cases , patients on pulse therapy and completely free of active lesions ) patients in group i , ii , iii , and iv were subjected to histopathologic , dif , and iif examination , and patients in group v were subjected to iif alone . group i v , included patients with age ranging from 28 to 60 years ( 42.5 years ) . group i , ii , and v showed male predominance and group iii and iv showed female predominance . group i iv showed site predilection for buccal mucosa , labial mucosa , tongue , gingival , and palate . i : histopathology showed suprabasilar cleft and acantholytic cells superficial to the basal cells which were attached to connective tissue ( tombstone appearance ) . both acute and chronic inflammatory cells and increased vascularity was seen within the connective tissue . ii : histopathology showed subepithelial split , red blood cells ( rbcs ) and inflammatory cells in the cleft . dif showed linear deposition of fluorescence outlining the basement mem brane zone ( bmz ) and extending irregularly into the superficial lamina propria as shaggy pattern . iv : histopathology showed hyperparakeratosis , thickening of the spinous cell layer , and degeneration of basal cell layer within the epithelium . dif showed deposition of one or more immunoreactants in a shaggy or granular band at the bmz . charts 1 and 2 shows the reliability of dif and iif as a confirmatory diagnostic test in patients with pemphigus , pemphigoid , lichen planus , and lupus erythematosus . oral mucosal vesiculobullous and ulcerative lesions can be broadly divided into those conditions with an acute onset and a self - limiting course and those with a chronic course . in general , acute ulcerations seldom present diagnostic problems and , being self - limiting may be ignored by the patient . chronic ulcerations , however , are often painful and persistent , causing patients to seek diagnosis and treatment . also , they frequently present diagnostic problems because the lesions may resemble each other clinically and routine biopsies may offer histological similarities and diagnosis of nonspecific inflammation . thus , immunofluorescence is increasingly being used with routine histology to accurately diagnose these lesions . so , this study was conducted with an attempt to diagnose , differentiate , and detect such vesiculo - bullous and ulcerative lesions by immunofluorescence . oral cavity was the first site to be involved in up to 70% of cases and is the only site affected in 50% of patients . buccal mucosa was most commonly involved.[35 ] also , in our study the clinical features were consistent with the above mentioned findings . williams in 1989 stated that dif performed on perilesional tissue reveals a uniform fishnet pattern of binding of igg localized to the intercellular spaces . parlowsky et al . in 2003 stated that dif reveals the deposition of complement ( c3 ) and igg , iga , or igm , within the intercellular spaces of epithelium resulting in a reticular pattern diagnostic of pemphigus . in our study also , out of seven patients , six patients showed intercellular fluorescence of igg ( 86% ) [ figure 1 ] , one case also showed complement ( c3 ) ( 14% ) [ figure 2 ] resulting in a fishnet or reticular pattern . based upon the clinico - pathologic correlation in conjunction with negative dif result , we suspected behcet 's syndrome . thus dif is essential for the diagnosis and must be performed to complement the clinical diagnosis . intercellular space deposition of igg in the epithelium ( dif ) pemphigus complement ( c3 ) found scattered in the wall of the bullae ( dif ) pemphigus sirois , et al in 2000 stated that circulating antibodies ( immunoglobulins ) are detected in 80%90% of patients with pemphigus vulgaris ( pv ) . iif performed on a monkey esophagus demonstrated the presence of circulating igg auto antibodies that bound to the epithelium with an intercellular staining pattern . mutasim et al . in 2001 stated that a punctate or granular fluorescence is appreciated at higher magnification . challacombe et al . in 2001 stated that assay of serum antibody titers by iif may also help to guide in prognostication and therapy . in our study also , circulating antibodies were detected in six patients ( 86% ) , who were symptomatic . iif performed on monkey esophagus demonstrated the presence of igg auto antibodies bound to the epithelium with an intercellular staining pattern [ figure 3 ] . a punctate or granular fluorescence was well appreciated . one case showed negative result which may be due to clinicopathologic correlation of the disease as behcet 's syndrome . six patients who were on pulse therapy for pemphigus , are completely free of lesions were grouped separately . these negative results indicate less severity of the disease , i.e. , good prognosis and helps to taper the drug dosage . granular fluorescence of igg in the intercellular space of spinosum ( iif ) pemphigus in our study , age of the patient ranged from 35 to 55 years with a mean age of 45 years . cicatricial pemphigoid ( cp ) occurs predominantly in females and bullous pemphigoid ( bp ) occurs in males . most commonly involved sites of the oral cavity are buccal mucosa , labial mucosa , gingival and palate . challacombe et al . in 2001 stated that dif using perilesional mucosa showed a linear continuous band at the bmz usually with igg and c3 but often with iga in virtually 100% of patients with clinical characteristics of pemphigoid [ bp and mucous membrane pemphigoid ( mmp ) ] . dif is essential for the diagnosis of mmp and must be performed to complement the clinical findings . jordan et al . in 2002 stated that deposition of c3 in the bmz is detected in almost all patients . in our study also , two cases showed a linear continuous band of c3 along the bmz ( 67% ) [ figure 4 ] and one case also showed fibrin in the same location ( 33% ) [ figure 5 ] . in one case , the dif result was negative which may be attributed to the mucosal peeling of the epithelium . linear and continuous band of c3 deposit along the basement membrane zone ( bmz ) ( dif ) pemphigoid linear deposits of fibrin along the bmz ( dif ) pemphigoid weinberg , et al in 1999 reported that iif studies are not reliable and may be negative or low in some cases . there is little correlation between the severity of the disease and the antibody titre , in contrast to pemphigus vulgaris in which iif studies are diagnostic . challacombe et al . in 2001 stated that the increased detection rate of circulatory antibodies by iif may be linked to the type of substrate , since salt - split skin was shown to be significantly better than intact skin , oral mucosa , or rabbit or monkey esophagus . in our study , iif performed on monkey esophagus in all the three cases showed negative results which may go in accordance with the results of few authors that selection of substrate plays an important role in the detection of circulating antibodies . like majority of authors , in our study also patients were in the age group of 3060 years and females were more commonly affected . buccal mucosa was the site most frequently involved and oral lesions in all the cases were bilateral . regezi and scuibba in 1998 stated that dif study demonstrated the presence of fibrinogen along the bmz in 90%100% of cases . jordan et al . in 2002 , stated that lp show a characteristic pattern of fibrinogen deposition outlining the bmz and extending irregularly into the superficial lamina propria , described as shaggy or fibrillar pattern . a fine granular deposition of c3 is frequently seen in bmz . in our study also , all the cases showed deposition of fibrinogen along the bmz ( 100% ) . out of six , one case showed the characteristic pattern of fibrinogen deposition outlining the bmz and extending irregularly into the superficial lamina propria as shaggy pattern [ figure 6 ] , four cases showed linear deposits of fibrin along the bmz [ figure 7 ] , and one case showed granular deposition of fibrin along the bmz . fibrin deposition along the bmz extending as irregular strands into the superficial lamina propria ( dif ) linear deposits of fibrin along the bmz ( dif ) iif may be a useful test if results of histopathologic and dif examinations are not specific . it was reported that none of the oral lesions showed the characteristic pattern of staining for lichen planus specific antigen ( lpsa ) by iif . in our study , all the patients showed negativity for iif . in our study , both the cases of systemic lupus erythematosus ( sle ) occurred in the age group of 2030 years and both were females . orally both the cases showed occurrence in the labial mucosa and one case also occurred in the buccal mucosa . in our study of two cases of sle , butterfly distribution over the malar region was seen in one case . in both the cases , generalized manifestations such as oral ulcers , histopathologically , oral lesions of sle are characterized by hyperkeratosis , alternating atrophy , thickening of the spinous cell layer , and degeneration of basal cell layer within the epithelium . subepithelial lymphocytic infiltration within the ct is also an important finding . in our study , both the cases showed nonspecific histopathologic features . in one case , in which the patient had malar rash and ana test was positive , the epithelium was denuded and the deeper stroma showed infiltration by lymphocytes . in other case , the histopathologic features showed acanthosis and slight edema in focal areas in the basal layer . dif testing of lesional tissue shows deposition of one or more immunoreactants ( usually igg , igm , or c3 ) in a shaggy or granular band at the bmz . in our study , dif results for both the cases were negative . approximately 95% of these patients have antibodies directed against multiple nuclear antigens ( antinuclear antibodies ) . antibodies directed against double stranded dna are noted in 70% patients with sle and are more specific for the disease . according to the results of our study , we could conclude that , in sle cases apart from dif , the diagnosis should be confirmed by ana and double stranded dna test only . in our study of two cases of dle , one patient is a male aged 32 years and the other is a female aged 29 years . distribution on the malar regions across the bridge of the nose . in our study , either of the two cases showed this appearance . vermillion border of the lower lip was involved in both the cases showing painful ulceration due to the crusting or bleeding which is a characteristic of dle . in our study of two cases of dle , only in one case the histopathologic features are suggestive of dle , showing features of hyperkeratosis , acanthosis , focal areas of liquefaction degeneration of basal layer , and basal zone shows deposits of pas positive material . subepithelial areas show focal collection of dense chronic inflammatory cell infiltrate . in another case , the histopathology features were suggestive of actinic chelitis showing atrophic stratified squamous epithelium , subepithelial zone of mild chronic inflammatory cells and a band of amorphous , a cellular , basophilic change known as solar elastosis . dif testing of lesional tissue shows deposition of one or more immunoreactants ( usually igg , or c3 ) in a shaggy or granular band at the bmz . also , dif testing of one case showed granular deposits of igg , c3 and faint deposits of igm along the bmz ( 25% ) [ figures 810 ] . linear deposition of igg along the bmz ( dif ) linear deposition of c3 along the bmz ( dif ) linear deposition of igm along the bmz ( dif ) few authors reported that in the recent years , substrate selection also plays a major role in the detection of circulating antibodies . if performed on human skin showed superior results when compared to oral mucosa . in our study , iif performed on monkey esophagus substrate in all four cases of lupus erythematosus showed negative results which may be attributed to substrate selection . oral cavity was the first site to be involved in up to 70% of cases and is the only site affected in 50% of patients . buccal mucosa was most commonly involved.[35 ] also , in our study the clinical features were consistent with the above mentioned findings . williams in 1989 stated that dif performed on perilesional tissue reveals a uniform fishnet pattern of binding of igg localized to the intercellular spaces . parlowsky et al . in 2003 stated that dif reveals the deposition of complement ( c3 ) and igg , iga , or igm , within the intercellular spaces of epithelium resulting in a reticular pattern diagnostic of pemphigus . in our study also , out of seven patients , six patients showed intercellular fluorescence of igg ( 86% ) [ figure 1 ] , one case also showed complement ( c3 ) ( 14% ) [ figure 2 ] resulting in a fishnet or reticular pattern . based upon the clinico - pathologic correlation in conjunction with negative dif result , we suspected behcet 's syndrome . thus dif is essential for the diagnosis and must be performed to complement the clinical diagnosis . intercellular space deposition of igg in the epithelium ( dif ) pemphigus complement ( c3 ) found scattered in the wall of the bullae ( dif ) pemphigus sirois , et al in 2000 stated that circulating antibodies ( immunoglobulins ) are detected in 80%90% of patients with pemphigus vulgaris ( pv ) . iif performed on a monkey esophagus demonstrated the presence of circulating igg auto antibodies that bound to the epithelium with an intercellular staining pattern . mutasim et al . in 2001 stated that a punctate or granular fluorescence is appreciated at higher magnification . challacombe et al . in 2001 stated that assay of serum antibody titers by iif may also help to guide in prognostication and therapy . in our study also , circulating antibodies were detected in six patients ( 86% ) , who were symptomatic . iif performed on monkey esophagus demonstrated the presence of igg auto antibodies bound to the epithelium with an intercellular staining pattern [ figure 3 ] . one case showed negative result which may be due to clinicopathologic correlation of the disease as behcet 's syndrome . six patients who were on pulse therapy for pemphigus , are completely free of lesions were grouped separately . these negative results indicate less severity of the disease , i.e. , good prognosis and helps to taper the drug dosage . in our study , age of the patient ranged from 35 to 55 years with a mean age of 45 years . cicatricial pemphigoid ( cp ) occurs predominantly in females and bullous pemphigoid ( bp ) occurs in males . most commonly involved sites of the oral cavity are buccal mucosa , labial mucosa , gingival and palate . challacombe et al . in 2001 stated that dif using perilesional mucosa showed a linear continuous band at the bmz usually with igg and c3 but often with iga in virtually 100% of patients with clinical characteristics of pemphigoid [ bp and mucous membrane pemphigoid ( mmp ) ] . dif is essential for the diagnosis of mmp and must be performed to complement the clinical findings . jordan et al . in 2002 stated that deposition of c3 in the bmz is detected in almost all patients . in our study also , two cases showed a linear continuous band of c3 along the bmz ( 67% ) [ figure 4 ] and one case also showed fibrin in the same location ( 33% ) [ figure 5 ] . in one case , the dif result was negative which may be attributed to the mucosal peeling of the epithelium . linear and continuous band of c3 deposit along the basement membrane zone ( bmz ) ( dif ) pemphigoid linear deposits of fibrin along the bmz ( dif ) pemphigoid weinberg , et al in 1999 reported that iif studies are not reliable and may be negative or low in some cases . there is little correlation between the severity of the disease and the antibody titre , in contrast to pemphigus vulgaris in which iif studies are diagnostic . challacombe et al . in 2001 stated that the increased detection rate of circulatory antibodies by iif may be linked to the type of substrate , since salt - split skin was shown to be significantly better than intact skin , oral mucosa , or rabbit or monkey esophagus . in our study , iif performed on monkey esophagus in all the three cases showed negative results which may go in accordance with the results of few authors that selection of substrate plays an important role in the detection of circulating antibodies . like majority of authors , in our study also patients were in the age group of 3060 years and females were more commonly affected . buccal mucosa was the site most frequently involved and oral lesions in all the cases were bilateral . regezi and scuibba in 1998 stated that dif study demonstrated the presence of fibrinogen along the bmz in 90%100% of cases . jordan et al . in 2002 , stated that lp show a characteristic pattern of fibrinogen deposition outlining the bmz and extending irregularly into the superficial lamina propria , described as shaggy or fibrillar pattern . a fine granular deposition of c3 is frequently seen in bmz . in our study also , all the cases showed deposition of fibrinogen along the bmz ( 100% ) . out of six , one case showed the characteristic pattern of fibrinogen deposition outlining the bmz and extending irregularly into the superficial lamina propria as shaggy pattern [ figure 6 ] , four cases showed linear deposits of fibrin along the bmz [ figure 7 ] , and one case showed granular deposition of fibrin along the bmz . fibrin deposition along the bmz extending as irregular strands into the superficial lamina propria ( dif ) linear deposits of fibrin along the bmz ( dif ) iif may be a useful test if results of histopathologic and dif examinations are not specific . it was reported that none of the oral lesions showed the characteristic pattern of staining for lichen planus specific antigen ( lpsa ) by iif . in our study , all the patients showed negativity for iif . in our study , both the cases of systemic lupus erythematosus ( sle ) occurred in the age group of 2030 years and both were females . orally both the cases showed occurrence in the labial mucosa and one case also occurred in the buccal mucosa . in our study of two cases of sle , butterfly distribution over the malar region was seen in one case . in both the cases , generalized manifestations such as oral ulcers , histopathologically , oral lesions of sle are characterized by hyperkeratosis , alternating atrophy , thickening of the spinous cell layer , and degeneration of basal cell layer within the epithelium . subepithelial lymphocytic infiltration within the ct is also an important finding . in our study , both the cases showed nonspecific histopathologic features . in one case , in which the patient had malar rash and ana test was positive , the epithelium was denuded and the deeper stroma showed infiltration by lymphocytes . in other case , the histopathologic features showed acanthosis and slight edema in focal areas in the basal layer . dif testing of lesional tissue shows deposition of one or more immunoreactants ( usually igg , igm , or c3 ) in a shaggy or granular band at the bmz . in our study , dif results for both the cases were negative . approximately 95% of these patients have antibodies directed against multiple nuclear antigens ( antinuclear antibodies ) . although this is a nonspecific finding , it is useful as a screening study . antibodies directed against double stranded dna are noted in 70% patients with sle and are more specific for the disease . according to the results of our study , we could conclude that , in sle cases apart from dif , the diagnosis should be confirmed by ana and double stranded dna test only . in our study , both the cases of systemic lupus erythematosus ( sle ) occurred in the age group of 2030 years and both were females . orally both the cases showed occurrence in the labial mucosa and one case also occurred in the buccal mucosa . in our study of two cases of sle , butterfly distribution over the malar region was seen in one case . in both the cases , generalized manifestations such as oral ulcers , histopathologically , oral lesions of sle are characterized by hyperkeratosis , alternating atrophy , thickening of the spinous cell layer , and degeneration of basal cell layer within the epithelium . subepithelial lymphocytic infiltration within the ct is also an important finding . in our study , both the cases showed nonspecific histopathologic features . in one case , in which the patient had malar rash and ana test was positive , the epithelium was denuded and the deeper stroma showed infiltration by lymphocytes . in other case , the histopathologic features showed acanthosis and slight edema in focal areas in the basal layer . dif testing of lesional tissue shows deposition of one or more immunoreactants ( usually igg , igm , or c3 ) in a shaggy or granular band at the bmz . in our study , dif results for both the cases were negative . approximately 95% of these patients have antibodies directed against multiple nuclear antigens ( antinuclear antibodies ) . although this is a nonspecific finding , it is useful as a screening study . antibodies directed against double stranded dna are noted in 70% patients with sle and are more specific for the disease . according to the results of our study , we could conclude that , in sle cases apart from dif , the diagnosis should be confirmed by ana and double stranded dna test only . in our study of two cases of dle , one patient is a male aged 32 years and the other is a female aged 29 years . distribution on the malar regions across the bridge of the nose . in our study , either of the two cases showed this appearance . vermillion border of the lower lip was involved in both the cases showing painful ulceration due to the crusting or bleeding which is a characteristic of dle . in our study of two cases of dle , only in one case the histopathologic features are suggestive of dle , showing features of hyperkeratosis , acanthosis , focal areas of liquefaction degeneration of basal layer , and basal zone shows deposits of pas positive material . subepithelial areas show focal collection of dense chronic inflammatory cell infiltrate . in another case , the histopathology features were suggestive of actinic chelitis showing atrophic stratified squamous epithelium , subepithelial zone of mild chronic inflammatory cells and a band of amorphous , a cellular , basophilic change known as solar elastosis . dif testing of lesional tissue shows deposition of one or more immunoreactants ( usually igg , or c3 ) in a shaggy or granular band at the bmz . complement components are present less frequently . in our study also , dif testing of one case showed granular deposits of igg , c3 and faint deposits of igm along the bmz ( 25% ) [ figures 810 ] . linear deposition of igg along the bmz ( dif ) linear deposition of c3 along the bmz ( dif ) linear deposition of igm along the bmz ( dif ) few authors reported that in the recent years , substrate selection also plays a major role in the detection of circulating antibodies . if performed on human skin showed superior results when compared to oral mucosa . in our study , iif performed on monkey esophagus substrate in all four cases of lupus erythematosus showed negative results which may be attributed to substrate selection . although histopathology remains gold standard for most of the diseases , it is recognized from this study that not all lesions are amenable to definitive histopathological diagnosis . dif can provide a valuable additional criterion in diagnosing chronic , ulcerative or erosive diseases of oral mucosa if the biopsy specimens are taken from appropriate sites and have attached epithelium . however , the consistency of dif can not be substantiated due to limited sample size which is attributed to cost effectiveness . iif can be used to detect circulating autoantibody in the blood which it does in approximately 80% of patients with pemphigus vulgaris . a negative result , however , does not exclude a diagnosis of pv . while monitoring the circulating pemphigus autoantibody titers via iif is not an essential part of the diagnosis of pv , it is useful in assessing therapeutic response and predicting relapse .

aim : to study the immunofluorescence pattern and to assess its reliability as a confirmatory diagnostic test in patients with pemphigus , pemphigoid , lichen planus , and lupus erythematosus and also to assess the disease activity by indirect immunofluorscence ( iif ) in patients with pemphigus only.materials and methods : twenty - six patients were included in the study group , out of which , 6 patients were clinically and histopathologically diagnosed as pemphigus , completely free of active lesions were subjected to iif only to assess the disease activity and were grouped separately . based on the clinical and provisional diagnosis , the remaining 20 patients who had active lesions were subjected to direct immunofluorscence ( dif ) and iif and were divided into four groups . biopsy specimens were taken from the periphery of the lesions and were examined by both conventional light microscopic and dif methods . five milliliters of venous blood was collected from each patient and were subjected to iif.results:histopathological diagnosis was consistent with direct immunofluorescence study in 15 cases ( 75% ) . the various immunofluorescence patterns observed in our study were consistent with those described by various authors in standard textbooks and articles.conclusion:histopathology remains gold standard for most of the diseases , it is recognized from this study that not all lesions are amenable to definitive histopathological diagnosis thus ; dif can provide a valuable additional criterion in diagnosis .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Pemphigus Pemphigoid Lichen planus Lupus Erythematosus Systemic lupus erythematosus Discoid lupus erythematosis CONCLUSION

oral mucocutaneous conditions are a group of disorders which are observed in the dental practice . however , vesiculo - bullous lesions frequently present diagnostic problems because the lesions often resemble each other clinically and routine histological examination sometimes can not differentiate between them . this study was carried out in the department of oral pathology , sri ramachandra dental college & hospital , chennai . the study population consisted of 26 patients with oral mucocutaneous diseases who were selected randomly . of the 26 patients,6 patients were under pulse therapy who were clinically and histopathologically diagnosed as pemphigus . these patients were free of active lesions and their disease activity was assessed by indirect immunofluorescence ( iif ) who were grouped separately . based on the clinical and provisional diagnosis , the remaining 20 patients who had active lesions were subjected to both direct immunofluorscence ( dif ) and iif and were divided into four groups . we carried out histopathology study with subsequent immunofluorescent technique to study the pattern of immunofluorescence in each group . whole blood was allowed to clot , centrifuged , and the serum was separated and transported to the laboratory . the study consisted of 26 patients of pemphigus , pemphigoid , lichen planus , and lupus erythematosus . they were grouped as follows : group i pemphigus ( seven cases , patients with active lesions ) group ii pemphigus ( six cases , patients on pulse therapy and completely free of active lesions ) patients in group i , ii , iii , and iv were subjected to histopathologic , dif , and iif examination , and patients in group v were subjected to iif alone . group i , ii , and v showed male predominance and group iii and iv showed female predominance . ii : histopathology showed subepithelial split , red blood cells ( rbcs ) and inflammatory cells in the cleft . dif showed linear deposition of fluorescence outlining the basement mem brane zone ( bmz ) and extending irregularly into the superficial lamina propria as shaggy pattern . iv : histopathology showed hyperparakeratosis , thickening of the spinous cell layer , and degeneration of basal cell layer within the epithelium . charts 1 and 2 shows the reliability of dif and iif as a confirmatory diagnostic test in patients with pemphigus , pemphigoid , lichen planus , and lupus erythematosus . oral mucosal vesiculobullous and ulcerative lesions can be broadly divided into those conditions with an acute onset and a self - limiting course and those with a chronic course . also , they frequently present diagnostic problems because the lesions may resemble each other clinically and routine biopsies may offer histological similarities and diagnosis of nonspecific inflammation . so , this study was conducted with an attempt to diagnose , differentiate , and detect such vesiculo - bullous and ulcerative lesions by immunofluorescence . [35 ] also , in our study the clinical features were consistent with the above mentioned findings . in our study also , out of seven patients , six patients showed intercellular fluorescence of igg ( 86% ) [ figure 1 ] , one case also showed complement ( c3 ) ( 14% ) [ figure 2 ] resulting in a fishnet or reticular pattern . thus dif is essential for the diagnosis and must be performed to complement the clinical diagnosis . intercellular space deposition of igg in the epithelium ( dif ) pemphigus complement ( c3 ) found scattered in the wall of the bullae ( dif ) pemphigus sirois , et al in 2000 stated that circulating antibodies ( immunoglobulins ) are detected in 80%90% of patients with pemphigus vulgaris ( pv ) . in our study also , circulating antibodies were detected in six patients ( 86% ) , who were symptomatic . one case showed negative result which may be due to clinicopathologic correlation of the disease as behcet 's syndrome . six patients who were on pulse therapy for pemphigus , are completely free of lesions were grouped separately . these negative results indicate less severity of the disease , i.e. granular fluorescence of igg in the intercellular space of spinosum ( iif ) pemphigus in our study , age of the patient ranged from 35 to 55 years with a mean age of 45 years . in 2001 stated that dif using perilesional mucosa showed a linear continuous band at the bmz usually with igg and c3 but often with iga in virtually 100% of patients with clinical characteristics of pemphigoid [ bp and mucous membrane pemphigoid ( mmp ) ] . dif is essential for the diagnosis of mmp and must be performed to complement the clinical findings . in our study also , two cases showed a linear continuous band of c3 along the bmz ( 67% ) [ figure 4 ] and one case also showed fibrin in the same location ( 33% ) [ figure 5 ] . in one case , the dif result was negative which may be attributed to the mucosal peeling of the epithelium . linear and continuous band of c3 deposit along the basement membrane zone ( bmz ) ( dif ) pemphigoid linear deposits of fibrin along the bmz ( dif ) pemphigoid weinberg , et al in 1999 reported that iif studies are not reliable and may be negative or low in some cases . there is little correlation between the severity of the disease and the antibody titre , in contrast to pemphigus vulgaris in which iif studies are diagnostic . in our study , iif performed on monkey esophagus in all the three cases showed negative results which may go in accordance with the results of few authors that selection of substrate plays an important role in the detection of circulating antibodies . like majority of authors , in our study also patients were in the age group of 3060 years and females were more commonly affected . in our study also , all the cases showed deposition of fibrinogen along the bmz ( 100% ) . out of six , one case showed the characteristic pattern of fibrinogen deposition outlining the bmz and extending irregularly into the superficial lamina propria as shaggy pattern [ figure 6 ] , four cases showed linear deposits of fibrin along the bmz [ figure 7 ] , and one case showed granular deposition of fibrin along the bmz . fibrin deposition along the bmz extending as irregular strands into the superficial lamina propria ( dif ) linear deposits of fibrin along the bmz ( dif ) iif may be a useful test if results of histopathologic and dif examinations are not specific . it was reported that none of the oral lesions showed the characteristic pattern of staining for lichen planus specific antigen ( lpsa ) by iif . in our study , all the patients showed negativity for iif . in our study , both the cases of systemic lupus erythematosus ( sle ) occurred in the age group of 2030 years and both were females . in our study of two cases of sle , butterfly distribution over the malar region was seen in one case . in both the cases , generalized manifestations such as oral ulcers , histopathologically , oral lesions of sle are characterized by hyperkeratosis , alternating atrophy , thickening of the spinous cell layer , and degeneration of basal cell layer within the epithelium . in our study , both the cases showed nonspecific histopathologic features . in other case , the histopathologic features showed acanthosis and slight edema in focal areas in the basal layer . in our study , dif results for both the cases were negative . antibodies directed against double stranded dna are noted in 70% patients with sle and are more specific for the disease . according to the results of our study , we could conclude that , in sle cases apart from dif , the diagnosis should be confirmed by ana and double stranded dna test only . in our study of two cases of dle , one patient is a male aged 32 years and the other is a female aged 29 years . distribution on the malar regions across the bridge of the nose . in our study , either of the two cases showed this appearance . vermillion border of the lower lip was involved in both the cases showing painful ulceration due to the crusting or bleeding which is a characteristic of dle . in our study of two cases of dle , only in one case the histopathologic features are suggestive of dle , showing features of hyperkeratosis , acanthosis , focal areas of liquefaction degeneration of basal layer , and basal zone shows deposits of pas positive material . in another case , the histopathology features were suggestive of actinic chelitis showing atrophic stratified squamous epithelium , subepithelial zone of mild chronic inflammatory cells and a band of amorphous , a cellular , basophilic change known as solar elastosis . dif testing of lesional tissue shows deposition of one or more immunoreactants ( usually igg , or c3 ) in a shaggy or granular band at the bmz . linear deposition of igg along the bmz ( dif ) linear deposition of c3 along the bmz ( dif ) linear deposition of igm along the bmz ( dif ) few authors reported that in the recent years , substrate selection also plays a major role in the detection of circulating antibodies . in our study , iif performed on monkey esophagus substrate in all four cases of lupus erythematosus showed negative results which may be attributed to substrate selection . [35 ] also , in our study the clinical features were consistent with the above mentioned findings . in 2003 stated that dif reveals the deposition of complement ( c3 ) and igg , iga , or igm , within the intercellular spaces of epithelium resulting in a reticular pattern diagnostic of pemphigus . in our study also , out of seven patients , six patients showed intercellular fluorescence of igg ( 86% ) [ figure 1 ] , one case also showed complement ( c3 ) ( 14% ) [ figure 2 ] resulting in a fishnet or reticular pattern . intercellular space deposition of igg in the epithelium ( dif ) pemphigus complement ( c3 ) found scattered in the wall of the bullae ( dif ) pemphigus sirois , et al in 2000 stated that circulating antibodies ( immunoglobulins ) are detected in 80%90% of patients with pemphigus vulgaris ( pv ) . in our study also , circulating antibodies were detected in six patients ( 86% ) , who were symptomatic . one case showed negative result which may be due to clinicopathologic correlation of the disease as behcet 's syndrome . six patients who were on pulse therapy for pemphigus , are completely free of lesions were grouped separately . these negative results indicate less severity of the disease , i.e. in our study , age of the patient ranged from 35 to 55 years with a mean age of 45 years . in 2001 stated that dif using perilesional mucosa showed a linear continuous band at the bmz usually with igg and c3 but often with iga in virtually 100% of patients with clinical characteristics of pemphigoid [ bp and mucous membrane pemphigoid ( mmp ) ] . in our study also , two cases showed a linear continuous band of c3 along the bmz ( 67% ) [ figure 4 ] and one case also showed fibrin in the same location ( 33% ) [ figure 5 ] . in one case , the dif result was negative which may be attributed to the mucosal peeling of the epithelium . linear and continuous band of c3 deposit along the basement membrane zone ( bmz ) ( dif ) pemphigoid linear deposits of fibrin along the bmz ( dif ) pemphigoid weinberg , et al in 1999 reported that iif studies are not reliable and may be negative or low in some cases . there is little correlation between the severity of the disease and the antibody titre , in contrast to pemphigus vulgaris in which iif studies are diagnostic . in our study , iif performed on monkey esophagus in all the three cases showed negative results which may go in accordance with the results of few authors that selection of substrate plays an important role in the detection of circulating antibodies . like majority of authors , in our study also patients were in the age group of 3060 years and females were more commonly affected . in our study also , all the cases showed deposition of fibrinogen along the bmz ( 100% ) . out of six , one case showed the characteristic pattern of fibrinogen deposition outlining the bmz and extending irregularly into the superficial lamina propria as shaggy pattern [ figure 6 ] , four cases showed linear deposits of fibrin along the bmz [ figure 7 ] , and one case showed granular deposition of fibrin along the bmz . fibrin deposition along the bmz extending as irregular strands into the superficial lamina propria ( dif ) linear deposits of fibrin along the bmz ( dif ) iif may be a useful test if results of histopathologic and dif examinations are not specific . it was reported that none of the oral lesions showed the characteristic pattern of staining for lichen planus specific antigen ( lpsa ) by iif . in our study , all the patients showed negativity for iif . in our study , both the cases of systemic lupus erythematosus ( sle ) occurred in the age group of 2030 years and both were females . in our study of two cases of sle , butterfly distribution over the malar region was seen in one case . in both the cases , generalized manifestations such as oral ulcers , histopathologically , oral lesions of sle are characterized by hyperkeratosis , alternating atrophy , thickening of the spinous cell layer , and degeneration of basal cell layer within the epithelium . in our study , both the cases showed nonspecific histopathologic features . in one case , in which the patient had malar rash and ana test was positive , the epithelium was denuded and the deeper stroma showed infiltration by lymphocytes . in other case , the histopathologic features showed acanthosis and slight edema in focal areas in the basal layer . dif testing of lesional tissue shows deposition of one or more immunoreactants ( usually igg , igm , or c3 ) in a shaggy or granular band at the bmz . in our study , dif results for both the cases were negative . although this is a nonspecific finding , it is useful as a screening study . antibodies directed against double stranded dna are noted in 70% patients with sle and are more specific for the disease . according to the results of our study , we could conclude that , in sle cases apart from dif , the diagnosis should be confirmed by ana and double stranded dna test only . in our study , both the cases of systemic lupus erythematosus ( sle ) occurred in the age group of 2030 years and both were females . orally both the cases showed occurrence in the labial mucosa and one case also occurred in the buccal mucosa . in our study of two cases of sle , butterfly distribution over the malar region was seen in one case . in both the cases , generalized manifestations such as oral ulcers , histopathologically , oral lesions of sle are characterized by hyperkeratosis , alternating atrophy , thickening of the spinous cell layer , and degeneration of basal cell layer within the epithelium . in our study , both the cases showed nonspecific histopathologic features . in other case , the histopathologic features showed acanthosis and slight edema in focal areas in the basal layer . dif testing of lesional tissue shows deposition of one or more immunoreactants ( usually igg , igm , or c3 ) in a shaggy or granular band at the bmz . in our study , dif results for both the cases were negative . although this is a nonspecific finding , it is useful as a screening study . antibodies directed against double stranded dna are noted in 70% patients with sle and are more specific for the disease . according to the results of our study , we could conclude that , in sle cases apart from dif , the diagnosis should be confirmed by ana and double stranded dna test only . in our study of two cases of dle , one patient is a male aged 32 years and the other is a female aged 29 years . distribution on the malar regions across the bridge of the nose . in our study , either of the two cases showed this appearance . vermillion border of the lower lip was involved in both the cases showing painful ulceration due to the crusting or bleeding which is a characteristic of dle . in our study of two cases of dle , only in one case the histopathologic features are suggestive of dle , showing features of hyperkeratosis , acanthosis , focal areas of liquefaction degeneration of basal layer , and basal zone shows deposits of pas positive material . in another case , the histopathology features were suggestive of actinic chelitis showing atrophic stratified squamous epithelium , subepithelial zone of mild chronic inflammatory cells and a band of amorphous , a cellular , basophilic change known as solar elastosis . in our study also , dif testing of one case showed granular deposits of igg , c3 and faint deposits of igm along the bmz ( 25% ) [ figures 810 ] . linear deposition of igg along the bmz ( dif ) linear deposition of c3 along the bmz ( dif ) linear deposition of igm along the bmz ( dif ) few authors reported that in the recent years , substrate selection also plays a major role in the detection of circulating antibodies . in our study , iif performed on monkey esophagus substrate in all four cases of lupus erythematosus showed negative results which may be attributed to substrate selection . although histopathology remains gold standard for most of the diseases , it is recognized from this study that not all lesions are amenable to definitive histopathological diagnosis . dif can provide a valuable additional criterion in diagnosing chronic , ulcerative or erosive diseases of oral mucosa if the biopsy specimens are taken from appropriate sites and have attached epithelium . however , the consistency of dif can not be substantiated due to limited sample size which is attributed to cost effectiveness . iif can be used to detect circulating autoantibody in the blood which it does in approximately 80% of patients with pemphigus vulgaris . while monitoring the circulating pemphigus autoantibody titers via iif is not an essential part of the diagnosis of pv , it is useful in assessing therapeutic response and predicting relapse .

the online version of this article ( doi:10.1007/s11605 - 014 - 2485 - 5 ) contains supplementary material , which is available to authorized users . while peptic ulcer disease has decreased in incidence over the past decades , the epidemiological pattern of the complications , including haemorrhage and perforation , have changed little.1 although outcomes from bleeding ulcers have improved with modern endoscopic and interventional radiological strategies,2 the outcomes of perforations have remained fairly unchanged.3 even in recent reports , the mortality from perforated peptic ulcer ( ppu ) remains up to 27 % 46 and complications are reported in 2050 % of the patients.6,7 a number of scoring systems for outcome prediction have been reported , yet none appear to be superior and most are investigated in isolation.8 among the most frequently used are the american society of anesthesiologists ( asa ) physical status classification system,7 the boey score9 and the more recently introduced peptic ulcer perforation ( pulp ) score.5 however , only the boey and pulp scores are designed specifically for the prediction of mortality for ppu patients . the boey score is the most frequently used score , but with varying degree of accuracy.7,10,11 the pulp score appears more accurate , yet it is more complex and has not been validated outside the original cohort . consequently , contemporary risk prediction in ppu patients is less well investigated with no universally agreed standard , and an optimal way of outcome prediction in this patient group is not known . thus , the aim of this study was to compare known risk scores and clinical and laboratory factors for the prediction of 30-day mortality in a consecutive cohort of patients surgically treated for perforated ulcer . the study was approved as a quality control assurance project according to the regional ethics committee ( rek vest # 2011/713 ) . the study was reported to comply with the strengthening the reporting of observational studies in epidemiology ( strobe ) statement as best applicable.12 the stavanger university hospital ( suh ) serves as the only hospital in the greater stavanger area and has a catchment area of about 340,000 inhabitants . all consecutive patients diagnosed with and operated for a perforated gastric or duodenal ulcer admitted between january 2001 and december 2010 were identified from the hospital administrative electronic database using icd-10 diagnostic codes ( k25 and k26 ) and relevant codes for surgical procedures ( i.e. jda 60 gastroraphy , jda 61 laparoscopic gastroraphy , jdh 70 duodenoraphy , jdh 71 laparoscopic duodenoraphy ) . patient demographics , including laboratory values and clinical data , were retrieved from hospital records and surgical notes . five patients with a malignant ulcer , three patients with ppu identified at autopsy , seven patients conservatively treated without operation and two patients operated on suspicion of ppu without finding any perforation were excluded . diagnosis was based on clinical symptoms and signs ( i.e presence of peritonitis ) supported by imaging ( mainly with abdominal computed tomography ( ct ) ) . a standard predefined set of laboratory values was obtained on admission for all patients with a suspected acute abdomen , including , among others , haemoglobin , c - reactive protein ( crp ) , liver enzymes , bilirubin , albumin and creatinine . the preferred surgical procedure in our institution is an open or laparoscopic primary closure of the perforation by interrupted sutures covered with a pedicled omentoplasty.3 in rare cases with no omentum ( or falciform ligament ) available , primary closure without omentoplasty was done . the primary endpoint of the study was mortality defined as death within 30 days of surgery . delay of treatment was measured as time from admittance to hospital until the start of surgery . sepsis was defined as the presence of two or more of the sepsis criteria ( i.e. temperature > 38.0 , pulse rate > 90 beats per minute , respiration rate > 20 per minute ) and in addition to infection being proved or likely . shock on admission was defined as a systolic blood pressure of < 100 mmhg and a heart rate of > 90 beats per minute . complications were graded using the clavien - dindo score.13 this classification separates complications into five categories : grades 1 and 2 are mild complications that can be medically treated ( e.g. pneumonia or urinary tract infections ) ; grade 3 complications require surgical , endoscopic or radiologic intervention ; and grade 4 complications are life - threatening complications . the boey score9 was calculated based upon the presence of shock , delay from admission to surgery of > 24 h and a high degree of co - morbidity , such as chronic obstructive pulmonary disease , heart failure and active cancer ( defined as current cancer under curative treatment or incurable cancer ) . the asa score14 was based on the patients pre - existing co - morbidity with present clinical condition at admission taken into consideration . accordingly , any acute deterioration of the patient at admission ( e.g. fulfilling the sepsis criteria or the presence of peritonitis or shock ) was incorporated in the asa score evaluation . the pulp score5 was based on age of > 65 years , co - morbidity including liver failure , aids and active cancer , concomitant use of steroids , shock on admission , time from admission to surgery of > 24 h , serum creatinine of > 130 ( mol / l ) and the above - mentioned asa score . the boey score originally measures delay of > 24 h from perforation to surgery , while the pulp score originally measures time from perforation to admission . since we measured time from admission to surgery , this time delay is used for the calculation of both the boey score and the pulp score . this means that a delay of > 24 h in this study , represents at least 24 h from perforation to surgery . optimal cut - off for each continuous variable and risk scores were calculated by the receiver operating characteristics ( roc ) curve analysis with assessment of the area under the curve ( auc ) and its 95 % confidence interval ( 95 % ci).15 an auc value of > 0.8 is considered excellent ( i.e. correctly classifies 80 % of , or four out of five , patients ) , while an auc of 0.700.80 is considered acceptable , and a value of 0.5 equals the flip of a coin.16 sensitivity and specificity with 95 % ci are given for the optimal cut - off value as defined by the roc analysis , and in addition , the corresponding positive or negative likelihood ratio ( lr+ or lr ) is given . data were analysed using the statistical package for social sciences ( v. 21 , spss inc . ) . roc analysis was performed by medcalc ( v. 12.7.5 , http://www.medcalc.org , medcalc software , ostend , belgium ) . a non - parametric distribution of data was assumed , and appropriate statistical tests were used for descriptive data . optimal cut - off values ( based on roc analyses ) were used for dichotomization of the variables for use in the binary regression analyses . clinical judgement was used for the cut - off for age , where a clinically defined cut - off was set to 60 years . logistic binary regression analysis was performed for mortality as the outcome to identify univariate risk factors . factors with a p value of < 0.20 in the univariate analyses were included in the multivariable logistic regression model , and all multivariable analyses were adjusted for gender . included factors were tested both for their continuous values and for the dichotomized variable , where applicable , to test the robustness of the model . for the final multivariable model , the corresponding predicted probability value given for each patient was tested by roc analysis to estimate the performance of the model by the auc . in addition , the same was run for each of the asa , pulp and boey scores in order to compare the accuracy performance across the models . in addition , the hosmer and lemeshow goodness - of - fit test was performed for the final multivariable regression model . for internal validation , boostrapping by 1,000 samples was performed on the final multivariable regression model . all tests are two - sided and p values of < 0.05 were regarded as statistically significant . the stavanger university hospital ( suh ) serves as the only hospital in the greater stavanger area and has a catchment area of about 340,000 inhabitants . all consecutive patients diagnosed with and operated for a perforated gastric or duodenal ulcer admitted between january 2001 and december 2010 were identified from the hospital administrative electronic database using icd-10 diagnostic codes ( k25 and k26 ) and relevant codes for surgical procedures ( i.e. jda 60 gastroraphy , jda 61 laparoscopic gastroraphy , jdh 70 duodenoraphy , jdh 71 laparoscopic duodenoraphy ) . patient demographics , including laboratory values and clinical data , were retrieved from hospital records and surgical notes . five patients with a malignant ulcer , three patients with ppu identified at autopsy , seven patients conservatively treated without operation and two patients operated on suspicion of ppu without finding any perforation were excluded . diagnosis was based on clinical symptoms and signs ( i.e presence of peritonitis ) supported by imaging ( mainly with abdominal computed tomography ( ct ) ) . a standard predefined set of laboratory values was obtained on admission for all patients with a suspected acute abdomen , including , among others , haemoglobin , c - reactive protein ( crp ) , liver enzymes , bilirubin , albumin and creatinine . the preferred surgical procedure in our institution is an open or laparoscopic primary closure of the perforation by interrupted sutures covered with a pedicled omentoplasty.3 in rare cases with no omentum ( or falciform ligament ) available , primary closure without omentoplasty was done . the primary endpoint of the study was mortality defined as death within 30 days of surgery . delay of treatment was measured as time from admittance to hospital until the start of surgery . sepsis was defined as the presence of two or more of the sepsis criteria ( i.e. temperature > 38.0 , pulse rate > 90 beats per minute , respiration rate > 20 per minute ) and in addition to infection being proved or likely . shock on admission was defined as a systolic blood pressure of < 100 mmhg and a heart rate of > 90 beats per minute . complications were graded using the clavien - dindo score.13 this classification separates complications into five categories : grades 1 and 2 are mild complications that can be medically treated ( e.g. pneumonia or urinary tract infections ) ; grade 3 complications require surgical , endoscopic or radiologic intervention ; and grade 4 complications are life - threatening complications . the boey score9 was calculated based upon the presence of shock , delay from admission to surgery of > 24 h and a high degree of co - morbidity , such as chronic obstructive pulmonary disease , heart failure and active cancer ( defined as current cancer under curative treatment or incurable cancer ) . the asa score14 was based on the patients pre - existing co - morbidity with present clinical condition at admission taken into consideration . accordingly , any acute deterioration of the patient at admission ( e.g. fulfilling the sepsis criteria or the presence of peritonitis or shock ) was incorporated in the asa score evaluation . the pulp score5 was based on age of > 65 years , co - morbidity including liver failure , aids and active cancer , concomitant use of steroids , shock on admission , time from admission to surgery of > 24 h , serum creatinine of > 130 ( mol / l ) and the above - mentioned asa score . the boey score originally measures delay of > 24 h from perforation to surgery , while the pulp score originally measures time from perforation to admission . since we measured time from admission to surgery , this time delay is used for the calculation of both the boey score and the pulp score . this means that a delay of > 24 h in this study , represents at least 24 h from perforation to surgery . optimal cut - off for each continuous variable and risk scores were calculated by the receiver operating characteristics ( roc ) curve analysis with assessment of the area under the curve ( auc ) and its 95 % confidence interval ( 95 % ci).15 an auc value of > 0.8 is considered excellent ( i.e. correctly classifies 80 % of , or four out of five , patients ) , while an auc of 0.700.80 is considered acceptable , and a value of 0.5 equals the flip of a coin.16 sensitivity and specificity with 95 % ci are given for the optimal cut - off value as defined by the roc analysis , and in addition , the corresponding positive or negative likelihood ratio ( lr+ or lr ) is given . the boey score9 was calculated based upon the presence of shock , delay from admission to surgery of > 24 h and a high degree of co - morbidity , such as chronic obstructive pulmonary disease , heart failure and active cancer ( defined as current cancer under curative treatment or incurable cancer ) . the asa score14 was based on the patients pre - existing co - morbidity with present clinical condition at admission taken into consideration . accordingly , any acute deterioration of the patient at admission ( e.g. fulfilling the sepsis criteria or the presence of peritonitis or shock ) was incorporated in the asa score evaluation . the pulp score5 was based on age of > 65 years , co - morbidity including liver failure , aids and active cancer , concomitant use of steroids , shock on admission , time from admission to surgery of > 24 h , serum creatinine of > 130 ( mol / l ) and the above - mentioned asa score . the boey score originally measures delay of > 24 h from perforation to surgery , while the pulp score originally measures time from perforation to admission . since we measured time from admission to surgery , this time delay is used for the calculation of both the boey score and the pulp score . this means that a delay of > 24 h in this study , represents at least 24 h from perforation to surgery . optimal cut - off for each continuous variable and risk scores were calculated by the receiver operating characteristics ( roc ) curve analysis with assessment of the area under the curve ( auc ) and its 95 % confidence interval ( 95 % ci).15 an auc value of > 0.8 is considered excellent ( i.e. correctly classifies 80 % of , or four out of five , patients ) , while an auc of 0.700.80 is considered acceptable , and a value of 0.5 equals the flip of a coin.16 sensitivity and specificity with 95 % ci are given for the optimal cut - off value as defined by the roc analysis , and in addition , the corresponding positive or negative likelihood ratio ( lr+ or lr ) is given . data were analysed using the statistical package for social sciences ( v. 21 , spss inc . ) . roc analysis was performed by medcalc ( v. 12.7.5 , http://www.medcalc.org , medcalc software , ostend , belgium ) . a non - parametric distribution of data was assumed , and appropriate statistical tests were used for descriptive data . optimal cut - off values ( based on roc analyses ) were used for dichotomization of the variables for use in the binary regression analyses . clinical judgement was used for the cut - off for age , where a clinically defined cut - off was set to 60 years . logistic binary regression analysis was performed for mortality as the outcome to identify univariate risk factors . factors with a p value of < 0.20 in the univariate analyses were included in the multivariable logistic regression model , and all multivariable analyses were adjusted for gender . included factors were tested both for their continuous values and for the dichotomized variable , where applicable , to test the robustness of the model . for the final multivariable model , the corresponding predicted probability value given for each patient was tested by roc analysis to estimate the performance of the model by the auc . in addition , the same was run for each of the asa , pulp and boey scores in order to compare the accuracy performance across the models . in addition , the hosmer and lemeshow goodness - of - fit test was performed for the final multivariable regression model . for internal validation , boostrapping by 1,000 samples was performed on the final multivariable regression model . all tests are two - sided and p values of < 0.05 were regarded as statistically significant . the study population comprised 172 patients with a median age of 68 ( range 18101 ) years . the 30-day mortality was 16.3 % ( 28/172 ) , and complications were encountered in 52 % ( 89/172 ) of the patients ( fig . 1 ) . there were no grade 1 complications recorded . among the complications recorded were ten suture leakages from the ulcer site , five in the laparotomy and five in the laparoscopy group ( p = 0.13 ) suture leaks were not associated with mortality ( p = 0.585).table 1clinicopathological characteristics of patients operated for perforated peptic ulcercharacteristics ( n = 172)age < 60 years ( n = 55)age 60 years ( n = 117 ) p value for differencegender , n ( % ) female20 ( 36)69 ( 59)0.006 male35 ( 64)48 ( 41)location of ulcer , n ( % ) duodenal18 ( 33)42 ( 36)0.606 gastric37 ( 67)75 ( 64)delay to surgery ( h ) , median ( range)6.3 ( 0.552.4)6.2 ( 1.1116.2)0.463operation time in min , median ( range)78 ( 41210)79 ( 34291)0.890laparoscopy16 ( 29)34 ( 29)0.997laparotomy39 ( 71)83 ( 71)asa score 1247 ( 85)52 ( 44)<0.001 38 ( 15)65 ( 56)boey score 147 ( 85)69 ( 59)0.001 > 18 ( 15)48 ( 41)pulp score < 648 ( 87)38 ( 32)<0.001 67 ( 13)79 ( 68)median ( range)3 ( 110)8 ( 114)albumin ( g / l ) median ( range)42 ( 2048)37 ( 1448)<0.001 hypoalbuminaemia ( 37 g / l ) , n ( % ) 9 ( 5)63 ( 37)<0.001bilirubin ( mol / l ) median ( range)8 ( 3197)12 ( 2481)0.004 hyperbilirubinaemia , n ( % ) 6 ( 4)18 ( 11)0.418creatinine ( mol / l ) median ( range)78 ( 27583)88 ( 34507)0.124 creatinine>1186 ( 4)32 ( 19)0.015 p value was calculated by chi - square test for categorical data and mann - whitney u test for continuous data p value represents differance between pulp scores < 6 and 6fig . 1distribution of complications according to the clavien - dindo complication grading systems clinicopathological characteristics of patients operated for perforated peptic ulcer p value was calculated by chi - square test for categorical data and mann - whitney u test for continuous data p value represents differance between pulp scores < 6 and 6 distribution of complications according to the clavien - dindo complication grading systems optimal cut - off values for each continuous variable based on roc are given in table 2 . a large variability in discrimination is shown , and none of the variables scored an auc over 0.8 . the lr + was consistently low , with none having a value over 4.table 2optimal cut - off based on receiver operating characteristics ( roc ) curve analysis for 30-day mortalityfactorcut - offsensitivity ( 95 % ci)specificity ( 95 % ci)auc p value of auclr+lrage ( years)>7953.6 ( 33.972.5)83.3 ( 76.289.0)0.74<0.0013.20.6delay ( h)>1448.2 ( 28.768.1)75.5 ( 67.682.3)0.580.222.00.7operation time ( min)8675.0 ( 55.189.3)41.6 ( 33.350.1)0.520.741.30.6asa score>385.7 ( 67.396.0)66.0 ( 57.673.7)0.79<0.0012.50.2boey score>164.3 ( 44.181.4)94.4 ( 89.397.6)0.75<0.0012.50.5pulp score>692.9 ( 76.599.1)58.3 ( 49.866.5)0.79<0.0012.30.1albumin ( g / l)3782.1 ( 63.193.9)65.5 ( 57.173.3)0.78<0.0012.40.3bilirubin ( mol / l)>1935.7 ( 18.655.9)90.2 ( 84.194.5)0.610.0963.70.7creatinine ( mol / l)>11842.9 ( 24.562.8)81.9 ( 74.787.9)0.520.782.40.7crp ( mg / l)>2178.6 ( 59.091.7)52.8 ( 44.361.1)0.69<0.0011.70.4 auc area under the curve , lr+ positive likelihood ratio , lr negative likelihood ratio , asa score american society of anesthesiologists score , pulp score peptic ulcer perforation score optimal cut - off based on receiver operating characteristics ( roc ) curve analysis for 30-day mortality auc area under the curve , lr+ positive likelihood ratio , lr negative likelihood ratio , asa score american society of anesthesiologists score , pulp score peptic ulcer perforation score univariate risk factors associated with mortality are displayed in table 3 . in addition , we analysed for ulcer site , method of operation , presence of cardiovascular disease , prednisolone use , smoking , sepsis , autoimmune disease and nsaid use , but none were statistically significantly associated with mortality . absence of peritonitis on admission was significantly associated with mortality ( p = 0.038 ) by univariate analysis , but not after multivariable regression analysis.table 3univariate regression analysis of factors associated with 30-day mortalityfactorsdeceasedalive p valueodds ratio ( 95 % ci)gendermale11720.2990.6 ( 0.31.5)female1772age ( years)>602592<0.0014.7 ( 1.416.4)60352delay ( h)>249220.0272.8 ( 1.16.9)2418121preoperative shockyes10270.0342.6 ( 1.16.2)no17117active canceryes910<0.0016.4 ( 2.317.6)no19134peritonitisyes13990.0340.4 ( 0.21.0)no1444asa score>32449<0.00111.6 ( 3.835.4)3495boey score>11838<0.0015.0 ( 2.111.8)110106pulp score>62660<0.00118.2 ( 4.279.6)6284albumin ( g / l)>37593<0.0018.7 ( 3.124.4)372349bilirubin ( mol / l)>191014<0.0015.1 ( 2.013.2)1918129creatinine ( mol / l)>11812260.0043.4 ( 1.48.1)11816118crp ( mg / l)>2122680.0024.1 ( 1.610.7)21676haemoglobin ( g / dl)>12.5141060.0132.8 ( 1.26.4)12.51438 asa score american society of anesthesiologists score , pulp score peptic ulcer perforation score univariate regression analysis of factors associated with 30-day mortality asa score american society of anesthesiologists score , pulp score peptic ulcer perforation score during multivariable modelling , non - significant variables were taken out of the regression model for optimization . adding or leaving the asa score out of the model only changed the model minimally , and the asa score was thus left out . the final multivariable regression model for mortality is presented in table 4 . the hosmer and lemeshow goodness - of - fit test ( p = 0.948 ) indicated a good model fit , and roc analyses of the predicted probability value gave an auc of 0.89 ( fig . the model was internally validated by bootstrapping , changing the p values only marginally , thus confirming the validity of the model ( see supplementary info , table 5).table 4multivariable regression analysis of factors associated with 30-day mortalityfactorswald p valueodds ratio ( 95 % ci)age10.20.0011.1 ( 1.01.1)delay > 24 h4.40.0353.5 ( 1.111.3)active cancer7.80.0057.6 ( 1.831.7)albumin 37 g / l5.60.0184.1 ( 1.313.8)bilirubin > 19 mol / l6.50.0115.1 ( 1.518.2)creatinine > 118 mol / l4.40.0363.5 ( 1.111.1)adjusted for genderfig . 2roc analysis of scores and current model in mortality prediction : a current model from the predictive probabilities of the multivariable regression model , b the pulp score , c the boey score and d the asa score multivariable regression analysis of factors associated with 30-day mortality roc analysis of scores and current model in mortality prediction : a current model from the predictive probabilities of the multivariable regression model , b the pulp score , c the boey score and d the asa score the predicted probability of each of the clinical scores , as well as the predicted probability used for variables included in the final model ( table 4 ) , is presented for comparison in fig . the final model had a better auc and more consistent 95 % ci ( fig . in addition , we analysed for ulcer site , method of operation , presence of cardiovascular disease , prednisolone use , smoking , sepsis , autoimmune disease and nsaid use , but none were statistically significantly associated with mortality . absence of peritonitis on admission was significantly associated with mortality ( p = 0.038 ) by univariate analysis , but not after multivariable regression analysis.table 3univariate regression analysis of factors associated with 30-day mortalityfactorsdeceasedalive p valueodds ratio ( 95 % ci)gendermale11720.2990.6 ( 0.31.5)female1772age ( years)>602592<0.0014.7 ( 1.416.4)60352delay ( h)>249220.0272.8 ( 1.16.9)2418121preoperative shockyes10270.0342.6 ( 1.16.2)no17117active canceryes910<0.0016.4 ( 2.317.6)no19134peritonitisyes13990.0340.4 ( 0.21.0)no1444asa score>32449<0.00111.6 ( 3.835.4)3495boey score>11838<0.0015.0 ( 2.111.8)110106pulp score>62660<0.00118.2 ( 4.279.6)6284albumin ( g / l)>37593<0.0018.7 ( 3.124.4)372349bilirubin ( mol / l)>191014<0.0015.1 ( 2.013.2)1918129creatinine ( mol / l)>11812260.0043.4 ( 1.48.1)11816118crp ( mg / l)>2122680.0024.1 ( 1.610.7)21676haemoglobin ( g / dl)>12.5141060.0132.8 ( 1.26.4)12.51438 asa score american society of anesthesiologists score , pulp score peptic ulcer perforation score univariate regression analysis of factors associated with 30-day mortality asa score american society of anesthesiologists score , pulp score peptic ulcer perforation score during multivariable modelling , non - significant variables were taken out of the regression model for optimization . adding or leaving the asa score out of the model only changed the model minimally , and the asa score was thus left out . the final multivariable regression model for mortality the hosmer and lemeshow goodness - of - fit test ( p = 0.948 ) indicated a good model fit , and roc analyses of the predicted probability value gave an auc of 0.89 ( fig . the model was internally validated by bootstrapping , changing the p values only marginally , thus confirming the validity of the model ( see supplementary info , table 5).table 4multivariable regression analysis of factors associated with 30-day mortalityfactorswald p valueodds ratio ( 95 % ci)age10.20.0011.1 ( 1.01.1)delay > 24 h4.40.0353.5 ( 1.111.3)active cancer7.80.0057.6 ( 1.831.7)albumin 37 g / l5.60.0184.1 ( 1.313.8)bilirubin > 19 mol / l6.50.0115.1 ( 1.518.2)creatinine > 118 mol / l4.40.0363.5 ( 1.111.1)adjusted for genderfig . 2roc analysis of scores and current model in mortality prediction : a current model from the predictive probabilities of the multivariable regression model , b the pulp score , c the boey score and d the asa score multivariable regression analysis of factors associated with 30-day mortality roc analysis of scores and current model in mortality prediction : a current model from the predictive probabilities of the multivariable regression model , b the pulp score , c the boey score and d the asa score the predicted probability of each of the clinical scores , as well as the predicted probability used for variables included in the final model ( table 4 ) , is presented for comparison in fig . the final model had a better auc and more consistent 95 % ci ( fig . in the current study , several clinical factors were predictive of 30-day post - operative mortality , of which the combination of increasing age , the presence of active cancer , the state of hypoalbuminaemia , presence of hyperbilirubinaemia , delay to surgery of > 24 h and increased creatinine represented the best predictive model . indicated by an auc of 0.89 , this model would correctly classify nine out of ten patients . notably , the included factors are all objective measures that are obtainable before surgery and could thus be used for improved risk prediction . while all clinical risk scores evaluated had reasonably accurate ability to predict mortality , none were excellent as deemed by the auc . moreover , the single most important factor , the state of hypoalbuminaemia , is not included in any of the three existing risk scores . an improved risk prediction model may be used for better communication with patients and next - of - kin before surgery for this disease with known high mortality . obviously , a single predictor can not be attributed to any individual patient , but the presence of several or all of the most detrimental factors may pose a much greater mortality risk compared to patients with few or none of these attributes . also , for clinical resource allocation and planning ( e.g. risk for prolonged icu or hospital stay , or need of prolonged care ) , the combined set of variables may be useful . finally , the combined score may better allow comparison of patients between studies and allow for case mix adjustments and , importantly , may also allow for potential risk stratification for future clinical trails . comparison between different patient cohorts from different regions may be valid , as all variables are objective and not influenced by subjective interpretation . hypoalbuminaemia was strongly associated with increased mortality , and this is in line with previous reports on perforated peptic ulcer.17 indeed , several past studies found a relation between preoperative hypoalbuminaemia and poor post - operative outcomes across several surgical disciplines.1820 this association may be due to the fact that a low serum albumin is closely correlated to a poor preoperative status of the patient , due to chronic disease , presence of underlying cancer , state of cachexia or other causes of malnutrition . hyperbilirubinaemia has been found to be associated with perforation in acute appendicitis , and this has been partly explained by a decrease in bile secretion as a consequence of bacteraemia.21 since similar mechanisms may apply for a perforated ulcer , hyperbilirubinaemia may also be of relevance in the management of ppu patients . mller et al . found a decrease in mortality from 27 to 17 % after initiating a care bundle protocol.22 in the current cohort , the mortality is 16 % , and the two cohorts appear comparable in most aspects . also , the mortality in our study is in the range of 1027 % as reported by recent studies from a number of countries , including the usa , denmark , scotland , israel , nigeria and ethiopia.8,2328 as our study includes all comers in a defined population with no selection in referral , we believe that the mortality rate is as would be expected for this group of patients . most of the deaths in the cohort were attributed to sepsis and multiorgane failure,6 which corroborates previous findings in ppu.29 however , the presence of sepsis preoperatively was not found to be significantly associated with mortality in the current study . notably , several other factors may likely be related to the sepsis syndrome and act as surrogates for the presence of sepsis , such as hyperbilirubinaemia and increased creatinine . increased creatinine levels may be an indicator of several conditions , including chronic renal failure ( known or unknown before diagnosis ) , the expression of pending renal failure ( due to the current disease ) , but may also be due to dehydration or reflect shock or sepsis per se . nevertheless , increased creatinine is a well - recognized risk factor for mortality both in ppu patients and in other patient groups.5,20 indeed , we recognize that several of the factors deemed to be of importance ( e.g. albumin , bilirubin and creatinine levels ) may be surrogates or indicators for other underlying factors , most likely attributed to pre - existing disease ( such as presence of cancer or severe chronic illness ) or the state of the acute disease ( e.g. reflecting dehydration , state of infection or sepsis , or altered physiology or pending organ failure ) . we did not attempt to define or investigate causality from the findings in this study . further investigation into the cause and consequence interactions for the better understanding of the nature of this disease is clearly warranted . incorporating delay as a risk factor is controversial and inconsistent across studies . the boey score originally measures delay as the time from perforation to surgery , which may be prone to error based on recall bias by the patient or record bias when prehospital data are to be obtained in retrospect . on the other side , the pulp score measures delay as the time from perforation to admission , thus not including the potential diagnostic delay that may occur in some patients prior to an established diagnosis and start of treatment . in the current study , we have obtained delay as the time interval from admission to surgery , as we believe this to be a more robust predictor , as admission to hospital and start of surgery are consistently recorded . hence , this was applied as the time delay variable for both the pulp score and the boey score . this means that a delay of > 24 h ( from admission ) represents at least 24 h of delay ( since symptom debut ) . notably , as our institution covers a region with fairly short travelling distances , the time from perforation to admission should not be considerable for most patients . however , we can not rule out an influence of this on the score parameters . even though the pulp score achieved both higher or and auc values , the boey score is a much simpler score with higher clinical usefulness than the more complex pulp score , ranging from 0 to 18 points . the asa score and the pulp score performed equally well by most aspects in our cohort . the reason for this can to some extent be attributed to our patients receiving higher asa grading . the asa grading is known to have interobserver variability , since it is not an objective system.30 the asa grade also varies according to whether or not the acute state is taken into consideration for the asa grading.31 most of the factors in the pulp score are related to preoperative status and could be included in the asa score alone , except of age and prednisolone use . since we included the acute state when grading asa , it is not so surprising that the pulp score and the asa score performed equally well . however , the asa grading which is only based upon pre - existing illness can also be problematic . clearly , a previous healthy patient presenting with septic shock and acute multiorgane failure is at high risk , but ignoring this in the asa grading can be misleading . the question remains how to best improve outcomes for the patients at high risk of dismal outcomes . as recently discussed , there are several important prognostic factors that are unmodifiable , such as age and the presence of active cancer.3 however , other important prognostic factors for mortality may be modifiable . as already addressed in the pulp study , the adherence to a sepsis - focused protocol could reduce the mortality by one third compared to conventional treatment,22 but when introducing this as a quality - of - care initiative , the factors that improved most were delay to surgery and monitoring of vital parameters , with no significant change in mortality.32 to decrease the time interval from perforation to operation appears particularly important as each hour of delay carries with it a worse prognosis.24 some limitations to this study deserve to be mentioned . however , we had little missing data in the variables obtained , likely due to a fairly consistent hospital system with electronic hospital files available for the majority of the study period . a larger study population may have revealed associations not seen in this study ; however , most of the results are in line with the recent national data from denmark,5 and as such , this study from the greater stavanger area should have a wide external validity . the combination of age , active cancer , hyperbilirubinaemia , hypoalbuminaemia , elevated creatinine and delay from perforation to surgery of > 24 h predicted mortality best . the new pulp score and the asa score predicted mortality equally well and better than the boey score , but none of them were optimal . hypoalbuminaemia was the strongest single predictor of mortality and may be included for improved risk estimation .

backgroundmortality rates in perforated peptic ulcer ( ppu ) have remained unchanged . the aim of this study was to compare known clinical factors and three scoring systems ( american society of anesthesiologists ( asa ) , boey and peptic ulcer perforation ( pulp ) ) in the ability to predict mortality in ppu.material and methodsthis is a consecutive , observational cohort study of patients surgically treated for perforated peptic ulcer over a decade ( january 2001 through december 2010 ) . primary outcome was 30-day mortality.resultsa total of 172 patients were included , of whom 28 ( 16 % ) died within 30 days . among the factors associated with mortality , the pulp score had an odds ratio ( or ) of 18.6 and the asa score had an or of 11.6 , both with an area under the curve ( auc ) of 0.79 . the boey score had an or of 5.0 and an auc of 0.75 . hypoalbuminaemia alone ( 37 g / l ) achieved an or of 8.7 and an auc of 0.78 . in multivariable regression , mortality was best predicted by a combination of increasing age , presence of active cancer and delay from admission to surgery of > 24 h , together with hypoalbuminaemia , hyperbilirubinaemia and increased creatinine values , for a model auc of 0.89.conclusionsix clinical factors predicted 30-day mortality better than available risk scores . hypoalbuminaemia was the strongest single predictor of mortality and may be included for improved risk estimation.electronic supplementary materialthe online version of this article ( doi:10.1007/s11605 - 014 - 2485 - 5 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Material and Methods Study Population Clinical Diagnosis and Surgical Treatment Study Outcomes Variables and Definitions Clinical Risk Scores Defining Cut-Offs for Optimal Sensitivity and Specificity Statistical Analysis Results Risk Factors for Mortality Discussion Conclusion Electronic supplementary material Conflict of Interest

the online version of this article ( doi:10.1007/s11605 - 014 - 2485 - 5 ) contains supplementary material , which is available to authorized users . while peptic ulcer disease has decreased in incidence over the past decades , the epidemiological pattern of the complications , including haemorrhage and perforation , have changed little.1 although outcomes from bleeding ulcers have improved with modern endoscopic and interventional radiological strategies,2 the outcomes of perforations have remained fairly unchanged.3 even in recent reports , the mortality from perforated peptic ulcer ( ppu ) remains up to 27 % 46 and complications are reported in 2050 % of the patients.6,7 a number of scoring systems for outcome prediction have been reported , yet none appear to be superior and most are investigated in isolation.8 among the most frequently used are the american society of anesthesiologists ( asa ) physical status classification system,7 the boey score9 and the more recently introduced peptic ulcer perforation ( pulp ) score.5 however , only the boey and pulp scores are designed specifically for the prediction of mortality for ppu patients . thus , the aim of this study was to compare known risk scores and clinical and laboratory factors for the prediction of 30-day mortality in a consecutive cohort of patients surgically treated for perforated ulcer . the boey score9 was calculated based upon the presence of shock , delay from admission to surgery of > 24 h and a high degree of co - morbidity , such as chronic obstructive pulmonary disease , heart failure and active cancer ( defined as current cancer under curative treatment or incurable cancer ) . the pulp score5 was based on age of > 65 years , co - morbidity including liver failure , aids and active cancer , concomitant use of steroids , shock on admission , time from admission to surgery of > 24 h , serum creatinine of > 130 ( mol / l ) and the above - mentioned asa score . the boey score originally measures delay of > 24 h from perforation to surgery , while the pulp score originally measures time from perforation to admission . since we measured time from admission to surgery , this time delay is used for the calculation of both the boey score and the pulp score . optimal cut - off for each continuous variable and risk scores were calculated by the receiver operating characteristics ( roc ) curve analysis with assessment of the area under the curve ( auc ) and its 95 % confidence interval ( 95 % ci).15 an auc value of > 0.8 is considered excellent ( i.e. the boey score9 was calculated based upon the presence of shock , delay from admission to surgery of > 24 h and a high degree of co - morbidity , such as chronic obstructive pulmonary disease , heart failure and active cancer ( defined as current cancer under curative treatment or incurable cancer ) . the pulp score5 was based on age of > 65 years , co - morbidity including liver failure , aids and active cancer , concomitant use of steroids , shock on admission , time from admission to surgery of > 24 h , serum creatinine of > 130 ( mol / l ) and the above - mentioned asa score . the boey score originally measures delay of > 24 h from perforation to surgery , while the pulp score originally measures time from perforation to admission . since we measured time from admission to surgery , this time delay is used for the calculation of both the boey score and the pulp score . optimal cut - off for each continuous variable and risk scores were calculated by the receiver operating characteristics ( roc ) curve analysis with assessment of the area under the curve ( auc ) and its 95 % confidence interval ( 95 % ci).15 an auc value of > 0.8 is considered excellent ( i.e. the boey score9 was calculated based upon the presence of shock , delay from admission to surgery of > 24 h and a high degree of co - morbidity , such as chronic obstructive pulmonary disease , heart failure and active cancer ( defined as current cancer under curative treatment or incurable cancer ) . the pulp score5 was based on age of > 65 years , co - morbidity including liver failure , aids and active cancer , concomitant use of steroids , shock on admission , time from admission to surgery of > 24 h , serum creatinine of > 130 ( mol / l ) and the above - mentioned asa score . the boey score originally measures delay of > 24 h from perforation to surgery , while the pulp score originally measures time from perforation to admission . since we measured time from admission to surgery , this time delay is used for the calculation of both the boey score and the pulp score . optimal cut - off for each continuous variable and risk scores were calculated by the receiver operating characteristics ( roc ) curve analysis with assessment of the area under the curve ( auc ) and its 95 % confidence interval ( 95 % ci).15 an auc value of > 0.8 is considered excellent ( i.e. correctly classifies 80 % of , or four out of five , patients ) , while an auc of 0.700.80 is considered acceptable , and a value of 0.5 equals the flip of a coin.16 sensitivity and specificity with 95 % ci are given for the optimal cut - off value as defined by the roc analysis , and in addition , the corresponding positive or negative likelihood ratio ( lr+ or lr ) is given . among the complications recorded were ten suture leakages from the ulcer site , five in the laparotomy and five in the laparoscopy group ( p = 0.13 ) suture leaks were not associated with mortality ( p = 0.585).table 1clinicopathological characteristics of patients operated for perforated peptic ulcercharacteristics ( n = 172)age < 60 years ( n = 55)age 60 years ( n = 117 ) p value for differencegender , n ( % ) female20 ( 36)69 ( 59)0.006 male35 ( 64)48 ( 41)location of ulcer , n ( % ) duodenal18 ( 33)42 ( 36)0.606 gastric37 ( 67)75 ( 64)delay to surgery ( h ) , median ( range)6.3 ( 0.552.4)6.2 ( 1.1116.2)0.463operation time in min , median ( range)78 ( 41210)79 ( 34291)0.890laparoscopy16 ( 29)34 ( 29)0.997laparotomy39 ( 71)83 ( 71)asa score 1247 ( 85)52 ( 44)<0.001 38 ( 15)65 ( 56)boey score 147 ( 85)69 ( 59)0.001 > 18 ( 15)48 ( 41)pulp score < 648 ( 87)38 ( 32)<0.001 67 ( 13)79 ( 68)median ( range)3 ( 110)8 ( 114)albumin ( g / l ) median ( range)42 ( 2048)37 ( 1448)<0.001 hypoalbuminaemia ( 37 g / l ) , n ( % ) 9 ( 5)63 ( 37)<0.001bilirubin ( mol / l ) median ( range)8 ( 3197)12 ( 2481)0.004 hyperbilirubinaemia , n ( % ) 6 ( 4)18 ( 11)0.418creatinine ( mol / l ) median ( range)78 ( 27583)88 ( 34507)0.124 creatinine>1186 ( 4)32 ( 19)0.015 p value was calculated by chi - square test for categorical data and mann - whitney u test for continuous data p value represents differance between pulp scores < 6 and 6fig . the lr + was consistently low , with none having a value over 4.table 2optimal cut - off based on receiver operating characteristics ( roc ) curve analysis for 30-day mortalityfactorcut - offsensitivity ( 95 % ci)specificity ( 95 % ci)auc p value of auclr+lrage ( years)>7953.6 ( 33.972.5)83.3 ( 76.289.0)0.74<0.0013.20.6delay ( h)>1448.2 ( 28.768.1)75.5 ( 67.682.3)0.580.222.00.7operation time ( min)8675.0 ( 55.189.3)41.6 ( 33.350.1)0.520.741.30.6asa score>385.7 ( 67.396.0)66.0 ( 57.673.7)0.79<0.0012.50.2boey score>164.3 ( 44.181.4)94.4 ( 89.397.6)0.75<0.0012.50.5pulp score>692.9 ( 76.599.1)58.3 ( 49.866.5)0.79<0.0012.30.1albumin ( g / l)3782.1 ( 63.193.9)65.5 ( 57.173.3)0.78<0.0012.40.3bilirubin ( mol / l)>1935.7 ( 18.655.9)90.2 ( 84.194.5)0.610.0963.70.7creatinine ( mol / l)>11842.9 ( 24.562.8)81.9 ( 74.787.9)0.520.782.40.7crp ( mg / l)>2178.6 ( 59.091.7)52.8 ( 44.361.1)0.69<0.0011.70.4 auc area under the curve , lr+ positive likelihood ratio , lr negative likelihood ratio , asa score american society of anesthesiologists score , pulp score peptic ulcer perforation score optimal cut - off based on receiver operating characteristics ( roc ) curve analysis for 30-day mortality auc area under the curve , lr+ positive likelihood ratio , lr negative likelihood ratio , asa score american society of anesthesiologists score , pulp score peptic ulcer perforation score univariate risk factors associated with mortality are displayed in table 3 . absence of peritonitis on admission was significantly associated with mortality ( p = 0.038 ) by univariate analysis , but not after multivariable regression analysis.table 3univariate regression analysis of factors associated with 30-day mortalityfactorsdeceasedalive p valueodds ratio ( 95 % ci)gendermale11720.2990.6 ( 0.31.5)female1772age ( years)>602592<0.0014.7 ( 1.416.4)60352delay ( h)>249220.0272.8 ( 1.16.9)2418121preoperative shockyes10270.0342.6 ( 1.16.2)no17117active canceryes910<0.0016.4 ( 2.317.6)no19134peritonitisyes13990.0340.4 ( 0.21.0)no1444asa score>32449<0.00111.6 ( 3.835.4)3495boey score>11838<0.0015.0 ( 2.111.8)110106pulp score>62660<0.00118.2 ( 4.279.6)6284albumin ( g / l)>37593<0.0018.7 ( 3.124.4)372349bilirubin ( mol / l)>191014<0.0015.1 ( 2.013.2)1918129creatinine ( mol / l)>11812260.0043.4 ( 1.48.1)11816118crp ( mg / l)>2122680.0024.1 ( 1.610.7)21676haemoglobin ( g / dl)>12.5141060.0132.8 ( 1.26.4)12.51438 asa score american society of anesthesiologists score , pulp score peptic ulcer perforation score univariate regression analysis of factors associated with 30-day mortality asa score american society of anesthesiologists score , pulp score peptic ulcer perforation score during multivariable modelling , non - significant variables were taken out of the regression model for optimization . 2roc analysis of scores and current model in mortality prediction : a current model from the predictive probabilities of the multivariable regression model , b the pulp score , c the boey score and d the asa score multivariable regression analysis of factors associated with 30-day mortality roc analysis of scores and current model in mortality prediction : a current model from the predictive probabilities of the multivariable regression model , b the pulp score , c the boey score and d the asa score the predicted probability of each of the clinical scores , as well as the predicted probability used for variables included in the final model ( table 4 ) , is presented for comparison in fig . absence of peritonitis on admission was significantly associated with mortality ( p = 0.038 ) by univariate analysis , but not after multivariable regression analysis.table 3univariate regression analysis of factors associated with 30-day mortalityfactorsdeceasedalive p valueodds ratio ( 95 % ci)gendermale11720.2990.6 ( 0.31.5)female1772age ( years)>602592<0.0014.7 ( 1.416.4)60352delay ( h)>249220.0272.8 ( 1.16.9)2418121preoperative shockyes10270.0342.6 ( 1.16.2)no17117active canceryes910<0.0016.4 ( 2.317.6)no19134peritonitisyes13990.0340.4 ( 0.21.0)no1444asa score>32449<0.00111.6 ( 3.835.4)3495boey score>11838<0.0015.0 ( 2.111.8)110106pulp score>62660<0.00118.2 ( 4.279.6)6284albumin ( g / l)>37593<0.0018.7 ( 3.124.4)372349bilirubin ( mol / l)>191014<0.0015.1 ( 2.013.2)1918129creatinine ( mol / l)>11812260.0043.4 ( 1.48.1)11816118crp ( mg / l)>2122680.0024.1 ( 1.610.7)21676haemoglobin ( g / dl)>12.5141060.0132.8 ( 1.26.4)12.51438 asa score american society of anesthesiologists score , pulp score peptic ulcer perforation score univariate regression analysis of factors associated with 30-day mortality asa score american society of anesthesiologists score , pulp score peptic ulcer perforation score during multivariable modelling , non - significant variables were taken out of the regression model for optimization . the model was internally validated by bootstrapping , changing the p values only marginally , thus confirming the validity of the model ( see supplementary info , table 5).table 4multivariable regression analysis of factors associated with 30-day mortalityfactorswald p valueodds ratio ( 95 % ci)age10.20.0011.1 ( 1.01.1)delay > 24 h4.40.0353.5 ( 1.111.3)active cancer7.80.0057.6 ( 1.831.7)albumin 37 g / l5.60.0184.1 ( 1.313.8)bilirubin > 19 mol / l6.50.0115.1 ( 1.518.2)creatinine > 118 mol / l4.40.0363.5 ( 1.111.1)adjusted for genderfig . 2roc analysis of scores and current model in mortality prediction : a current model from the predictive probabilities of the multivariable regression model , b the pulp score , c the boey score and d the asa score multivariable regression analysis of factors associated with 30-day mortality roc analysis of scores and current model in mortality prediction : a current model from the predictive probabilities of the multivariable regression model , b the pulp score , c the boey score and d the asa score the predicted probability of each of the clinical scores , as well as the predicted probability used for variables included in the final model ( table 4 ) , is presented for comparison in fig . in the current study , several clinical factors were predictive of 30-day post - operative mortality , of which the combination of increasing age , the presence of active cancer , the state of hypoalbuminaemia , presence of hyperbilirubinaemia , delay to surgery of > 24 h and increased creatinine represented the best predictive model . hypoalbuminaemia was strongly associated with increased mortality , and this is in line with previous reports on perforated peptic ulcer.17 indeed , several past studies found a relation between preoperative hypoalbuminaemia and poor post - operative outcomes across several surgical disciplines.1820 this association may be due to the fact that a low serum albumin is closely correlated to a poor preoperative status of the patient , due to chronic disease , presence of underlying cancer , state of cachexia or other causes of malnutrition . most of the deaths in the cohort were attributed to sepsis and multiorgane failure,6 which corroborates previous findings in ppu.29 however , the presence of sepsis preoperatively was not found to be significantly associated with mortality in the current study . even though the pulp score achieved both higher or and auc values , the boey score is a much simpler score with higher clinical usefulness than the more complex pulp score , ranging from 0 to 18 points . the asa grading is known to have interobserver variability , since it is not an objective system.30 the asa grade also varies according to whether or not the acute state is taken into consideration for the asa grading.31 most of the factors in the pulp score are related to preoperative status and could be included in the asa score alone , except of age and prednisolone use . the combination of age , active cancer , hyperbilirubinaemia , hypoalbuminaemia , elevated creatinine and delay from perforation to surgery of > 24 h predicted mortality best . the new pulp score and the asa score predicted mortality equally well and better than the boey score , but none of them were optimal . hypoalbuminaemia was the strongest single predictor of mortality and may be included for improved risk estimation .

the control of human prehensile movements has been widely studied ( for reviews see castiello 2005 ; smeets and brenner , 1999 ) . the number of degrees of freedom in the arm exceeds the number of degrees of freedom necessary to specify the contact points of the digits . moreover , for most objects , the contact points themselves can be chosen from many possibilities . this redundancy makes the system flexible ( robertson and miall 1997 ) , so regularities in the postures that are observed when subjects perform reach - to - grasp tasks are not trivial . studies on 3d pointing have shown that final arm postures largely obey donders law ( miller et al . 1992 ) , although deviations of a few degrees are often observed ( gielen et al . grasping postures toward objects in the sagittal plane are even very consistent after perturbations , suggesting that the central nervous system uses final posture as a control variable ( desmurget and prablanc 1997 ; grea et al . final postures of reach - to - grasp movements also tend to be very consistent across repetitions in non - human primates ( helms et al . ideas that have been put forward for the choice of final postures for pointing movements are the minimization of travel costs ( rosenbaum et al . 1989 ) , and avoiding extreme joint angles ( cruse and bruwer 1987 ) to prevent degraded position signals ( rossetti et al . studies on grasping have been interpreted as showing an invariant grasp orientation in body - centered coordinates ( paulignan et al . 1997 ) or as a function of movement direction ( bennis and roby - brami 2002 ; roby - brami et al . 2000 , 2003 ) . however , in these studies , only the grip orientation was measured and not the configuration of the whole arm . because the studies on whole - arm posture mentioned previously have focused on pointing and studies on grasping have been limited to the grasp orientation or to a small range of object positions , it remains unclear what determines the final arm posture in grasping . in the current study , we set out to determine which factors determine the grasp posture in an unconstrained reach - to - grasp movement . we wanted subjects to be free to use all the degrees of freedom at their disposal including translating the shoulder and rotating the wrist . therefore , we did not restrict torso movement and we did not use a splint on the wrist . we used a sphere as the target to leave the grasp orientation as free as possible . by varying the position of the sphere , as well as the starting position of the hand and the position at which the sphere was to be placed , the role of all these factors one might expect considerable effects of movement direction ( e.g. starting and placing position ) on the grip orientation and posture of the arm , because most theories state that movement - related costs are minimized . on the other hand , if the effect of starting position is as small as in pointing [ only small deviations from donders law ( gielen et al . 1997 ) ] , the effect of starting position may be small . we found that the latter prediction turned out to be correct ; effects of movement direction were observed , but they were small compared to the effects of the position of the sphere . this study involved six subjects ( two of whom were authors ) and was part of a program that was approved by the local ethics committee . all subjects signed an informed consent form before participating . in the experiment , subjects made reach - to - grasp movements toward a glass opaque sphere with a 4.5 cm diameter . they were seated comfortably behind a table on which a board with indentations on a three by seven grid was mounted ( fig . 1 ) . the indentations were spaced 10 cm apart and indicated the 21 possible target positions . movements could start at one of two positions , with thumb and index finger touching each other . the starting position was either near the body ( 10 cm from the edge of the table ) or far from the body ( 40 cm further away ) at the same lateral position as the body midline . because the workspace was quite large and the starting and placing positions were on opposite sides of the grid , we tested a wide range of movement directions . the largest gap between sampled movement directions was 37 ( 18.5 to either side of the lateral direction).fig . 1overview of the experiment . a top view of a single trial : a subject moved her hand from the starting position to pick up a sphere ( indicated in yellow ) and put it at the placing position . if the placing position was different from the starting position , she then brought her hand back to the starting position . the movement path of the thumb is shown in red and the path of the index finger in blue . are wrist angle 2 , elbow angle 3 , shoulder angle 4 , and trunkhead rotation 6 . b definition of the grasp angle 1 , azimuth from the shoulder ( a ) , and distance from the shoulder ( d ) . a top view of a single trial : a subject moved her hand from the starting position to pick up a sphere ( indicated in yellow ) and put it at the placing position . if the placing position was different from the starting position , she then brought her hand back to the starting position . the movement path of the thumb is shown in red and the path of the index finger in blue . are wrist angle 2 , elbow angle 3 , shoulder angle 4 , and trunkhead rotation 6 . b definition of the grasp angle 1 , azimuth from the shoulder ( a ) , and distance from the shoulder ( d ) . c definition of shoulder elevation 5 movements were recorded at 100 hz with an optotrak motion recording system ( northern digital , waterloo , ontario , canada ) . clusters of three infrared emitting diodes ( ireds ) were attached to the nails of the thumb and index finger . single markers were attached to the outer edge of the acromion , the epicondylus lateralis humerus , the proc . a calibration trial was performed to be able to reconstruct the approximate positions of the contact points of the thumb and the index finger from the positions of the clusters of markers attached to the nails . during this trial , the subject held an ired between thumb and index finger such that the ired was approximately at the position of the contact points of the thumb and index finger with the sphere . if the starting position was near the body , the subject grasped the sphere at the target location and then placed it at the far location before returning to the starting position ( as in fig . 1 ) , and if the starting position was far from the body , the sphere was to be placed at the near location before returning to the starting position . in the second session , the sphere was to be grasped and then placed at the starting position of the hand . five blocks with the starting position nearby and five blocks with the starting position far away . each block consisted of one trial for each possible position of the sphere , in random order . all 21 positions could be used in the blocks with the starting position close by . when starting far away , the trial starts with the arm reaching over the table . positions that were occluded by the arm in the starting posture could not be used , so blocks with the starting position far away consisted of fewer than 21 trials . before a trial started , the subject moved his right hand to the starting position with thumb and index finger touching each other . the subject subsequently closed his eyes so that the starting posture could not be influenced by seeing the position of the sphere . after the experimenter placed the sphere at the target position , she gave a verbal signal to the subject . upon this signal , the subject opened his eyes , grasped the sphere , and placed it at the appropriate location . in the first session , the subject then had to move his hand back to the starting position to start the next trial . in the second session , the subject had to put the sphere at the start position and wait for the experimenter to remove it before placing the hand for the next trial . we used the relationship between the clusters of three markers attached to the nails and the single marker held between finger and thumb during the calibration to determine the position of the digits . the beginning of the reach - to - grasp movement was defined as the first moment the speed of the wrist exceeded 0.1 m / s . the end of the reach - to - grasp movement was determined using the msi - method that has been described in detail elsewhere ( schot et al . this method finds the moment that can best be considered as the end of the movement . for this , we required the digits sagittal position to be between 6 cm and 34 cm from the near starting position ( the sphere could be placed at 10 , 20 or 30 cm ) and their height to be lower than the sphere s height . additionally , the grip size should be decreasing and decelerating . from the time points that remained , the time point with the most convincing combination of a grip size close to the sphere s diameter ( with an additional penalty on larger apertures ) and a low speed of the wrist was selected as the movement endpoint . for a full account of how this was implemented and of the robustness of the method see schot et al . we were interested in the posture at the end of the reach - to - grasp movement , so we calculated the grasp orientation , wrist angle , elbow angle , shoulder angle , shoulder elevation , trunk - head rotation , and sagittal and lateral head position at the end of the reach - to - grasp movement . grasp orientation is defined as the angle in the horizontal plane between the projection of the opposition axis and the reference axis pointing rightward . the wrist angle is the smallest angle in space between the forearm and the digits as defined by the positions of the elbow , wrist and the average position of the thumb and index finger ( aligned is 0 , flexion is positive ) . the elbow angle is defined as the smallest angle in space between the upper arm and the forearm as defined by the positions of the shoulder , elbow and wrist ( fully extended is 0 , flexion is positive ) . the shoulder angle is defined as the angle between the projection in the horizontal plane of the upper arm and the line connecting the shoulder and the head . shoulder elevation is defined as the smallest angle between the upper arm ( as defined by the shoulder and elbow positions ) and the vertical ( upper arm straight down is 0 ) . finally , trunk - head rotation is defined as the angle in the horizontal plane between the projection of the vector from the shoulder to the head and the reference axis pointing rightward . because one of the ireds used to calculate trunk - head rotation was placed on the forehead and the other on the shoulder , this measure contains trunk rotation as well as head - on - trunk rotation . we estimated that the sd of the contribution of head - on - trunk rotation when calculating trunk - head rotation was 3.5 ( see appendix for details ) . this is much smaller than the within - subject sd for trunk - head rotation ( 11.2 ) , so this variation is mainly caused by trunk rotation rather than head - on - trunk rotation . we wanted to examine how well grasp orientation , wrist angle , elbow angle , shoulder angle , shoulder elevation , trunk - head rotation , and sagittal and lateral head position at the end of the reach - to - grasp movement could be predicted from starting position , placing position , and the sphere s azimuth and distance from the subject before the movement . to achieve this , we encoded start position and place position as 0 ( far ) or 1 ( close ) . the encoding is arbitrary , but using 0 and 1 has the advantage that the resulting coefficient directly matches the size of the effect . we used a binary value rather than a continuous one such as movement direction , because a continuous measure would correlate strongly with other variables such as target azimuth . azimuth is defined as the angle between the projection in the horizontal plane of the vector connecting the subject s shoulder position with the sphere , and a reference axis pointing rightward in the horizontal plane ( a in fig . distance is the length of the vector from the subject s shoulder position to the sphere ( d in fig . for this , we took each subject s average shoulder position at the beginning of the movement . we did so because as the position of the shoulder was not fixed , shoulder position at movement onset could vary systematically across conditions ( e.g. the shoulder was further forward when the starting position was far from the body ) . this would influence the calculation of azimuth and distance from the shoulder and therewith confound the analysis in terms of the positions relative to the chair . we therefore used the average of all shoulder positions at the beginning of the movement instead of the actual shoulder position in each trial . for each subject , we performed a stepwise multiple regression analyses for each angle or position with starting position , placing position , azimuth and distance as independent variables . the distance and azimuth of the sphere expressed relative to the subjects average shoulder position is obviously correlated with its distance and azimuth relative to the subjects average head position . to be sure , we would not obtain a better fit by expressing azimuth and distance relative to the average head position rather than the average shoulder position , we also did the regression with azimuth and distance calculated relative to the average head position . because it hardly mattered for the quality of the fits whether the azimuth and distance were expressed relative to the shoulder or relative to the head ( the r values were slightly larger when azimuth and distance were calculated relative to the shoulder , but the largest r difference was only 0.06 ) , we will only report the results of the analyses with the sphere s azimuth and distance defined relative to the shoulder ( and refer to this as azimuth and distance ) . however , as we can not clearly distinguish between the two accounts , it should be noted that we are not committed to the expression relative to the shoulder . this study involved six subjects ( two of whom were authors ) and was part of a program that was approved by the local ethics committee . all subjects signed an informed consent form before participating . in the experiment , subjects made reach - to - grasp movements toward a glass opaque sphere with a 4.5 cm diameter . they were seated comfortably behind a table on which a board with indentations on a three by seven grid was mounted ( fig . 1 ) . the indentations were spaced 10 cm apart and indicated the 21 possible target positions . movements could start at one of two positions , with thumb and index finger touching each other . the starting position was either near the body ( 10 cm from the edge of the table ) or far from the body ( 40 cm further away ) at the same lateral position as the body midline . because the workspace was quite large and the starting and placing positions were on opposite sides of the grid , we tested a wide range of movement directions . the largest gap between sampled movement directions was 37 ( 18.5 to either side of the lateral direction).fig . 1overview of the experiment . a top view of a single trial : a subject moved her hand from the starting position to pick up a sphere ( indicated in yellow ) and put it at the placing position . if the placing position was different from the starting position , she then brought her hand back to the starting position . the movement path of the thumb is shown in red and the path of the index finger in blue . are wrist angle 2 , elbow angle 3 , shoulder angle 4 , and trunkhead rotation 6 . b definition of the grasp angle 1 , azimuth from the shoulder ( a ) , and distance from the shoulder ( d ) . a top view of a single trial : a subject moved her hand from the starting position to pick up a sphere ( indicated in yellow ) and put it at the placing position . if the placing position was different from the starting position , she then brought her hand back to the starting position . the movement path of the thumb is shown in red and the path of the index finger in blue . are wrist angle 2 , elbow angle 3 , shoulder angle 4 , and trunkhead rotation 6 . b definition of the grasp angle 1 , azimuth from the shoulder ( a ) , and distance from the shoulder ( d ) . c definition of shoulder elevation 5 movements were recorded at 100 hz with an optotrak motion recording system ( northern digital , waterloo , ontario , canada ) . clusters of three infrared emitting diodes ( ireds ) were attached to the nails of the thumb and index finger . single markers were attached to the outer edge of the acromion , the epicondylus lateralis humerus , the proc . a calibration trial was performed to be able to reconstruct the approximate positions of the contact points of the thumb and the index finger from the positions of the clusters of markers attached to the nails . during this trial , the subject held an ired between thumb and index finger such that the ired was approximately at the position of the contact points of the thumb and index finger with the sphere . data were collected in two sessions . in the first session , if the starting position was near the body , the subject grasped the sphere at the target location and then placed it at the far location before returning to the starting position ( as in fig . 1 ) , and if the starting position was far from the body , the sphere was to be placed at the near location before returning to the starting position . in the second session , the sphere was to be grasped and then placed at the starting position of the hand . five blocks with the starting position nearby and five blocks with the starting position far away . each block consisted of one trial for each possible position of the sphere , in random order . all 21 positions could be used in the blocks with the starting position close by . when starting far away , the trial starts with the arm reaching over the table . positions that were occluded by the arm in the starting posture could not be used , so blocks with the starting position far away consisted of fewer than 21 trials . before a trial started , the subject moved his right hand to the starting position with thumb and index finger touching each other . the subject subsequently closed his eyes so that the starting posture could not be influenced by seeing the position of the sphere . after the experimenter placed the sphere at the target position , she gave a verbal signal to the subject . upon this signal , the subject opened his eyes , grasped the sphere , and placed it at the appropriate location . in the first session , the subject then had to move his hand back to the starting position to start the next trial . in the second session , the subject had to put the sphere at the start position and wait for the experimenter to remove it before placing the hand for the next trial . we used the relationship between the clusters of three markers attached to the nails and the single marker held between finger and thumb during the calibration to determine the position of the digits . the beginning of the reach - to - grasp movement was defined as the first moment the speed of the wrist exceeded 0.1 m / s . the end of the reach - to - grasp movement was determined using the msi - method that has been described in detail elsewhere ( schot et al . this method finds the moment that can best be considered as the end of the movement . for this , we required the digits sagittal position to be between 6 cm and 34 cm from the near starting position ( the sphere could be placed at 10 , 20 or 30 cm ) and their height to be lower than the sphere s height . points that remained , the time point with the most convincing combination of a grip size close to the sphere s diameter ( with an additional penalty on larger apertures ) and a low speed of the wrist was selected as the movement endpoint . for a full account of how this was implemented and of the robustness of the method see schot et al . we were interested in the posture at the end of the reach - to - grasp movement , so we calculated the grasp orientation , wrist angle , elbow angle , shoulder angle , shoulder elevation , trunk - head rotation , and sagittal and lateral head position at the end of the reach - to - grasp movement . grasp orientation is defined as the angle in the horizontal plane between the projection of the opposition axis and the reference axis pointing rightward . the wrist angle is the smallest angle in space between the forearm and the digits as defined by the positions of the elbow , wrist and the average position of the thumb and index finger ( aligned is 0 , flexion is positive ) . the elbow angle is defined as the smallest angle in space between the upper arm and the forearm as defined by the positions of the shoulder , elbow and wrist ( fully extended is 0 , flexion is positive ) . the shoulder angle is defined as the angle between the projection in the horizontal plane of the upper arm and the line connecting the shoulder and the head . shoulder elevation is defined as the smallest angle between the upper arm ( as defined by the shoulder and elbow positions ) and the vertical ( upper arm straight down is 0 ) . finally , trunk - head rotation is defined as the angle in the horizontal plane between the projection of the vector from the shoulder to the head and the reference axis pointing rightward . because one of the ireds used to calculate trunk - head rotation was placed on the forehead and the other on the shoulder , this measure contains trunk rotation as well as head - on - trunk rotation . we estimated that the sd of the contribution of head - on - trunk rotation when calculating trunk - head rotation was 3.5 ( see appendix for details ) . this is much smaller than the within - subject sd for trunk - head rotation ( 11.2 ) , so this variation is mainly caused by trunk rotation rather than head - on - trunk rotation . we wanted to examine how well grasp orientation , wrist angle , elbow angle , shoulder angle , shoulder elevation , trunk - head rotation , and sagittal and lateral head position at the end of the reach - to - grasp movement could be predicted from starting position , placing position , and the sphere s azimuth and distance from the subject before the movement . to achieve this , we encoded start position and place position as 0 ( far ) or 1 ( close ) . the encoding is arbitrary , but using 0 and 1 has the advantage that the resulting coefficient directly matches the size of the effect . we used a binary value rather than a continuous one such as movement direction , because a continuous measure would correlate strongly with other variables such as target azimuth . azimuth is defined as the angle between the projection in the horizontal plane of the vector connecting the subject s shoulder position with the sphere , and a reference axis pointing rightward in the horizontal plane ( a in fig . distance is the length of the vector from the subject s shoulder position to the sphere ( d in fig . 1b ) . in the definition of distance and azimuth , we used shoulder position . for this , we took each subject s average shoulder position at the beginning of the movement . we did so because as the position of the shoulder was not fixed , shoulder position at movement onset could vary systematically across conditions ( e.g. the shoulder was further forward when the starting position was far from the body ) . this would influence the calculation of azimuth and distance from the shoulder and therewith confound the analysis in terms of the positions relative to the chair . we therefore used the average of all shoulder positions at the beginning of the movement instead of the actual shoulder position in each trial . for each subject , we performed a stepwise multiple regression analyses for each angle or position with starting position , placing position , azimuth and distance as independent variables . the distance and azimuth of the sphere expressed relative to the subjects average shoulder position is obviously correlated with its distance and azimuth relative to the subjects average head position . to be sure , we would not obtain a better fit by expressing azimuth and distance relative to the average head position rather than the average shoulder position , we also did the regression with azimuth and distance calculated relative to the average head position . because it hardly mattered for the quality of the fits whether the azimuth and distance were expressed relative to the shoulder or relative to the head ( the r values were slightly larger when azimuth and distance were calculated relative to the shoulder , but the largest r difference was only 0.06 ) , we will only report the results of the analyses with the sphere s azimuth and distance defined relative to the shoulder ( and refer to this as azimuth and distance ) . however , as we can not clearly distinguish between the two accounts , it should be noted that we are not committed to the expression relative to the shoulder . the lengths of each subject s upper arm , forearm , and hand are shown in table 1 , as are the mean sd of the grasp orientation , joint angles and head position . we see considerable differences in average postures across subjects but these can not be easily attributed to differences in segment lengths.table 1various measures for each subjectsubjectupper armforearmhandgrasp orientationwrist angleelbow angleshoulder angleshoulder elevationtrunk - head rotationlateral head positionsagittal head position131.923.615.178.2 23.136.3 7.965.0 17.9103.7 20.741.0 8.629.5 12.33.8 3.912.5 8.5231.826.417.355.8 25.427.6 6.168.9 16.392.7 21.545.0 5.841.9 8.810.4 5.17.9 3.2332.524.517.172.6 25.427.6 7.572.2 18.193.8 21.842.9 6.634.7 11.08.4 4.510.5 3.6428.521.714.472.7 26.524.7 5.768.8 15.997.5 17.654.3 3.931.9 12.94.4 5.39.1 3.7528.823.415.962.4 25.824.3 4.481.9 15.977.0 30.037.8 3.032.3 11.64.1 5.43.7 2.0633.627.619.362.5 22.424.6 4.671.4 14.787.2 20.643.4 4.931.5 10.46.2 4.110.3 2.4upper arm , forearm , and hand length ( in cm ) and the means sd s at the time of grasping of the angles ( in degrees ) and positions ( in cm , from the near starting position ) various measures for each subject upper arm , forearm , and hand length ( in cm ) and the means sd s at the time of grasping of the angles ( in degrees ) and positions ( in cm , from the near starting position ) the average grasp orientations for spheres at all 21 positions and the average postures at the end of reach - to - grasp movements to five of these positions are shown in fig . they were further counterclockwise when the sphere was further to the left and when the movement had started from far . this can result in different positions of the more distal joints without differences in their joint angles.fig . the four combinations of starting and placing positions are indicated by different colors . a average axes connecting index finger and thumb for all object positions ( not to scale ) b average postures for five selected positions . lines connect the markers on the head , shoulder , elbow , and wrist and connect the marker on the wrist to the calculated positions of the thumb and index finger grasping postures of one subject . the four combinations of starting and placing positions are indicated by different colors . a average axes connecting index finger and thumb for all object positions ( not to scale ) b average postures for five selected positions . lines connect the markers on the head , shoulder , elbow , and wrist and connect the marker on the wrist to the calculated positions of the thumb and index finger to test to what extent the grasp orientation , wrist angle , elbow angle , shoulder angle , shoulder elevation , trunk - head rotation , and the lateral and sagittal position of the head could be predicted from the distance ( d ) , azimuth ( a ) , and starting and placing positions ( s and p , respectively ; with values of 0 for far and 1 for near ) , eight stepwise multiple regression analyses were performed for each subject to find the best coefficients for eq . 1 , where is the prediction of one of the dependent measures.1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{y } = y_{0 } + c_{1 } s + c_{2 } p + c_{3 } a + c_{4 } d $ $ \end{document } the mean sd of the coefficients that we found by fitting eq . 1 to the data of each subject are shown in table 2 of the angular measures and in table 3 for head position , as are the proportions of variance explained by the individual predictors ( r ) , the total variance explained by the predictors ( rtotal2 ) , and the residual standard deviations after fitting eq . 1 . the rtotal2 values show that grasp orientation is very predictable , as are shoulder angle , trunk - head rotation , and elbow angle . the sizes of the mean individual r values indicate how much a predictor contributed to the prediction of a particular angle or position . for instance , grasp orientation mainly depended on the objects azimuth , followed by distance and starting position . placing position only has a small contribution of 1.7 and does not reach significance in 2 out of 6 subjects . similarly , wrist angle mainly depended on starting position.table 2results of the regression analyses for angular measuresindependentdependentgrasp orientationwrist angleelbow angleshoulder angleshoulder elevationtrunk - head rotationcoefficientrcoefficientrcoefficientrcoefficientrcoefficientrcoefficientry022.92 16.9340.84 13.76135.43 10.5522.66 23.4726.69 9.8989.69 8.76start ( )c19.50 6.040.066.09 1.550.255.40 6.590.043.35 6.380.020.94 3.840.110.74 5.260.04place ( )c21.70 2.960.001.09 3.020.042.42 2.260.011.05 3.820.011.84 2.020.030.16 2.720.01azimuthc31.06 0.100.850.01 0.040.030.21 0.060.060.46 0.050.610.00 0.020.080.26 0.040.65distance ( /cm)c40.39 0.120.150.20 0.100.081.62 0.130.721.24 0.370.670.30 0.120.290.53 0.110.59rtotal20.930.410.840.880.440.86residual ( )6.46 1.374.54 0.806.43 1.577.00 1.724.08 1.533.88 0.83coefficients are the fit values in eq . 1 the number in brackets indicates the number of subjects for whom this coefficient was statistically significant . residual gives the standard deviations of the data s deviations from the predictions ( mean sd of the six subjects)table 3results of the regression analyses for head positionindependentdependentlateral head positionsagittal head positioncoefficientrcoefficientry015.99 23.998.59 34.33start ( cm)c10.16 0.490.004.02 5.570.23place ( cm)c20.36 0.960.011.58 1.120.05azimuth ( cm/)c30.13 0.020.690.01 0.010.01distancec40.14 0.030.440.06 0.080.04rtotal20.770.31residual ( cm)2.23 0.382.65 0.52see table 2 for further details results of the regression analyses for angular measures coefficients are the fit values in eq . 1 . the number in brackets indicates the number of subjects for whom this coefficient was statistically significant . residual gives the standard deviations of the data s deviations from the predictions ( mean sd of the six subjects ) results of the regression analyses for head position see table 2 for further details the results of the analyses show that even though there are differences between the individual subjects , the factors that have most predictive power have it in all the individual subjects . correlating total limb length ( the sum of upper arm , forearm , and hand length given in table 1 ) with the coefficients obtained in the regression showed that differences between subjects could not easily be attributed to differences in limb size ( all p > 0.15 ) . correlating the coefficients with upper arm , forearm or hand length did not give more significant results than could be expected by chance ( 5% ) either . the only significant correlation was of upper arm length and the coefficient of distance when predicting shoulder elevation . 3 . in this figure , we plot the measured angles and positions as a function of azimuth and distance . we also plot the average fit . as s and p are both binary variables , the equation describes four planes ( one for each combination of starting and placing position ) . the separation between the planes indicates that on average the grasp orientation is 9.5 more counterclockwise when the starting position was far rather than near , and only 1.7 more counterclockwise when the place position was far rather than near . the fact that the planes are only slightly tilted along the distance axis indicates that distance did not explain much of the variance ( after considering the predictive power of the azimuth ) . except in the case of the sagittal head position and the wrist angle , the separation between the planes is much smaller than the change in height along the distance and/or the azimuth axis . this means that most parameters are mainly determined by the sphere s position , independent of the start and place positions.fig . as there are four combinations of starting and placing position , we drew a plane for each combination visualization of the means of the eight regression analyses done per subject . as there are four combinations of starting and placing position , we drew a plane for each combination because the difference in movement direction between the two starting positions is larger for the central part of the workspace , one might expect that the effect of starting position on grasp orientation was larger for movements to targets that were in the center of the workspace . to test this the result showed that for the center of the workspace , the effect of starting position on grasp orientation was slightly larger ( 12.2 ) than the effect over the whole workspace ( 9.5 ) . in this study , we explored the factors that determine the posture of the arm when grasping a sphere in order to place it elsewhere . we tested whether grasp postures were more predictable when the sphere s position was expressed relative to the subject s head or shoulder . we found that both methods yielded about the same r for all angles and positions we wanted to predict . given this result , we only report to what extent the grasp orientation , wrist angle , elbow angle , shoulder angle , shoulder elevation , trunk - head rotation , and the lateral and sagittal position of the head were predictable given the sphere s position in terms of the azimuth and distance from the shoulder and the starting and placing positions . however , as we could not distinguish between the two accounts , we would like to mention again that we are not committed to the expression of azimuth and distance relative to the shoulder . we found only small effects of placing position on the final posture of the reach - to - grasp movement . this suggests that a subsequent movement is only very modestly taken into account when people plan the final posture of the previous movement . this is in seeming contrast with observations that subjects adjust the grip orientation of a movement by 180 in order to avoid uncomfortable postures in a subsequent movement ( rosenbaum et al . our results suggest that a considerable effect of a subsequent movement may only have considerable effects when the movement involves uncomfortable postures . for instance , although all objects could be reached using fixed angles of elbow and shoulder ( by moving the trunk to different positions ) , it makes sense to extend the elbow and rotate and elevate the shoulder to reach objects that are further away ( see table 2 ) . we found that the variance in the wrist angle could best be explained by the starting position . the effect of start position on sagittal head position arises because subjects leaned forward more when starting far away ( not shown ) and did not completely move back to grasp the sphere . starting position had a marginal effect on shoulder elevation ; trunk - head rotation and shoulder angle are slightly smaller when the start position was near . these effects indicate that the elbow was positioned further forward ( which is clearly visible in fig . 2 ) and the relationship between sagittal head position ( presumably caused by hip flexion ) and wrist angle implies that there are synergies between joints that are quite distant from each other . when reaching for targets at different positions , the flexibility of the wrist was hardly used ( see table 1 ) . rather , people used the freedom in their shoulder and elbow to reach the different targets . the tendency not to use the full range of motion of the wrist has been reported previously in pointing in the horizontal plane and grasping in the sagittal plane ( cruse 1986 ; wang 1999 ) . it is in contradiction with the idea that the final posture is planned to minimize the work ( soechting et al . 1995 ) because in most cost functions wrist movements are associated with lower costs than elbow movements ( soechting et al . 1995 ; wang 1999 ) , so wrist movements should be preferred over elbow movements . it may be that people prefer to move the elbow ( rather than the wrist ) because they need a smaller angular displacement at the elbow to obtain the same amount of hand displacement . similar violations of minimizing work have been found in pointing in the saggital plane ( vaughan et al . ( 1997 ) , but seemingly in contradiction with the study by bennis and roby - brami ( 2002 ) who found a strong correlation between grasp orientation and movement direction . the latter authors also found that rotating the trunk has no effect on the grasp orientation . they therefore concluded that grasp orientation can not be planned in a shoulder - centered frame of reference fixed to the trunk , because rotating the trunk gives the same grasp orientations . we propose that it is not a shoulder - centered frame of reference fixed to the trunk , but rather the direction of the line connecting the shoulder and the object ( azimuth ) that determines the grasp orientation . as was stated in the data analysis section , the shoulder position is not an egocentric reference that varied with the initial posture of the subject . by taking the average shoulder position of each subject at the beginning of the trial this proposal is in line with bennis and roby - brami s finding that rotating the body without moving the shoulder relative to the body has no effect on grasp orientation . to test whether our proposal can really explain their data , we reanalyzed their data expressing the grasp orientations as a function of azimuth from the shoulder ; we find an r of 0.94 ( fig . this is higher than when expressing the grasp orientation as a function of movement direction ( r = 0.86 ; fig . this indicates that grasp orientation can be predicted even better from the direction of the line connecting the shoulder to the object than from the movement direction . the relationship between grasp orientation and azimuth conforms to the slope that we found in the present study ( 1.06 ; slope of lines in fig . the effect of movement direction expected based on the present results is the difference between the two lines . 3a ) , azimuth had a strong influence and starting position a weak but reliable influence on grasp orientation . as bennis and roby - brami used different starting positions , a cylinder rather than a sphere as a target , and a different task ( subjects were to grasp and lift the cylinder rather than place it somewhere else ) , this suggests that our result holds for a wider variety of tasks than the one we let the subjects perform.fig . 4our interpretation of the data presented by bennis and robybrami ( 2002 ) a the relationship between grasp orientation and movement direction as reported by bennis and robybrami ( replotted from their fig . 5 ) . b same data showing the relationship between grasp orientation and azimuth ( angle between the projection in the horizontal plane of the vector from the shoulder to the sphere and a reference axis pointing rightward in the horizontal plane ) . the solid line has the slope that we obtained for this relationship from the regression analysis on our data ( 0.98 ) . since grasp orientation was defined differently in their study and could not easily be converted , the intercept was fit to the data points for their start 1 ( this starting position was close to our near starting position ; solid line ) . the grasp orientation in our study was roughly 9 further counterclockwise when the starting position was further away ( dashed line ) . this nicely fits bennis and robybrami s ( 2002 ) data for start 2 and 3 which were further away than their start 1 our interpretation of the data presented by bennis and robybrami ( 2002 ) a the relationship between grasp orientation and movement direction as reported by bennis and robybrami ( replotted from their fig . 5 ) . b same data showing the relationship between grasp orientation and azimuth ( angle between the projection in the horizontal plane of the vector from the shoulder to the sphere and a reference axis pointing rightward in the horizontal plane ) . the solid line has the slope that we obtained for this relationship from the regression analysis on our data ( 0.98 ) . since grasp orientation was defined differently in their study and could not easily be converted , the intercept was fit to the data points for their start 1 ( this starting position was close to our near starting position ; solid line ) . the grasp orientation in our study was roughly 9 further counterclockwise when the starting position was further away ( dashed line ) . this nicely fits bennis and robybrami s ( 2002 ) data for start 2 and 3 which were further away than their start 1 the 9.5 effect of start position on grasp orientation is rather small compared to the 1030 effect observed by roby - brami et al . in another study ( 2000 ) . this could be caused by the smaller workspace they used , consistent with our finding that the effect of starting position was slightly larger in the center of the workspace . for some movement directions , this might mean that subjects have to move around the cylinder in order to avoid knocking it over . in another study , we found evidence that the effect of starting position is indeed larger for a tall cylinder than for a smaller sphere ( voudouris et al . altogether , the results offer some support for the idea that the final posture is planned on the basis of the target s position ( cruse 1986 ) . although this does not hold for any of the angles or positions measured , for all but wrist angle and sagittal head position , the effects of movement direction ( starting and placing position ) were much smaller than those of object position ( distance and azimuth form the shoulder ) . taken together , our data suggest that the choice of grasping points and the final posture after a reach - to - grasp movement toward a spherical object to place it elsewhere can be predicted from the position of the object that is to be grasped . this is in line with studies on 3d pointing that have shown that movements largely obey donders law , but with deviations of a few degrees ( gielen et al . to measure trunk - head rotation , we used the ired s on the subjects shoulder and forehead to measure trunk rotation . this measure accurately reflects trunk rotation when subjects do not rotate their heads ( i.e. when the angle between the center of rotation ( r ) , shoulder ( s ) , and forehead ( f ) in fig . when subjects do rotate their heads , a contribution of head - on - trunk rotation ( ) is added to the measure of trunk - head rotation . also , the length of the vector between the forehead and the shoulder changes from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \overline{l } $ $ \end{document } to lm . we used this to estimate the contribution of head - on - trunk rotation to head - trunk rotation . we calculated the average distance ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \overline{l } $ $ \end{document } ) between the forehead and the shoulder in the horizontal plane for the first sample of the measurement for each subject ) . this is well before movement onset when the subjects still had their eyes closed and were assumed to we could estimate the length of the vectors connecting the shoulder and the forehead to the center of rotation . given these lengths , we could calculate the angles 1 and 2 for each trial . the difference between these angles gives the estimated contribution of head - on - trunk rotation ( ) . the standard deviation of all these contributions was 3.5. this is small compared to the mean within - subject standard deviation in trunk - head rotation of 11.2 ( see table 1 ) so trunk - head rotation is mainly caused by trunk rotation rather than by head rotation.fig . 5illustration of the values used to calculate the error in the measure of trunk rotation introduced by head on trunk movement illustration of the values used to calculate the error in the measure of trunk rotation introduced by head on trunk movement

despite the infinitely many ways to grasp a spherical object , regularities have been observed in the posture of the arm and the grasp orientation . in the present study , we set out to determine the factors that predict the grasp orientation and the final joint angles of reach - to - grasp movements . subjects made reach - to - grasp movements toward a sphere to pick it up and place it at an indicated location . we varied the position of the sphere and the starting and placing positions . multiple regression analysis showed that the sphere s azimuth from the subject was the best predictor of grasp orientation , although there were also smaller but reliable contributions of distance , starting position , and perhaps even placing position . the sphere s initial distance from the subject was the best predictor of the final elbow angle and shoulder elevation . a combination of the sphere s azimuth and distance from the subject was required to predict shoulder angle , trunk - head rotation , and lateral head position . the starting position best predicted the final wrist angle and sagittal head position . we conclude that the final posture of the arm when grasping a sphere to place it elsewhere is determined to a larger extend by the initial position of the object than by effects of starting and placing position .

Introduction Materials and methods Subjects and setup Procedure Data analysis Results Discussion Appendix

this redundancy makes the system flexible ( robertson and miall 1997 ) , so regularities in the postures that are observed when subjects perform reach - to - grasp tasks are not trivial . final postures of reach - to - grasp movements also tend to be very consistent across repetitions in non - human primates ( helms et al . in the current study , we set out to determine which factors determine the grasp posture in an unconstrained reach - to - grasp movement . by varying the position of the sphere , as well as the starting position of the hand and the position at which the sphere was to be placed , the role of all these factors one might expect considerable effects of movement direction ( e.g. starting and placing position ) on the grip orientation and posture of the arm , because most theories state that movement - related costs are minimized . we found that the latter prediction turned out to be correct ; effects of movement direction were observed , but they were small compared to the effects of the position of the sphere . in the experiment , subjects made reach - to - grasp movements toward a glass opaque sphere with a 4.5 cm diameter . because the workspace was quite large and the starting and placing positions were on opposite sides of the grid , we tested a wide range of movement directions . a top view of a single trial : a subject moved her hand from the starting position to pick up a sphere ( indicated in yellow ) and put it at the placing position . if the placing position was different from the starting position , she then brought her hand back to the starting position . b definition of the grasp angle 1 , azimuth from the shoulder ( a ) , and distance from the shoulder ( d ) . a top view of a single trial : a subject moved her hand from the starting position to pick up a sphere ( indicated in yellow ) and put it at the placing position . if the placing position was different from the starting position , she then brought her hand back to the starting position . b definition of the grasp angle 1 , azimuth from the shoulder ( a ) , and distance from the shoulder ( d ) . during this trial , the subject held an ired between thumb and index finger such that the ired was approximately at the position of the contact points of the thumb and index finger with the sphere . if the starting position was near the body , the subject grasped the sphere at the target location and then placed it at the far location before returning to the starting position ( as in fig . 1 ) , and if the starting position was far from the body , the sphere was to be placed at the near location before returning to the starting position . in the second session , the sphere was to be grasped and then placed at the starting position of the hand . positions that were occluded by the arm in the starting posture could not be used , so blocks with the starting position far away consisted of fewer than 21 trials . the subject subsequently closed his eyes so that the starting posture could not be influenced by seeing the position of the sphere . we used the relationship between the clusters of three markers attached to the nails and the single marker held between finger and thumb during the calibration to determine the position of the digits . the beginning of the reach - to - grasp movement was defined as the first moment the speed of the wrist exceeded 0.1 m / s . the end of the reach - to - grasp movement was determined using the msi - method that has been described in detail elsewhere ( schot et al . we were interested in the posture at the end of the reach - to - grasp movement , so we calculated the grasp orientation , wrist angle , elbow angle , shoulder angle , shoulder elevation , trunk - head rotation , and sagittal and lateral head position at the end of the reach - to - grasp movement . the wrist angle is the smallest angle in space between the forearm and the digits as defined by the positions of the elbow , wrist and the average position of the thumb and index finger ( aligned is 0 , flexion is positive ) . the elbow angle is defined as the smallest angle in space between the upper arm and the forearm as defined by the positions of the shoulder , elbow and wrist ( fully extended is 0 , flexion is positive ) . the shoulder angle is defined as the angle between the projection in the horizontal plane of the upper arm and the line connecting the shoulder and the head . finally , trunk - head rotation is defined as the angle in the horizontal plane between the projection of the vector from the shoulder to the head and the reference axis pointing rightward . we estimated that the sd of the contribution of head - on - trunk rotation when calculating trunk - head rotation was 3.5 ( see appendix for details ) . we wanted to examine how well grasp orientation , wrist angle , elbow angle , shoulder angle , shoulder elevation , trunk - head rotation , and sagittal and lateral head position at the end of the reach - to - grasp movement could be predicted from starting position , placing position , and the sphere s azimuth and distance from the subject before the movement . azimuth is defined as the angle between the projection in the horizontal plane of the vector connecting the subject s shoulder position with the sphere , and a reference axis pointing rightward in the horizontal plane ( a in fig . this would influence the calculation of azimuth and distance from the shoulder and therewith confound the analysis in terms of the positions relative to the chair . for each subject , we performed a stepwise multiple regression analyses for each angle or position with starting position , placing position , azimuth and distance as independent variables . to be sure , we would not obtain a better fit by expressing azimuth and distance relative to the average head position rather than the average shoulder position , we also did the regression with azimuth and distance calculated relative to the average head position . because it hardly mattered for the quality of the fits whether the azimuth and distance were expressed relative to the shoulder or relative to the head ( the r values were slightly larger when azimuth and distance were calculated relative to the shoulder , but the largest r difference was only 0.06 ) , we will only report the results of the analyses with the sphere s azimuth and distance defined relative to the shoulder ( and refer to this as azimuth and distance ) . in the experiment , subjects made reach - to - grasp movements toward a glass opaque sphere with a 4.5 cm diameter . because the workspace was quite large and the starting and placing positions were on opposite sides of the grid , we tested a wide range of movement directions . a top view of a single trial : a subject moved her hand from the starting position to pick up a sphere ( indicated in yellow ) and put it at the placing position . if the placing position was different from the starting position , she then brought her hand back to the starting position . b definition of the grasp angle 1 , azimuth from the shoulder ( a ) , and distance from the shoulder ( d ) . a top view of a single trial : a subject moved her hand from the starting position to pick up a sphere ( indicated in yellow ) and put it at the placing position . b definition of the grasp angle 1 , azimuth from the shoulder ( a ) , and distance from the shoulder ( d ) . during this trial , the subject held an ired between thumb and index finger such that the ired was approximately at the position of the contact points of the thumb and index finger with the sphere . in the first session , if the starting position was near the body , the subject grasped the sphere at the target location and then placed it at the far location before returning to the starting position ( as in fig . 1 ) , and if the starting position was far from the body , the sphere was to be placed at the near location before returning to the starting position . in the second session , the sphere was to be grasped and then placed at the starting position of the hand . positions that were occluded by the arm in the starting posture could not be used , so blocks with the starting position far away consisted of fewer than 21 trials . the subject subsequently closed his eyes so that the starting posture could not be influenced by seeing the position of the sphere . we used the relationship between the clusters of three markers attached to the nails and the single marker held between finger and thumb during the calibration to determine the position of the digits . the beginning of the reach - to - grasp movement was defined as the first moment the speed of the wrist exceeded 0.1 m / s . the end of the reach - to - grasp movement was determined using the msi - method that has been described in detail elsewhere ( schot et al . for this , we required the digits sagittal position to be between 6 cm and 34 cm from the near starting position ( the sphere could be placed at 10 , 20 or 30 cm ) and their height to be lower than the sphere s height . we were interested in the posture at the end of the reach - to - grasp movement , so we calculated the grasp orientation , wrist angle , elbow angle , shoulder angle , shoulder elevation , trunk - head rotation , and sagittal and lateral head position at the end of the reach - to - grasp movement . the wrist angle is the smallest angle in space between the forearm and the digits as defined by the positions of the elbow , wrist and the average position of the thumb and index finger ( aligned is 0 , flexion is positive ) . the elbow angle is defined as the smallest angle in space between the upper arm and the forearm as defined by the positions of the shoulder , elbow and wrist ( fully extended is 0 , flexion is positive ) . the shoulder angle is defined as the angle between the projection in the horizontal plane of the upper arm and the line connecting the shoulder and the head . finally , trunk - head rotation is defined as the angle in the horizontal plane between the projection of the vector from the shoulder to the head and the reference axis pointing rightward . because one of the ireds used to calculate trunk - head rotation was placed on the forehead and the other on the shoulder , this measure contains trunk rotation as well as head - on - trunk rotation . we wanted to examine how well grasp orientation , wrist angle , elbow angle , shoulder angle , shoulder elevation , trunk - head rotation , and sagittal and lateral head position at the end of the reach - to - grasp movement could be predicted from starting position , placing position , and the sphere s azimuth and distance from the subject before the movement . azimuth is defined as the angle between the projection in the horizontal plane of the vector connecting the subject s shoulder position with the sphere , and a reference axis pointing rightward in the horizontal plane ( a in fig . this would influence the calculation of azimuth and distance from the shoulder and therewith confound the analysis in terms of the positions relative to the chair . for each subject , we performed a stepwise multiple regression analyses for each angle or position with starting position , placing position , azimuth and distance as independent variables . to be sure , we would not obtain a better fit by expressing azimuth and distance relative to the average head position rather than the average shoulder position , we also did the regression with azimuth and distance calculated relative to the average head position . because it hardly mattered for the quality of the fits whether the azimuth and distance were expressed relative to the shoulder or relative to the head ( the r values were slightly larger when azimuth and distance were calculated relative to the shoulder , but the largest r difference was only 0.06 ) , we will only report the results of the analyses with the sphere s azimuth and distance defined relative to the shoulder ( and refer to this as azimuth and distance ) . the lengths of each subject s upper arm , forearm , and hand are shown in table 1 , as are the mean sd of the grasp orientation , joint angles and head position . we see considerable differences in average postures across subjects but these can not be easily attributed to differences in segment lengths.table 1various measures for each subjectsubjectupper armforearmhandgrasp orientationwrist angleelbow angleshoulder angleshoulder elevationtrunk - head rotationlateral head positionsagittal head position131.923.615.178.2 23.136.3 7.965.0 17.9103.7 20.741.0 8.629.5 12.33.8 3.912.5 8.5231.826.417.355.8 25.427.6 6.168.9 16.392.7 21.545.0 5.841.9 8.810.4 5.17.9 3.2332.524.517.172.6 25.427.6 7.572.2 18.193.8 21.842.9 6.634.7 11.08.4 4.510.5 3.6428.521.714.472.7 26.524.7 5.768.8 15.997.5 17.654.3 3.931.9 12.94.4 5.39.1 3.7528.823.415.962.4 25.824.3 4.481.9 15.977.0 30.037.8 3.032.3 11.64.1 5.43.7 2.0633.627.619.362.5 22.424.6 4.671.4 14.787.2 20.643.4 4.931.5 10.46.2 4.110.3 2.4upper arm , forearm , and hand length ( in cm ) and the means sd s at the time of grasping of the angles ( in degrees ) and positions ( in cm , from the near starting position ) various measures for each subject upper arm , forearm , and hand length ( in cm ) and the means sd s at the time of grasping of the angles ( in degrees ) and positions ( in cm , from the near starting position ) the average grasp orientations for spheres at all 21 positions and the average postures at the end of reach - to - grasp movements to five of these positions are shown in fig . lines connect the markers on the head , shoulder , elbow , and wrist and connect the marker on the wrist to the calculated positions of the thumb and index finger to test to what extent the grasp orientation , wrist angle , elbow angle , shoulder angle , shoulder elevation , trunk - head rotation , and the lateral and sagittal position of the head could be predicted from the distance ( d ) , azimuth ( a ) , and starting and placing positions ( s and p , respectively ; with values of 0 for far and 1 for near ) , eight stepwise multiple regression analyses were performed for each subject to find the best coefficients for eq . 1 to the data of each subject are shown in table 2 of the angular measures and in table 3 for head position , as are the proportions of variance explained by the individual predictors ( r ) , the total variance explained by the predictors ( rtotal2 ) , and the residual standard deviations after fitting eq . the rtotal2 values show that grasp orientation is very predictable , as are shoulder angle , trunk - head rotation , and elbow angle . as s and p are both binary variables , the equation describes four planes ( one for each combination of starting and placing position ) . except in the case of the sagittal head position and the wrist angle , the separation between the planes is much smaller than the change in height along the distance and/or the azimuth axis . this means that most parameters are mainly determined by the sphere s position , independent of the start and place positions.fig . as there are four combinations of starting and placing position , we drew a plane for each combination visualization of the means of the eight regression analyses done per subject . as there are four combinations of starting and placing position , we drew a plane for each combination because the difference in movement direction between the two starting positions is larger for the central part of the workspace , one might expect that the effect of starting position on grasp orientation was larger for movements to targets that were in the center of the workspace . to test this the result showed that for the center of the workspace , the effect of starting position on grasp orientation was slightly larger ( 12.2 ) than the effect over the whole workspace ( 9.5 ) . in this study , we explored the factors that determine the posture of the arm when grasping a sphere in order to place it elsewhere . given this result , we only report to what extent the grasp orientation , wrist angle , elbow angle , shoulder angle , shoulder elevation , trunk - head rotation , and the lateral and sagittal position of the head were predictable given the sphere s position in terms of the azimuth and distance from the shoulder and the starting and placing positions . we found only small effects of placing position on the final posture of the reach - to - grasp movement . we found that the variance in the wrist angle could best be explained by the starting position . starting position had a marginal effect on shoulder elevation ; trunk - head rotation and shoulder angle are slightly smaller when the start position was near . as was stated in the data analysis section , the shoulder position is not an egocentric reference that varied with the initial posture of the subject . this indicates that grasp orientation can be predicted even better from the direction of the line connecting the shoulder to the object than from the movement direction . the relationship between grasp orientation and azimuth conforms to the slope that we found in the present study ( 1.06 ; slope of lines in fig . b same data showing the relationship between grasp orientation and azimuth ( angle between the projection in the horizontal plane of the vector from the shoulder to the sphere and a reference axis pointing rightward in the horizontal plane ) . b same data showing the relationship between grasp orientation and azimuth ( angle between the projection in the horizontal plane of the vector from the shoulder to the sphere and a reference axis pointing rightward in the horizontal plane ) . the grasp orientation in our study was roughly 9 further counterclockwise when the starting position was further away ( dashed line ) . this could be caused by the smaller workspace they used , consistent with our finding that the effect of starting position was slightly larger in the center of the workspace . in another study , we found evidence that the effect of starting position is indeed larger for a tall cylinder than for a smaller sphere ( voudouris et al . although this does not hold for any of the angles or positions measured , for all but wrist angle and sagittal head position , the effects of movement direction ( starting and placing position ) were much smaller than those of object position ( distance and azimuth form the shoulder ) . taken together , our data suggest that the choice of grasping points and the final posture after a reach - to - grasp movement toward a spherical object to place it elsewhere can be predicted from the position of the object that is to be grasped . to measure trunk - head rotation , we used the ired s on the subjects shoulder and forehead to measure trunk rotation .

multiple sclerosis ( ms ) is considered a chronic inflammatory disease of the central nervous system of autoimmune origin . onset of disease is most frequently in young adulthood , between 20 to 40 years of age.1 although many aspects of ms pathogenesis have been elucidated , the exact causal mechanisms are still not fully understood . an interplay between environmental factors in genetically susceptible individuals is assumed.2 this concept is supported by a wealth of research findings.35 unambiguously , once the disease has developed , it continues lifelong , and there is still no cure . the course of the disease can be relapsing remitting , which means that episodes with exacerbation of neurological symptoms alternate with periods of remission.6 over time , these relapses often do not fully resolve , leading to a stepwise accumulation of disability . moreover , after 1015 years , one - half of patients with initial relapsing remitting disease course will develop a secondary progressive disease7 that is characterized by a progressive increase of disability , independent of relapses and predominantly caused by deterioration of walking ability . only about 10%15% of patients exhibit a primary progressive disease course , which is defined by at least one year disease progression from onset on.8 a clinically isolated syndrome ( cis ) is diagnosed in patients with a first clinical event suggestive of ms and evidence of disease dissemination in space , but not yet evidence of dissemination in time , thus chronicity of disease . one of the hallmarks of ms is a high variability of the natural disease course . while some patients , apart from relapses , never experience any handicap , the majority of patients will ultimately develop disability to a variable degree and after a variable time interval after onset of disease . as first shown by confavreux et al9 and based on a concept of a two - stage disease with focal inflammation in the early stage and diffuse inflammation and neurodegeneration in a second - disease stage , leray et al recently confirmed highly interesting data on time to disability accumulation in ms:10 while duration of the first stage from onset of disease until reaching a milestone of kurtzke disability status scale ( dss ) 311 ( corresponding to moderate disability ) ranged from less than 3 years to more than 15 years , duration of phase ii ( kurtzke dss 3 to dss 6 , the latter is defined by requiring unilateral assistance to walk 100 meters ) remained nearly identical in all patients with 69 years , irrespective of the duration of phase i. this observation suggests that , once a clinical threshold of irreversible disability is reached , further progression of disability becomes inevitable . support of this hypothesis can be derived from two typical findings of the chronic progressive phase of ms . even in the absence of new or contrast - enhancing magnetic resonance imaging ( mri ) lesions , disability usually increases in the chronic progressive phase of the disease , and once the chronic progressive phase is reached , efficacy of disease - modifying therapies often declines.12,13 although relapse rate and progression of the expanded disability status score ( edss)11 are widespread parameters of disease activity , many patients with ms do not only suffer from physical disability ; they also suffer from neurocognitive decline , fatigue , or depression.14,15 hence , the diagnosis of ms is a life - changing event with a significant impact on family , society , and the social welfare system . fortunately , during the last decades , several disease - modifying therapies ( dmt ) have been licensed that ameliorate the course of the disease.16 the aim of these dmt is to reduce inflammation , disease activity as measured by mri , and relapse rate . thereby , a delay of transition into the secondary progressive phase and prevention of permanent disability ought to be achieved . due to the chronic nature of ms , dmts are usually applied continuously over many years , or even decades . to keep in mind , dmts are applied in a preventive manner , since neuronal damage can not be reversed . ms is the most frequent nontraumatic cause for disability among young adults in north america and europe.17 worldwide , about 2.5 million people are affected by ms ; although traditionally , the incidence is supposed to vary by geographical latitude.18 the highest prevalence with more than 30 ms patients per 100,000 inhabitants can be found in northern europe and north america . a medium prevalence of 530 per 100,000 is observed in southern europe and the southern united states ; whereas , a low prevalence of fewer than 5 per 100,000 has been reported for asia and south america.19 population genetics , the geographically determined physical environment , and socioeconomic structures have been proposed as possible reasons for continental differences in ms prevalence.20 in the united states alone , about 400,000 individuals suffer from ms ; each week , more than 200 persons are newly diagnosed with the disease.18 according to estimates for 2010 , ms has been diagnosed in 540,000 subjects of the european population.21 however , recent epidemiological studies found an increasing incidence and prevalence of ms , especially in women.20 this seems to be the case also in areas of the world that were formerly classified as low - prevalence regions . for example , ms prevalence was recently reported as 3155 per 100,000 inhabitants in the arabian gulf region and 0.8321.5 per 100,000 individuals in latin america and the caribbean.22,23 factors contributing to an increasing incidence of ms have not been fully elucidated . earlier ms diagnosis , due to revised diagnostic criteria ; better ascertainment of diagnosis ; less sunlight exposure ; higher standards in hygiene ; and cigarette smoking , have all been proposed as likely candidates.20,24 the economic impact of ms can be divided into direct costs and indirect costs . direct costs account for health care costs , such as pharmaceuticals , inpatient care costs , outpatient care costs , additional treatments like physiotherapy , and medical aids . in contrast , indirect costs are caused by productivity loss of patients and caregivers.25 this might be due to sick leave , reduced daily working time , unemployment , or premature retirement . in europe , mean annual cost per person with ms is estimated to be around 27,000 , ranging from 7,227 in bulgaria to 44,565 in luxembourg . on average , about two - thirds of the totals are caused by direct costs , while approximately one - third are derived from indirect costs.21 in 1998 , all - over costs of ms in the united states were us$6.8$11.9 billion annually , which averages about us$34,000 per patient , per year.26 in a survey among patients receiving dmt in the united states in 2004 , total average costs per patient and year were estimated as high as us$47,000.27 of these , 63% accounted for direct costs , including 34% of total costs ( us$16,000 ) for dmt . of note , patients having suffered a relapse were found to have higher costs than patients without relapse.27,28 furthermore , several studies have shown that total costs rise with increasing disability.2832 in line with this result , a secondary and primary progressive disease course was found to be more costly than a relapsing remitting one , due to a rise of indirect costs . besides degree of physical disability , age , depression , and fatigue have been identified as independent factors of an increase of costs.32 quality of life remarkably and constantly declines with advancing stages of disability , and the occurrence of relapses negatively affects quality of life measures.28,31 it was in 1993 that interferon beta-1b was introduced as the first dmt of ms . still , it represents a very common first - line treatment that is widely used . remitting ms , and secondary progressive ms with superimposed relapses.33 there are three different recombinant interferon beta preparations on the market . interferon beta-1b ( betaferon , bayer healthcare pharmaceuticals , berlin , germany ; betaseron , bayer healthcare pharmaceuticals , berlin , germany ; extavia , novartis , basel , switzerland ) is recombinantly expressed in escherichia coli and is subcutaneously injected every other day . interferon beta-1a is recombinantly expressed in chinese hamster ovary cells and is identical to the human interferon beta.34 there are two preparations of interferon beta-1a . rebif ( merck serono , geneva , switzerland ) is subcutaneously administered three times a week . avonex ( biogen idec , cambridge , ma , usa ) is applied once weekly as an intramuscular injection . placebo - controlled trials have demonstrated a significant reduction of disease activity for all three interferon beta preparations.3537 the pivotal trial on efficacy of interferon beta-1b in ms , which led to the initial approval of the drug for treatment of ms , demonstrated an annual relapse rate of 1.27 for placebo and 0.84 for 250 g interferon beta-1b.35 similar results were observed for interferon beta-1a . in the prevention of relapses and disability by interferon beta-1a subcutaneously in multiple sclerosis ( prisms ) trial on subcutaneous interferon beta-1a , the annual relapse rate was 1.28 for placebo , 0.91 for 22 g , and 0.87 for 44 g.37 intramuscular interferon beta-1a reduced the annual relapse rate to 0.61 , compared with 0.90 in the placebo group.36 these findings are supported by significant reduction of mri disease activity in all three trials , while only in the latter two trials , a beneficial effect on disability progression was recorded during this 2-year study period . while these therapies have been introduced , a better understanding of ms pathophysiology especially the knowledge of unrecoverable axonal loss already in early stages of the disease has prompted a fundamental shift in the ms treatment approach and provided the rational for early ms treatment.33,34 based on this consideration , dmts are initiated early in the course of the disease and before irreversible neuronal damage and disability have occurred . following the concept of early ms treatment , interferon beta treatment has been investigated in patients with cis and has shown a beneficial effect in terms of conversion to definite ms , edss progression , and mri disease activity.3841 in the betaferon/betaseron in newly emerging multiple sclerosis for initial treatment ( benefit ) trial,40 patients with cis were randomized to receive interferon beta1-b or placebo for 2 years , or until clinically definite multiple sclerosis ( cdms ) was diagnosed , according to the poser et al criteria.42 within the 2-year study period , 45% of patients in the placebo group versus 28% of the interferon beta group progressed to cdms , according to poser et al ; and 85% of the placebo versus 69% of the interferon beta group were diagnosed with definite ms , according to the mcdonald criteria of 2001.43 both results reached statistically significant difference . these data revealed a significant lower number of newly active lesions , cumulative number of new t2-weighted mri lesions and gadolinium - enhancing lesions , as well as cumulative volume of gadolinium - enhancing lesions in the interferon beta - treated patients group . of note , there was no change of health - related quality of life observed during the 2 years . after having finished the placebo - controlled core study , patients were offered to enroll in an open - label interferon beta-1b 250 g follow - up trial to investigate effects of early treatment in cis , versus delayed treatment after diagnosis of cdms or after 2 years on study . three years after initial randomization , 37% of the early treatment group versus 51% of the delayed - treatment group have developed cdms ( risk reduction of 41% ) ; 16% of the early treatment group versus 24% of the delayed - treatment group experienced confirmed edss progression ( risk reduction 40%).44 a subgroup analysis revealed lower treatment effects in patients with less clinical or mri activity at the time of the first event ; patients with initial multifocal presentation and high lesion load would benefit more from early treatment . in the 5-year active treatment extension follow - up study , risk reduction of cdms remained 37% in the early treatment group ; whereas , no statistically significant difference in confirmed disability progression was observed any more between early and delayed interferon beta treatment.45 in both treatment groups , median edss remained 1.5 during the 5-year study period . finally , in the benefit extension trial , a long - term international observational study of patients having completed the initial placebo - controlled core study , data on follow - up for more than 8 years are available.46 at the discretion of the physician and according to local standards , patients were treated with any of the disease - modifying drugs or received no treatment . during the study periods , 5.1% of patients received no treatment ; 77.6% received no other drug than interferon beta-1b ; 6% , interferon beta-1a ; 5.8% , glatiramer acetate ; and 6.6% of patients received any escalation therapy . in the overall study population , disability as quantified by the edss changed only slightly over 8 years , with the median edss remaining 1.5 at year 8 . however , 55.5% of patients , who had received early interferon beta treatment in the core study , developed clinically definite ms , while clinically definite ms was diagnosed in 65.8% of patients with delayed interferon beta treatment . moreover , a difference of the overall annualized relapse rate was still observed over the 8-year period 0.20 early treatment versus 0.26 delayed treatment.47 placebo - controlled clinical trials on interferon beta-1a in cis have yielded very similar results,38,41 suggesting a class effect of interferon beta treatment in early ms and cis . by numerous investigational trials and the clinical experience in interferon beta for two decades , safety and tolerability profile of this drug is well - known and has proven favorable , also , in long - term application . side effects mainly include injection site reactions , flu - like symptoms , leukopenia , and liver - enzyme elevation.48 moreover , up to 40% of patients develop antibodies against interferon beta , which antagonize bioactivity in a significant proportion of patients.4951 if neutralizing antibodies against interferon beta persistently occur , therapeutic efficacy of the drug is reduced or abolished.5256 besides interferon beta glatiramer acetate ( [ ga ] , copaxone , teva , petah tikva , israel ) represents a common first - line therapy with daily subcutaneous injections , which is approved for treatment of patients with relapsing remitting ms , as well as for patients with cis and typical mri features of ms . the precise ( early glatiramer acetate treatment in delaying conversion to clinically definite multiple sclerosis in subjects presenting with a clinically isolated syndrome ) study demonstrated that ga reduced the risk of developing clinically definite multiple sclerosis by 45% , compared with placebo during a 3-year study period.57 interferon beta and ga are the only agents that are licensed for treatment of cis . however , for the sake of completeness , it needs to be mentioned that there are several other therapeutics licensed for baseline and escalation therapy in definite ms , and more agents are likely to enter the market.16 approval of all these agents is usually based on efficacy and safety data of a 2-year clinical trial . in ms phase ii and phase iii interventional clinical trials , a duration of about 6 months to a few years has become common , since demonstration of efficacy usually needs several months of treatment . on the other hand , duration of clinical trials longer than 2 years will make a well - controlled trial less feasible to accomplish and might hold back an effective treatment from ms patient care . given an increasing number of available dmt in ms , trials investigating the long - term efficacy are needed . this is to briefly summarize the terms and types of pharmacoeconomic evaluations used in the studies reviewed here ( adapted from clifford goodman unless cited otherwise).25 cost - effectiveness analyses ( cea ) measure costs in monetary units and consequences in natural units , such as life - years gained , or relapses avoided.58 in cost - utility analyses ( cua ) , a variant of cea , consequences are measured in terms of preference - based measures of health,58 most commonly based on quality - adjusted life - years ( qaly).59 a qaly is a unit that adjusts gained or lost life - years subsequent to an intervention by the quality of life during those years . gained or lost life - years are multiplied by a weighting factor ( utilities ) ranging from 0.00 ( representing death ) to 1.00 ( representing a perfect health state ) . cea and cua always involve comparison of alternative interventions , expressed as incremental cost - effectiveness ratio ( icer ) and incremental cost - utility ratio ( icur ) , respectively . therefore , incremental costs are divided by the change of effectiveness or utility ( eg , qualys gained ) . depending on the perspective of pharmacoeconomic evaluations ( eg , society overall , third - party payers , patients ) , different types of costs are of interest . costs need to be corrected for effects of inflation ; both costs and outcomes should be discounted . discounting reflects the fact that costs and benefits tend to have less value in the future than in present . the former approach involves collection of original data ( eg , randomized controlled studies ) . quantitative modeling - based pharmacoeconomic analyses simulate costs and consequences of pharmacological interventions , based on existing data and estimates of key variables where there are no data available . in this type of analysis it is based on predefined sets of health states ( eg , edss levels ) . its time horizon is divided into equal increments of time , referred to as markov cycles . for each cycle , costs , outcomes , and transition probabilities of moving from one state to another or of remaining in the same state are estimated.60 finally , sensitivity analyses determine robustness of results of model - based analyses by varying the estimates of key variables within plausible ranges ( eg , by applying a multivariate approach ) . we conducted a systematic literature search in medline ( pubmed ) on december 14 , 2012 , applying the medical subject headings ( mesh ) terms multiple sclerosis and costs and cost analysis . we deliberately chose the comprehensive mesh term costs and cost analysis to cover all potentially relevant studies . four hundred fourteen publications met our search criteria , including 40 non - english publications , which were excluded ; titles and abstracts of the resulting 374 publications were reviewed by fc and db . inclusion criteria were as follows : ( 1 ) original research on the pharmacoeconomics of dmt in ms ; ( 2 ) comparative study ; and ( 3 ) comparators ( initiation of dmt at the time of the first demyelinating event versus treatment after cdms ) . because our search strategy resulted in no more than four relevant studies,6164 we did not exclude the study by curkendall et al,62 although the authors only report on costs and not on health - outcome measures of dmt in cis . the remaining three studies were rated for quality of health - economic analyses , based on the quantitative scoring system that has been proposed by chiou et al ( range 0100 ; 0 representing the lowest ; 100 representing the highest quality).65 the 16-item weighted scoring system was independently applied by fc and db . our scoring results were in accordance with the results reported by yamamoto and campbell , who chose the same approach ( table 1).66 as outlined before , early initiation of dmt with interferon beta or ga has been proven to reduce time to cdms , relapse rate , and disease progression . this observation led to the approval of interferon beta and ga for treatment of relapsing remitting ms , as well as cis . therefore , initiation of dmt may be started even before definite diagnosis of ms is established . to date , four studies have been published that address the economic impact of starting dmt at the time of the incident cis versus after conversion to cdms ( table 1).6164 the swedish study by caloyeras et al estimates cost - effectiveness of interferon beta-1b if initiated in cis compared with delayed treatment after the diagnosis of cdms has been made.61 the authors applied a markov model analysis , based on twelve health states defined by edss scores , the assigned diagnosis ( ie , cis versus cdms ) , and the course of disease ( ie , relapsing or nonrelapsing forms of ms , and death ) . transition probabilities after 6-month cycles were estimated for a lifetime time horizon of 50 years . the authors assumed that treatment discontinuation rates would not differ between both treatment arms ; all hypothetical patients would discontinue dmt after roughly 25 years . switching to another dmt and the core input data for the statistical model were derived from the benefit trial.40,44,45 moreover , extrapolations of benefit data and best available data from the published literature were used as model parameters . results of the cua demonstrate that , during a 50-year time horizon , patients assigned to the early treatment group gained 12.9 qalys , as compared with 12.4 qualys in the delayed - treatment group . on average , patients in the delayed - treatment group progressed 2 years earlier to cdms ( estimated conversion rate 99.54% ) , compared with patients in the early treatment group ( estimated conversion rate 99.31% ) . direct medical costs ( ie , interferon beta-1b drug costs , costs for inpatient and outpatient care , and costs related to testing , other drugs , and relapse events ) were higher in the early treatment arm , whereas indirect costs ( ie , costs related to early retirement and short - term absence ) and direct nonmedical costs ( ie , informal care , services , investments ) in the delayed - treatment group exceeded those in the early treatment group . when all costs were summed up over the 50-year time horizon , total costs in the delayed - treatment arm were higher than those in the early treatment arm ( difference of approximately 344,000 sek per patient ) . in summary , the markov model - based estimation revealed that early interferon beta-1b treatment may decrease total costs and increase qalys . however , sensitivity analyses revealed that costs per qaly gained were very high for short time horizons . in fact , only for time horizons longer than 10 years , early treatment was less costly and more effective with respect to qalys gained . this study reached the highest quality of health economic studies ( qhes ) score of the articles reviewed here ( 99 points ) . curkendall et al applied a different approach to assess the pharmacoeconomics of early dmt.62 their analysis was based on insurance claims data in the united states for the years 20002008 , extracted from the thomson reuters marketscan commercial and medicare supplemental databases ; therefore , only health care expenditures and utilizations were analyzed . the core inclusion and exclusion criteria were as follows : ( 1 ) no diagnosis of ms during 12 months before and at the time of a cerebral mri ; this mri scan was defined as start date of the 12-months observation period ; ( 2 ) at least one symptom that was documented 6 months before or 1 month after the index date and that was likely to be attributable to ms ; ( 3 ) initiation of treatment with interferon beta-1a , interferon beta-1b , or ga sometime during the follow - up period . thus , cis patients who may have received dmt after the follow - up period were not included . patients were categorized into two groups an early dmt and a delayed dmt group depending on whether they received dmt before or after the diagnosis of ms . ms diagnosis was defined as one inpatient claim or two outpatients claims coded with icd-9-cm diagnosis code 340 . three thousand nine hundred fifty one patients were enrolled ; 227 and 3,724 were assigned to the early and delayed - treatment cohort , respectively . the mean follow - up was approximately 3 years in both groups , yet the pharmacoeconomic analysis was restricted to the first year after the index mri . the mean time interval between index mri and initiation of dmt was , as expected , significantly shorter in the early treatment group than in the delayed - treatment group ( 122 days versus 184 days ) . however , this difference was small , and more than 90% of the patients assigned to the delayed - treatment group received dmt within the first year after index mri . within the observation period , the early dmt cohort had significantly fewer hospitalizations compared with the delayed dmt cohort ( 0.20 versus 0.33 ) . total drug expenditures were higher in the early dmt cohort ; whereas , total ms - related expenditures were significantly lower in the early dmt group , compared with the delayed dmt group after taking out costs for dmt . both all - cause and ms - related total financial expenditures were not significantly different between the two groups ( all - cause total expenditures us$31,184 in the early dmt group , us$30,051 in the delayed dmt group ) . the authors conclude that the high drug costs of early dmt might be compensated by savings in other medical expenditures . however , the observation period was short , and the analysis was conducted from a third - party payer s perspective alone . the qhes quality score could not be applied to this study , as it does not report on outcome measures , thus not fulfilling the criteria of pharmacoeconomic analyses . in contrast to the other articles reviewed here , the strength of this study lies in the analysis of real costs , albeit restricted to health care expenditures . lazzaro et al applied an open cohort epidemiological model from an italian perspective.63 two treatment arms were analyzed . one group of patients received interferon beta-1b after diagnosis of cis ; the other group was treated with interferon beta-1b after diagnosis of cdms . observation period lasted 25 years with 2,000 cis patients being added to each treatment arm every year , resulting in two cohorts of 50,000 patients each . the conversion rates for years 12 were based on the benefit data,40 while the conversion rates for the remaining 22 years were assumed to decline asymptotically . nineteen years after cdms diagnosis , approximately 50% of the patients would progress to secondary progressive ms . moreover , the observation period was arbitrarily divided into four periods ( year 0 ; years 110 ; years 1120 ; and years 2124 ) . based on the report by kobelt et al,67 edss scores were converted into utility parameters , which were assumed to stay constant within each period . costs were adopted from both the italian national health service ( inhs ) and the societal viewpoints . most of the cost estimates were based on the italian cost - of - illness study by amato et al;68 all costs were reported in 2006 euro . according to this analysis , patients gained 7.84 and 7.49 qalys in the early and delayed treatment arm , respectively ; the difference of 0.35 qalys reached the level of significance . the cua showed that , from the inhs perspective ( ie , only health care resources were considered ) , the icur for early versus delayed interferon treatment was 2,575 per qualy gained and lay considerably below a recently proposed italian willingness to pay of 12,00060,000 per qaly.69 when patient and family resources ( ie , the societal perspective ) were additionally taken into account , early treatment would be even less costly than delayed treatment ( 220,416 versus 226,022 per patient for early versus delayed treatment , respectively ) . the authors conclude that , from the health service and societal perspective , early treatment with interferon beta-1b may be cost - effective , compared with treatment initiation after cdms . however , this interpretation did not hold true for short time horizons as there was a sharp decline in icur estimates over the first years ; eg , from the societal perspective , the icur started from approximately 135,000 per qualy gained at year 1 , decreased below 60,000 after year 3 , and , finally , became negative after year 6 onward . the qhes score of this study reached 75 points and lay considerably below the quality score of the swedish study reviewed here . iskedjian et al performed a pharmacoeconomic analysis of treatment with interferon beta-1a administered intramuscularly once weekly in cis patients ; the study was conducted from a canadian perspective.64 the aim of this study was to investigate : ( 1 ) cost - effectiveness of interferon beta-1a treatment based on the gain of additional monosymptomatic life - years ( mlys ) ; and ( 2 ) cost - utility of long - term interferon beta treatment based on the gain of quality - adjusted monosymptomatic life - years ( qamlys ) . monosymptomatic life - years were defined as years after diagnosis of cis and before diagnosis of cdms . for cis patients , high - dose intravenous steroid pulse therapy ( plus tapering ) for the index event ( current treatment ) and high dose intravenous steroid pulse therapy ( plus tapering ) for the index event plus interferon beta-1a . once the diagnosis of cdms was established , all patients received the same treatment regime with interferon and high dose steroids for treatment of relapse . the time horizon of the cea and cua was 12 and 15 years , respectively , starting from diagnosis of cis and extending to various edss stages of cdms . for the cea and cua , two markov models were developed : the state of cis and various edss states defined the markov cycles ( cycle length , 1 year ) . the conversion rate to cdms was derived from the efficacy results of the controlled high risk avonex multiple sclerosis study ( champs ) study.38 transition probabilities through the various stages of edss were derived from a canadian ms study by weinshenker et al.7 cost analyses , including unemployment rates and the average hospital length of stay within each edss level , were based on data from the literature7072 and canadian health authorities . costs were reported in 2002 canadian dollars ( ca$ ) ; total costs were estimated from the perspective of the canadian ministry of health ( moh ) and from the canadian societal perspective . the former perspective was limited to direct medical costs ; the latter additionally covered direct nonmedical and indirect costs . the input parameters tested in the sensitivity analyses comprised the progression rate to cdms , the indirect costs associated with the duration from cis diagnosis to cdms , the time horizon , the discount rate , and the utilities . the cea demonstrates that the incremental cost - effectiveness ratio ( icer ) of interferon beta per mly gained was ca$53,110 and ca$44,789 from the moh and societal perspectives , respectively . as opposed to the moh perspective , interferon beta treatment was considered cost - effective from the societal perspective , as costs of current treatment per mly gained were higher ( ca$75,444 ) than the icer of interferon beta treatment . the cua was conducted , based on the utility estimates derived from both a canadian and a swedish study.30,70 from the moh perspective , incremental cost of interferon beta per qamly gained ( icur ) was ca$227,586 ( ca$116,071 for the swedish utility estimates ) ; whereas , from the societal perspective , incremental cost of interferon beta reached ca$189,286 ( ca$91,228 , swedish utility estimates ) . the sensitivity analyses showed that both the cea and cua were sensitive to variations of the time horizon with shorter horizons producing higher icer / ucer and vice versa . the multivariate sensitivity analysis of the cea showed that in 87% and 6% of the resulting scenarios the icer of interferon beta therapy would be lower than the costs of current treatment per mly gained from the societal and moh perspective , respectively . therefore , the authors conclude that interferon beta treatment in cis may be cost - effective . however , this conclusion could be drawn from the cea of the societal perspective , only . moreover , concerning the results of the cost - utility analysis , the authors omit to address the issue of decision makers willingness to pay . of the three pharmacoeconomic studies reviewed here , ms is a chronic disease with high economic burden on patients , families , the health system , and society . worldwide incidence and prevalence seems to be increasing with a total of about 2.5 million individuals currently being affected by ms . as reviewed here , several studies have demonstrated that progression of disease severity is accompanied by a rise of total costs as well as a change in the distribution of costs . during early stages of the disease , direct costs , which are mainly caused by dmt , dominate the total costs ; whereas , with accumulation of disability during later stages of the disease especially indirect costs significantly increase.73 moreover , occurrence of relapses has been associated with a peak of costs . noteworthy , quality of life is substantially reduced with increase of disability and during relapse . due to a better understanding of ms pathogenesis and the experience that efficacy of available dmt is high in early stages of ms but declines in the progressive phase of the disease , the concept of early ms treatment has been established . therefore , dmts are usually initiated after diagnosis of relapsing remitting ms or even before definite diagnosis of ms is made at the stage of cis . since dmts are , in general , considered as rather expensive treatments , there has been much debate whether it is economically justifiable to widely prescribe these drugs . a major concern is that clinical trials for approval of dmt even though they have clearly demonstrated efficacy on parameters such as relapses , mri activity , and sometimes also on edss progression during the trial have hardly addressed long - term disability . numerous studies have investigated the pharmacoeconomics of dmt in ms , though only few have explicitly analyzed the pharmacoeconomic effect of early initiation of dmt in cis.6164 these studies indicate that early versus delayed treatment with interferon beta may be overall cost - effective in the long term . reduction of relapses , hospitalization , and indirect costs and a gain of qalys seem to outweigh the costs of dmts . however , to keep in mind , all these analyses and models highly depend on estimates of the applied input parameters . for instance , becker et al74 recently demonstrated the impact of cohort selection by replicating a previously published model - based analysis75 on the cost - effectiveness of interferon beta in ms . based on a different patient cohort with longer follow - up , costs per relapse avoided turned out to be approximately 45% lower than in the original model . in their study reviewed here , iskedjian et al showed that , from the canadian societal perspective , estimated incremental costs of early interferon beta treatment would be 189,286 cad per qamly gained . when applying different utility estimates , the same analysis resulted in 91,228 cad , which is less than 50% of the former result.64 however , it seems to be ambiguous which of the applied utility estimates may be more valid than the other . thus , results of pharmacoeconomic studies and inferences that may be drawn by decision makers largely depend on the analysis approach , the applied simulations and estimations , the perspective of the analysis , and particularly on the quality of the pharmacoeconomic analysis . as shown in our review and for instance in the recent work by yamamoto et al , the latter has turned out to be considerably variable.66 we believe that this is especially critical in a chronic , heterogeneous , and complex disease , such as ms . in this context , one should note , that several pharmacoeconomic studies in ms , including the four studies reviewed here , have been funded by manufacturers of dmt ( table 1 ) . obviously , the results of all four reviewed studies can not be readily generalized to other national settings as several key input data ( eg , costs ) were country - specific.76 besides interferon beta and ga , new dmts have been introduced , which are used as baseline and/or escalation therapy . the wider range of dmts , as well as escalation strategies , needs to be taken into account in future pharmacoeconomic studies , which will be even more challenging . therefore , we need more data on long - term efficacy of dmt and costs in ms in the real - life setting . biomarkers , which will help to stratify patients at early stages with respect to severity of the disease course and response to therapy , are of great need from an economic point of view and even more so for patient care .

multiple sclerosis ( ms ) is a common neurological disease with increasing incidence and prevalence . onset of disease is most frequently in young adulthood when productivity is usually highest ; it is of chronic nature and , in the majority of patients , it will result in accumulation of disability . due to loss of productivity in patients and caregivers as well as high expenses for medical treatment , ms is considered a disease with high economic burden for patients and society . several drugs have been approved for treatment of ms . while treatment ameliorates the course of the disease , it is very costly ; therefore , pharmacoeconomics , evaluating costs and effects of disease - modifying treatment in ms , has become an important issue . here , we review the economic impact and treatment strategies of ms and discuss recent studies on pharmacoeconomics of early treatment with interferon beta .

Introduction Epidemiology and economic impact of MS Efficacy of early initiation of DMT in MS using interferon beta Methodological aspects of pharmacoeconomic analyses Literature search and quality assessment Early treatment with interferon beta from a pharmacoeconomic perspective Conclusion

multiple sclerosis ( ms ) is considered a chronic inflammatory disease of the central nervous system of autoimmune origin . onset of disease is most frequently in young adulthood , between 20 to 40 years of age.1 although many aspects of ms pathogenesis have been elucidated , the exact causal mechanisms are still not fully understood . the course of the disease can be relapsing remitting , which means that episodes with exacerbation of neurological symptoms alternate with periods of remission.6 over time , these relapses often do not fully resolve , leading to a stepwise accumulation of disability . only about 10%15% of patients exhibit a primary progressive disease course , which is defined by at least one year disease progression from onset on.8 a clinically isolated syndrome ( cis ) is diagnosed in patients with a first clinical event suggestive of ms and evidence of disease dissemination in space , but not yet evidence of dissemination in time , thus chronicity of disease . one of the hallmarks of ms is a high variability of the natural disease course . while some patients , apart from relapses , never experience any handicap , the majority of patients will ultimately develop disability to a variable degree and after a variable time interval after onset of disease . as first shown by confavreux et al9 and based on a concept of a two - stage disease with focal inflammation in the early stage and diffuse inflammation and neurodegeneration in a second - disease stage , leray et al recently confirmed highly interesting data on time to disability accumulation in ms:10 while duration of the first stage from onset of disease until reaching a milestone of kurtzke disability status scale ( dss ) 311 ( corresponding to moderate disability ) ranged from less than 3 years to more than 15 years , duration of phase ii ( kurtzke dss 3 to dss 6 , the latter is defined by requiring unilateral assistance to walk 100 meters ) remained nearly identical in all patients with 69 years , irrespective of the duration of phase i. this observation suggests that , once a clinical threshold of irreversible disability is reached , further progression of disability becomes inevitable . even in the absence of new or contrast - enhancing magnetic resonance imaging ( mri ) lesions , disability usually increases in the chronic progressive phase of the disease , and once the chronic progressive phase is reached , efficacy of disease - modifying therapies often declines.12,13 although relapse rate and progression of the expanded disability status score ( edss)11 are widespread parameters of disease activity , many patients with ms do not only suffer from physical disability ; they also suffer from neurocognitive decline , fatigue , or depression.14,15 hence , the diagnosis of ms is a life - changing event with a significant impact on family , society , and the social welfare system . fortunately , during the last decades , several disease - modifying therapies ( dmt ) have been licensed that ameliorate the course of the disease.16 the aim of these dmt is to reduce inflammation , disease activity as measured by mri , and relapse rate . due to the chronic nature of ms , dmts are usually applied continuously over many years , or even decades . a medium prevalence of 530 per 100,000 is observed in southern europe and the southern united states ; whereas , a low prevalence of fewer than 5 per 100,000 has been reported for asia and south america.19 population genetics , the geographically determined physical environment , and socioeconomic structures have been proposed as possible reasons for continental differences in ms prevalence.20 in the united states alone , about 400,000 individuals suffer from ms ; each week , more than 200 persons are newly diagnosed with the disease.18 according to estimates for 2010 , ms has been diagnosed in 540,000 subjects of the european population.21 however , recent epidemiological studies found an increasing incidence and prevalence of ms , especially in women.20 this seems to be the case also in areas of the world that were formerly classified as low - prevalence regions . for example , ms prevalence was recently reported as 3155 per 100,000 inhabitants in the arabian gulf region and 0.8321.5 per 100,000 individuals in latin america and the caribbean.22,23 factors contributing to an increasing incidence of ms have not been fully elucidated . earlier ms diagnosis , due to revised diagnostic criteria ; better ascertainment of diagnosis ; less sunlight exposure ; higher standards in hygiene ; and cigarette smoking , have all been proposed as likely candidates.20,24 the economic impact of ms can be divided into direct costs and indirect costs . in contrast , indirect costs are caused by productivity loss of patients and caregivers.25 this might be due to sick leave , reduced daily working time , unemployment , or premature retirement . on average , about two - thirds of the totals are caused by direct costs , while approximately one - third are derived from indirect costs.21 in 1998 , all - over costs of ms in the united states were us$6.8$11.9 billion annually , which averages about us$34,000 per patient , per year.26 in a survey among patients receiving dmt in the united states in 2004 , total average costs per patient and year were estimated as high as us$47,000.27 of these , 63% accounted for direct costs , including 34% of total costs ( us$16,000 ) for dmt . besides degree of physical disability , age , depression , and fatigue have been identified as independent factors of an increase of costs.32 quality of life remarkably and constantly declines with advancing stages of disability , and the occurrence of relapses negatively affects quality of life measures.28,31 it was in 1993 that interferon beta-1b was introduced as the first dmt of ms . placebo - controlled trials have demonstrated a significant reduction of disease activity for all three interferon beta preparations.3537 the pivotal trial on efficacy of interferon beta-1b in ms , which led to the initial approval of the drug for treatment of ms , demonstrated an annual relapse rate of 1.27 for placebo and 0.84 for 250 g interferon beta-1b.35 similar results were observed for interferon beta-1a . in the prevention of relapses and disability by interferon beta-1a subcutaneously in multiple sclerosis ( prisms ) trial on subcutaneous interferon beta-1a , the annual relapse rate was 1.28 for placebo , 0.91 for 22 g , and 0.87 for 44 g.37 intramuscular interferon beta-1a reduced the annual relapse rate to 0.61 , compared with 0.90 in the placebo group.36 these findings are supported by significant reduction of mri disease activity in all three trials , while only in the latter two trials , a beneficial effect on disability progression was recorded during this 2-year study period . while these therapies have been introduced , a better understanding of ms pathophysiology especially the knowledge of unrecoverable axonal loss already in early stages of the disease has prompted a fundamental shift in the ms treatment approach and provided the rational for early ms treatment.33,34 based on this consideration , dmts are initiated early in the course of the disease and before irreversible neuronal damage and disability have occurred . following the concept of early ms treatment , interferon beta treatment has been investigated in patients with cis and has shown a beneficial effect in terms of conversion to definite ms , edss progression , and mri disease activity.3841 in the betaferon/betaseron in newly emerging multiple sclerosis for initial treatment ( benefit ) trial,40 patients with cis were randomized to receive interferon beta1-b or placebo for 2 years , or until clinically definite multiple sclerosis ( cdms ) was diagnosed , according to the poser et al criteria.42 within the 2-year study period , 45% of patients in the placebo group versus 28% of the interferon beta group progressed to cdms , according to poser et al ; and 85% of the placebo versus 69% of the interferon beta group were diagnosed with definite ms , according to the mcdonald criteria of 2001.43 both results reached statistically significant difference . these data revealed a significant lower number of newly active lesions , cumulative number of new t2-weighted mri lesions and gadolinium - enhancing lesions , as well as cumulative volume of gadolinium - enhancing lesions in the interferon beta - treated patients group . after having finished the placebo - controlled core study , patients were offered to enroll in an open - label interferon beta-1b 250 g follow - up trial to investigate effects of early treatment in cis , versus delayed treatment after diagnosis of cdms or after 2 years on study . three years after initial randomization , 37% of the early treatment group versus 51% of the delayed - treatment group have developed cdms ( risk reduction of 41% ) ; 16% of the early treatment group versus 24% of the delayed - treatment group experienced confirmed edss progression ( risk reduction 40%).44 a subgroup analysis revealed lower treatment effects in patients with less clinical or mri activity at the time of the first event ; patients with initial multifocal presentation and high lesion load would benefit more from early treatment . in the 5-year active treatment extension follow - up study , risk reduction of cdms remained 37% in the early treatment group ; whereas , no statistically significant difference in confirmed disability progression was observed any more between early and delayed interferon beta treatment.45 in both treatment groups , median edss remained 1.5 during the 5-year study period . finally , in the benefit extension trial , a long - term international observational study of patients having completed the initial placebo - controlled core study , data on follow - up for more than 8 years are available.46 at the discretion of the physician and according to local standards , patients were treated with any of the disease - modifying drugs or received no treatment . however , 55.5% of patients , who had received early interferon beta treatment in the core study , developed clinically definite ms , while clinically definite ms was diagnosed in 65.8% of patients with delayed interferon beta treatment . moreover , a difference of the overall annualized relapse rate was still observed over the 8-year period 0.20 early treatment versus 0.26 delayed treatment.47 placebo - controlled clinical trials on interferon beta-1a in cis have yielded very similar results,38,41 suggesting a class effect of interferon beta treatment in early ms and cis . side effects mainly include injection site reactions , flu - like symptoms , leukopenia , and liver - enzyme elevation.48 moreover , up to 40% of patients develop antibodies against interferon beta , which antagonize bioactivity in a significant proportion of patients.4951 if neutralizing antibodies against interferon beta persistently occur , therapeutic efficacy of the drug is reduced or abolished.5256 besides interferon beta glatiramer acetate ( [ ga ] , copaxone , teva , petah tikva , israel ) represents a common first - line therapy with daily subcutaneous injections , which is approved for treatment of patients with relapsing remitting ms , as well as for patients with cis and typical mri features of ms . the precise ( early glatiramer acetate treatment in delaying conversion to clinically definite multiple sclerosis in subjects presenting with a clinically isolated syndrome ) study demonstrated that ga reduced the risk of developing clinically definite multiple sclerosis by 45% , compared with placebo during a 3-year study period.57 interferon beta and ga are the only agents that are licensed for treatment of cis . however , for the sake of completeness , it needs to be mentioned that there are several other therapeutics licensed for baseline and escalation therapy in definite ms , and more agents are likely to enter the market.16 approval of all these agents is usually based on efficacy and safety data of a 2-year clinical trial . given an increasing number of available dmt in ms , trials investigating the long - term efficacy are needed . this is to briefly summarize the terms and types of pharmacoeconomic evaluations used in the studies reviewed here ( adapted from clifford goodman unless cited otherwise).25 cost - effectiveness analyses ( cea ) measure costs in monetary units and consequences in natural units , such as life - years gained , or relapses avoided.58 in cost - utility analyses ( cua ) , a variant of cea , consequences are measured in terms of preference - based measures of health,58 most commonly based on quality - adjusted life - years ( qaly).59 a qaly is a unit that adjusts gained or lost life - years subsequent to an intervention by the quality of life during those years . costs need to be corrected for effects of inflation ; both costs and outcomes should be discounted . discounting reflects the fact that costs and benefits tend to have less value in the future than in present . we conducted a systematic literature search in medline ( pubmed ) on december 14 , 2012 , applying the medical subject headings ( mesh ) terms multiple sclerosis and costs and cost analysis . inclusion criteria were as follows : ( 1 ) original research on the pharmacoeconomics of dmt in ms ; ( 2 ) comparative study ; and ( 3 ) comparators ( initiation of dmt at the time of the first demyelinating event versus treatment after cdms ) . this observation led to the approval of interferon beta and ga for treatment of relapsing remitting ms , as well as cis . therefore , initiation of dmt may be started even before definite diagnosis of ms is established . to date , four studies have been published that address the economic impact of starting dmt at the time of the incident cis versus after conversion to cdms ( table 1).6164 the swedish study by caloyeras et al estimates cost - effectiveness of interferon beta-1b if initiated in cis compared with delayed treatment after the diagnosis of cdms has been made.61 the authors applied a markov model analysis , based on twelve health states defined by edss scores , the assigned diagnosis ( ie , cis versus cdms ) , and the course of disease ( ie , relapsing or nonrelapsing forms of ms , and death ) . results of the cua demonstrate that , during a 50-year time horizon , patients assigned to the early treatment group gained 12.9 qalys , as compared with 12.4 qualys in the delayed - treatment group . on average , patients in the delayed - treatment group progressed 2 years earlier to cdms ( estimated conversion rate 99.54% ) , compared with patients in the early treatment group ( estimated conversion rate 99.31% ) . when all costs were summed up over the 50-year time horizon , total costs in the delayed - treatment arm were higher than those in the early treatment arm ( difference of approximately 344,000 sek per patient ) . curkendall et al applied a different approach to assess the pharmacoeconomics of early dmt.62 their analysis was based on insurance claims data in the united states for the years 20002008 , extracted from the thomson reuters marketscan commercial and medicare supplemental databases ; therefore , only health care expenditures and utilizations were analyzed . the core inclusion and exclusion criteria were as follows : ( 1 ) no diagnosis of ms during 12 months before and at the time of a cerebral mri ; this mri scan was defined as start date of the 12-months observation period ; ( 2 ) at least one symptom that was documented 6 months before or 1 month after the index date and that was likely to be attributable to ms ; ( 3 ) initiation of treatment with interferon beta-1a , interferon beta-1b , or ga sometime during the follow - up period . in contrast to the other articles reviewed here , the strength of this study lies in the analysis of real costs , albeit restricted to health care expenditures . one group of patients received interferon beta-1b after diagnosis of cis ; the other group was treated with interferon beta-1b after diagnosis of cdms . the authors conclude that , from the health service and societal perspective , early treatment with interferon beta-1b may be cost - effective , compared with treatment initiation after cdms . iskedjian et al performed a pharmacoeconomic analysis of treatment with interferon beta-1a administered intramuscularly once weekly in cis patients ; the study was conducted from a canadian perspective.64 the aim of this study was to investigate : ( 1 ) cost - effectiveness of interferon beta-1a treatment based on the gain of additional monosymptomatic life - years ( mlys ) ; and ( 2 ) cost - utility of long - term interferon beta treatment based on the gain of quality - adjusted monosymptomatic life - years ( qamlys ) . once the diagnosis of cdms was established , all patients received the same treatment regime with interferon and high dose steroids for treatment of relapse . the conversion rate to cdms was derived from the efficacy results of the controlled high risk avonex multiple sclerosis study ( champs ) study.38 transition probabilities through the various stages of edss were derived from a canadian ms study by weinshenker et al.7 cost analyses , including unemployment rates and the average hospital length of stay within each edss level , were based on data from the literature7072 and canadian health authorities . the multivariate sensitivity analysis of the cea showed that in 87% and 6% of the resulting scenarios the icer of interferon beta therapy would be lower than the costs of current treatment per mly gained from the societal and moh perspective , respectively . therefore , the authors conclude that interferon beta treatment in cis may be cost - effective . of the three pharmacoeconomic studies reviewed here , ms is a chronic disease with high economic burden on patients , families , the health system , and society . as reviewed here , several studies have demonstrated that progression of disease severity is accompanied by a rise of total costs as well as a change in the distribution of costs . during early stages of the disease , direct costs , which are mainly caused by dmt , dominate the total costs ; whereas , with accumulation of disability during later stages of the disease especially indirect costs significantly increase.73 moreover , occurrence of relapses has been associated with a peak of costs . due to a better understanding of ms pathogenesis and the experience that efficacy of available dmt is high in early stages of ms but declines in the progressive phase of the disease , the concept of early ms treatment has been established . therefore , dmts are usually initiated after diagnosis of relapsing remitting ms or even before definite diagnosis of ms is made at the stage of cis . numerous studies have investigated the pharmacoeconomics of dmt in ms , though only few have explicitly analyzed the pharmacoeconomic effect of early initiation of dmt in cis.6164 these studies indicate that early versus delayed treatment with interferon beta may be overall cost - effective in the long term . for instance , becker et al74 recently demonstrated the impact of cohort selection by replicating a previously published model - based analysis75 on the cost - effectiveness of interferon beta in ms . in their study reviewed here , iskedjian et al showed that , from the canadian societal perspective , estimated incremental costs of early interferon beta treatment would be 189,286 cad per qamly gained . when applying different utility estimates , the same analysis resulted in 91,228 cad , which is less than 50% of the former result.64 however , it seems to be ambiguous which of the applied utility estimates may be more valid than the other . in this context , one should note , that several pharmacoeconomic studies in ms , including the four studies reviewed here , have been funded by manufacturers of dmt ( table 1 ) . therefore , we need more data on long - term efficacy of dmt and costs in ms in the real - life setting .

fanconi anemia ( fa ) is a chromosomal instability syndrome characterized by developmental abnormalities , progressive bone marrow failure , and increased cancer susceptibility ( dandrea , 2010 ) . bone marrow failure is the main cause of childhood fatality in fa patients , and unfortunately , most children with fa develop myelodysplastic syndrome ( mds ) or acute myeloid leukemia ( aml ) ( garaycoechea and patel , 2014 ; kee et al . , 2012 ) . moreover , patients who live into adulthood are predisposed to additional malignancies , including head and neck , gynecological , and gastrointestinal cancers . thus , the study of fa has provided important insights into the pathogenesis of cancer and could lead to the development of anti - cancer therapeutics for the general population . fa is caused by germ - line mutations in one of the 17 currently known fa genes whose gene products participate in a common dna repair pathway called the fa pathway ( kim and dandrea , 2012 ) . the pathway functions to resolve dna interstrand cross - links ( icls ) , lethal dna lesions that block dna replication and transcription ( clauson et al . , 2013 ; deans and west , 2011 ) . it is also required to buffer against the genotoxic effects of reactive aldehydes naturally produced inside the cells ( langevin et al . , 2011 ) . the fa pathway coordinates various steps in icl repair : recognition and unhooking of icls , translesion dna synthesis ( tls ) , and homologous recombination ( hr ) ( kottemann and smogorzewska , 2013 ) . central to the fa pathway is the conjugation of monoubiquitin to fancd2 at lys561 and , to a lesser extent , its binding partner fanci at lys523 in human ( garcia - higuera et al . , 2001 ; smogorzewska et al . , 2007 ) the ubiquitinated fancd2-fanci complex promotes downstream nucleolytic incisions near the icl and tls past the lesion ( knipscheer et al . , 2009 ) . this step is activated by the multi - subunit ubiquitin e3 ligase complex , also known as the fa core complex , which is composed of eight fanc proteins ( fanca / b / c / e / f / g / l / m ) along with the accessory proteins , faap100 , faap20 , and faap24 ( walden and deans , 2014 ) . the fa pathway senses an icl - mediated stalled replication fork , and the fancm - faap24 heterodimer in the complex recognizes dna damage caused by the dna icl and induces ataxia - telangiectasia and rad3-related ( atr)-dependent activation of the fa core complex ( collis et al . , 2008 ) . the auxiliary proteins , histone - fold containing kinetochore protein mhf1 and mhf2 , promote constitutive association of fancm to chromatin ( singh et al . , 2010 ; yan et al . , 2010 ) . fancm contains a deah translocase domain at its n - terminus ( meetei et al . , 2005 ) . although not required for fancd2 monoubiquitination , atpase activity of fancm is essential for remodeling and stabilization of replication forks , and activation of the cell cycle checkpoint ( blackford et al . , 2012 ; gari et al . , 2008 ; schwab et al although the mechanisms of icl repair are not yet fully understood , the most detailed model derived from studies in xenopus egg extracts suggests that two dna replication forks converge on an icl to initiate repair ( fig . the ubiquitinated fancd2-fanci heterodimer is relocalized to a converged fork near the icl and controls nucleolytic incision to release the icl from one of the two strands ( knipscheer et al . , 2009 ) . fancd2-monoubiquitin ( fancd2-ub ) has been implicated in the recruitment of several structure - specific nucleases , which are responsible for unhooking the dna icl ( fig . fancp / slx4 functions as a scaffold that targets structure - specific nucleases such as the ercc1-fancq / xpf heterodimer and slx1 to a lesion , utilizing its ubz4 ( ubiquitin - binding zinc finger 4 ) motif that recognizes fancd2-ub ( cybulski and howlett , 2011 ; yamamoto et al . , 2011 ) . recent biochemical and genetic studies indicated that nuclease activity of the ercc1-xpf complex plays a prominent role in carrying out unhooking incisions , which is controlled by the presence of slx4 ( hodskinson et al . interestingly , mutations in fa - q patients disrupt the function of xpf in icl repair , without altering its function in nucleotide excision repair ( ner ) , highlighting the multifunctional activity of the xpf endonuclease ( bogliolo et al . , 2013 ) . the mus81-eme1 nuclease complex also associates with slx4 and functions in icl repair , but is dispensable for initial icl processing in xenopus egg extracts ( klein douwel et al . , 2014 ; rschle et al . , 2015 ) , suggesting that the complex may process different intermediates in icl repair such as holliday junction ( hj ) resolution during double - strand break ( dsb ) repair ( chen et al . , 2001 ) . fan1 , another structure - specific nuclease implicated in icl repair , contains a ubz4 motif required for interaction with fancd2-ub ( smogorzewska et al . , 2010 ) . however , fan1 deficiency leads not to fa , but rather to a rare kidney disease called karyomegalic interstitial nephritis ( kin ) , suggesting that fan1 may play a redundant role with other nucleases in the fa pathway or deal with lesions independently of the fa pathway ( zhou et al . , slx4 also participates in diverse genome maintenance pathways including telomere maintenance and regulation of dna damage checkpoints ( kim , 2014 ) . interestingly , the slx4 complex was recently shown to function as a sumo e3 ligase that sumoylates xpf and counteracts replication stress ( guervilly et al . , 2015 ) . tls , a dna damage tolerance mechanism mediated by specialized tls polymerases ( chang and cimprich , 2009 ) , allows a nascent leading strand to bypass the unhooked adduct and extend from primer - template termini to resume replication ( fig . the 5 exonuclease snm1a may promote this process by digesting the unhooked cross - link intermediate , generating a preferred substrate for tls polymerases ( wang et al . , 2011 ) . the active site for tls polymerases is less restricted , and tls polymerases can thus accommodate base mismatches and distorted base - parings . the precise mechanism for the recruitment of tls polymerases to dna icl lesions remains elusive , but the fa core complex has been shown to facilitate this process ( budzowska et al . , 2015 ; kim et al . , 2012 ) . among tls polymerases , pol ( a rev3-rev7 heterodimer ) and rev1 play a key role in replication - dependent icl repair . previous genetic studies in chicken dt40 cells indicated that rev1 and rev3 are epistatic with fancc in cisplatin sensitivity ( niedzwiedz et al . , 2004 ) . pol has been shown to execute the lesion bypass step of icl repair that occurs in xenopus egg extracts ( raschle et al . , 2008 ) . although rev1 has deoxycytidyl transferase activity to insert dcmp opposite an icl , it plays a more structural role to facilitate polymerase switching between different tls polymerases , and coordinates insertion and extension steps ( lehmann et al . indeed , recent structural analysis revealed the formation of a quaternary tls polymerase complex consisting of the c - terminal domain ( ctd ) of rev1 , heterodimeric pol and pol , thereby highlighting the role of the rev1 ctd in a scaffold that simultaneously binds these polymerases ( wojtaszek et al . , 2012 ) . given the diverse structures formed by distinct icl - inducing agents , each icl lesion may be processed by a combination of specific tls polymerases with unique substrate preferences ( guainazzi and schrer , 2010 ) . another important step following nucleolytic incision is repairing replication - associated dsbs , which is mediated by hr . a sister chromatid restored by tls is used as a template for strand invasion by the 3 overhang of a lagging strand to restore information lost during the incision process ( fig . fancd2 physically interacts with ctip , a protein required for end resection , to channel repair to the hr process ( unno et al . , 2014 ) . rad51 coats a single - stranded dna to initiate strand invasion , and fancd1/brca2 is required for its loading onto stalled forks ( moynahan et al . , 2001 ) . fancn / palb2 interacts with brca1 to promote this process ( xia et al . , 2006 ) . the rad51 paralog fanco / rad51c also contributes to replication - associated dsb repair by participating in strand invasion and hj resolution ( liu et al . , 2007 ; vaz et al . , 2010 ) . biallelic mutations in brca1 were recently found in a breast cancer patient with a fa - like disorder , and thus brca1 has been designated as a new fa gene , fancs ( sawyer et al . , 2015 ) . it associates with brca2 and promotes resection of the double - stranded dna ends for rad51 loading ( zhang et al . , 2009 ) . it is also required for unloading of the cdc45-mcm - gins ( cmg ) helicase complex from stalled forks and loading of fancd2-ub onto dna lesions , which functions independently of hr ( bunting et al . , 2012 ; long et al . , 2014 copying information from a sister chromatid through hr restores a replication fork , and the unhooked adduct is believed to be removed by ner . the deubiquitinating enzyme usp1 regulates the level of fancd2-ub ( nijman et al . , 2005 ) . usp1 associates with its activating factor uaf1 , and the usp1-uaf1 complex removes monoubiquitin from fancd2 to complete the repair ( cohn et al . , 2007 ) in addition to its stimulatory role , uaf1 is also necessary for recruiting the fancd2-fanci complex to usp1 ( yang et al . , 2011 ) . the usp1 knockout mouse exhibits fa phenotypes , and usp1 cells are hypersensitive to dna cross - linking agents , indicating that timely deubiquitination process is essential for the integrity of the fa pathway ( kim et al . , 2009 ) . however , the precise timing and location of deubiquitination of fancd2 remain to be resolved . fancd2 monoubiquitination by the fa core complex is a key regulatory step in the fa pathway as it connects an upstream dna damage response ( ddr ) to downstream enzymatic dna repair processes . a variety of posttranslational modifications and protein - protein interactions maintain the integrity of the complex and regulate its enzymatic activity ( fig . 2 ) . among the components of the complex , only the fancl subunit has known catalytic activity mediated by a ring domain ( meetei et al . , 2003 ) . notably , disruption of the interaction between fancl and its ubiquitin e2 conjugating enzyme ube2 t caused by ube2 t mutations in fa patients leads to compromised fancd2 monoubiquitination , suggesting that ube2 t ( fanct ) mutations define a new fa subtype ( hira et al . , recent biochemical and genetic experiments classified the fa core complex into three distinct modules , namely , the fancb - fancl - faap100 core catalytic unit , and the fanca - fancg - faap20 and fancc - fance - fancf auxiliary units ( huang et al . , 2014 ; rajendra et al . , 2014 ) . a minimal fancb - fancl - faap100 subcomplex is sufficient to monoubiquitinate fancd2 in vitro ( rajendra et al . , 2014 ) . the other modules are necessary for stabilizing the fa core complex and achieving its maximal activity . for instance , the n - terminus of fancf connects three modules to the fancm anchor complex ( deans and west , 2009 ) . the c - terminus of fance is required for recruiting the fancd2-fanci heterodimer to the fa core complex to facilitate fancd2 monoubiquitination ( polito et al . , 2014 ) . the n - terminus of faap20 interacts with fanca and prevents it from undergoing uncontrolled degradation ( kim et al . , 2012 ; deficiency in fanca also results in destabilization of its binding partners fancg and faap20 , and hypersensitivity to icl - inducing agents , highlighting its role as a scaffold to preserve the integrity of the complex . however , it remains unclear why the fa core complex consists of at least ten subunits that do not appear to have any homology or evolutionary connections , and what precise roles each module exerts . recognition of icl - stalled replication forks by the fancm - faap24 heterodimer , which recruits replication protein a ( rpa ) and the human homolog of the caenorhabditis elegans biological clock protein clk-2 ( hclk2 ) , initiates the activation of the atr - mediated checkpoint and the fa core complex ( collins et al . multiple components of the fa core complex are activated by phosphorylation during the stress response . for instance , disruption of atr - dependent fancm phosphorylation at ser1045 compromises chk1 activation and fancd2 monoubiquitination ( singh et al . , 2013 ) . chk1-mediated phosphorylation of fance at thr346 and ser374 , and atr - dependent fanca phosphorylation at ser1449 are required for the functional integrity of the fa pathway ( collins et al . fancd2 phosphorylation at thr691 and ser717 promotes fancd2 monoubiquitination and the intra - s - phase checkpoint ( ho et al . , 2006 ) . fanci contains a cluster of sq / tq phosphorylation sites near its ubiquitination site , and mutations of these residues abrogate fancd2 monoubiquitination ( ishiai et al . , 2008 ) . conversely , fanci phosphomimetic mutations enhance fancd2 activation , indicating that the level of fanci phosphorylation dictates fancd2 activation . interestingly , the three - dimensional structure of the fancd2-fanci heterodimer revealed a unique fancd2 interacting domain in fanci , which undergoes a conformational change that stabilizes its interaction with fancd2 ( joo et al . , 2011 ) . structural reorganization of the heterodimer mediated by atr - dependent fanci phosphorylation may render fancd2 suitable for monoubiquitination , although how fancd2 monoubiquitination is coordinated with fanci phosphorylation is unclear . the fancd2-fanci heterodimer is sumoylated following replication stress in an atr - dependent manner , and sumoylated fancd2 is a target for the sumo - targeted ubiquitin e3 ligase ( stubl ) rnf4 , which results in polyubiquitination and removal of fancd2 from damage sites via the dvc1-p97 ubiquitin segregase complex ( gibbs - seymour et al . , 2015 ) . improper clearance of fancd2 due to disruption of sumo signaling compromises cellular survival against replication stress , suggesting that timely inactivation of fancd2 is required for the functional integrity of the fa pathway . characterization of a breast cancer patient with a unique fanca mutation that disrupts the interaction with faap20 revealed that in the absence of faap20 interaction , a fanca sumoylation site becomes exposed , which initiates proteasome - dependent fanca degradation mediated by rnf4 ( xie et al . , 2015 ) . aberrant accumulation of fanca at sites of repair by the loss of rnf4 may prevent replication fork restarting and thus inhibit completion of dna repair . notably , sumo modification has been shown to occur simultaneously at multiple sites of several proteins during dsb repair and stabilize physical interactions between the proteins ( psakhye and jentsch , 2012 ) . this protein group sumoylation may be relevant to the activation of the fa core complex as well ; pervasive sumoylation may stabilize the fa core complex assembly and promote its activation , which is followed by selective degradation of its components by a stubl such as rnf4 . several other components of the fa core complex in addition to fanca are expected to be sumoylated in a similar manner ( xie et al . , 2015 ) , and thus the role of sumoylation in regulating the fa pathway awaits further characterization . several factors have been implicated in the regulation of fancd2 activation in addition to the fa core complex . deficiency of rad18 , an ubiquitin e3 ligase involved in postreplication repair of uv - damaged dna , delays the kinetics of fancd2 monoubiquitination ( williams et al . , 2011a ) . the muts complex , a damage sensor required for mismatch repair , has been shown to facilitate the recruitment of the fa core complex to chromatin , thereby playing a redundant role with the fancm complex ( huang et al . , 2011 ; williams et al . ubiquitin - like with phd and ring finger domains 1 ( uhrf1 ) , a key epigenetic regulator of chromatin modification at replication forks , was recently identified as a dna icl recognition factor that initiates icl repair ( liang et al . , 2015 ; tian et al . , 2015 uhrf1 is rapidly targeted to a dna icl , which promotes recruitment of fancd2 and structure - specific nucleases required for icl processing . it will be interesting to determine if these factors crosstalk with the fa core complex to fine - tune the steps of fancd2 activation . fancd2 monoubiquitination by the fa core complex is a key regulatory step in the fa pathway as it connects an upstream dna damage response ( ddr ) to downstream enzymatic dna repair processes . a variety of posttranslational modifications and protein - protein interactions maintain the integrity of the complex and regulate its enzymatic activity ( fig . 2 ) . among the components of the complex , only the fancl subunit has known catalytic activity mediated by a ring domain ( meetei et al . , 2003 ) . notably , disruption of the interaction between fancl and its ubiquitin e2 conjugating enzyme ube2 t caused by ube2 t mutations in fa patients leads to compromised fancd2 monoubiquitination , suggesting that ube2 t ( fanct ) mutations define a new fa subtype ( hira et al . , recent biochemical and genetic experiments classified the fa core complex into three distinct modules , namely , the fancb - fancl - faap100 core catalytic unit , and the fanca - fancg - faap20 and fancc - fance - fancf auxiliary units ( huang et al . , 2014 ; rajendra et al . , 2014 ) . a minimal fancb - fancl - faap100 subcomplex is sufficient to monoubiquitinate fancd2 in vitro ( rajendra et al . , 2014 ) . the other modules are necessary for stabilizing the fa core complex and achieving its maximal activity . for instance , the n - terminus of fancf connects three modules to the fancm anchor complex ( deans and west , 2009 ) . the c - terminus of fance is required for recruiting the fancd2-fanci heterodimer to the fa core complex to facilitate fancd2 monoubiquitination ( polito et al . , 2014 ) . the n - terminus of faap20 interacts with fanca and prevents it from undergoing uncontrolled degradation ( kim et al . , 2012 ; deficiency in fanca also results in destabilization of its binding partners fancg and faap20 , and hypersensitivity to icl - inducing agents , highlighting its role as a scaffold to preserve the integrity of the complex . however , it remains unclear why the fa core complex consists of at least ten subunits that do not appear to have any homology or evolutionary connections , and what precise roles each module exerts . recognition of icl - stalled replication forks by the fancm - faap24 heterodimer , which recruits replication protein a ( rpa ) and the human homolog of the caenorhabditis elegans biological clock protein clk-2 ( hclk2 ) , initiates the activation of the atr - mediated checkpoint and the fa core complex ( collins et al . multiple components of the fa core complex are activated by phosphorylation during the stress response . for instance , disruption of atr - dependent fancm phosphorylation at ser1045 compromises chk1 activation and fancd2 monoubiquitination ( singh et al . , 2013 ) . chk1-mediated phosphorylation of fance at thr346 and ser374 , and atr - dependent fanca phosphorylation at ser1449 are required for the functional integrity of the fa pathway ( collins et al . , 2009 ; wang et al . , 2007 ) fancd2 phosphorylation at thr691 and ser717 promotes fancd2 monoubiquitination and the intra - s - phase checkpoint ( ho et al . , 2006 ) . fanci contains a cluster of sq / tq phosphorylation sites near its ubiquitination site , and mutations of these residues abrogate fancd2 monoubiquitination ( ishiai et al . , 2008 ) . conversely , fanci phosphomimetic mutations enhance fancd2 activation , indicating that the level of fanci phosphorylation dictates fancd2 activation . interestingly , the three - dimensional structure of the fancd2-fanci heterodimer revealed a unique fancd2 interacting domain in fanci , which undergoes a conformational change that stabilizes its interaction with fancd2 ( joo et al . , 2011 ) . structural reorganization of the heterodimer mediated by atr - dependent fanci phosphorylation may render fancd2 suitable for monoubiquitination , although how fancd2 monoubiquitination is coordinated with fanci phosphorylation is unclear . the fancd2-fanci heterodimer is sumoylated following replication stress in an atr - dependent manner , and sumoylated fancd2 is a target for the sumo - targeted ubiquitin e3 ligase ( stubl ) rnf4 , which results in polyubiquitination and removal of fancd2 from damage sites via the dvc1-p97 ubiquitin segregase complex ( gibbs - seymour et al . , 2015 ) . improper clearance of fancd2 due to disruption of sumo signaling compromises cellular survival against replication stress , suggesting that timely inactivation of fancd2 is required for the functional integrity of the fa pathway characterization of a breast cancer patient with a unique fanca mutation that disrupts the interaction with faap20 revealed that in the absence of faap20 interaction , a fanca sumoylation site becomes exposed , which initiates proteasome - dependent fanca degradation mediated by rnf4 ( xie et al . , 2015 ) . aberrant accumulation of fanca at sites of repair by the loss of rnf4 may prevent replication fork restarting and thus inhibit completion of dna repair . notably , sumo modification has been shown to occur simultaneously at multiple sites of several proteins during dsb repair and stabilize physical interactions between the proteins ( psakhye and jentsch , 2012 ) . this protein group sumoylation may be relevant to the activation of the fa core complex as well ; pervasive sumoylation may stabilize the fa core complex assembly and promote its activation , which is followed by selective degradation of its components by a stubl such as rnf4 . several other components of the fa core complex in addition to fanca are expected to be sumoylated in a similar manner ( xie et al . , 2015 ) , and thus the role of sumoylation in regulating the fa pathway awaits further characterization . several factors have been implicated in the regulation of fancd2 activation in addition to the fa core complex . deficiency of rad18 , an ubiquitin e3 ligase involved in postreplication repair of uv - damaged dna , delays the kinetics of fancd2 monoubiquitination ( williams et al . , 2011a ) . the muts complex , a damage sensor required for mismatch repair , has been shown to facilitate the recruitment of the fa core complex to chromatin , thereby playing a redundant role with the fancm complex ( huang et al . ubiquitin - like with phd and ring finger domains 1 ( uhrf1 ) , a key epigenetic regulator of chromatin modification at replication forks , was recently identified as a dna icl recognition factor that initiates icl repair ( liang et al . , 2015 ; tian et al . , 2015 ) uhrf1 is rapidly targeted to a dna icl , which promotes recruitment of fancd2 and structure - specific nucleases required for icl processing . it will be interesting to determine if these factors crosstalk with the fa core complex to fine - tune the steps of fancd2 activation . the dna repair system functions as a critical tumor suppressor network to preserve the integrity of the genome and prevent malignancy . germ - line mutations or promoter hyper - methylation of dna repair genes confer increased risk for multiple cancers ( negrini et al . , 2010 ) . in addition , replication stress resulting from high levels of dna damage that interfere with dna replication and progression is augmented by faulty dna repair mechanisms caused by selection of somatic mutations that disrupt dna repair process ( gaillard et al . , 2015 ) . for instance , dna hyper - replication induced by oncogene activation triggers ddr as a natural barrier to prevent malignancy ; thus , inactivation of ddr promotes cellular transformation , and mutations in ddr factors are frequently found in various human cancers ( bartkova et al . , 2006 ; di micco et al . , 2006 ; kandoth et al . , however , although defects in dna repair confer survival advantages on cancer cells by increasing their adaptability , these defects may reveal a weakness that can be therapeutically exploited . this is possible because cancer cells either become more susceptible to conventional chemotherapy that causes dna damage due to a decreased capacity to address genotoxicity , or become hyperdependent on another compensatory dna repair pathway , which provides a therapeutic window for specific killing of cancer cells . hence , icl - inducing agents such as nitrogen mustards and platinum compounds are some of the most widely used chemotherapeutic regimens to treat leukemia as well as a variety of solid tumors . in - depth analysis of the fa signaling pathway has helped to understand the molecular mechanism of the chemotherapeutic effects of dna icl - inducing agents and allowed for targeted anti - cancer therapy . this effect could be achieved either by inhibiting the intact or upregulated fa pathway to chemosensitize cancer cells , or by exploiting the synthetic lethality of cancer cells that are defective in the fa pathway ( fig . 3 ) . resistance to icl - inducing chemotherapy constitutes a significant barrier to improving patient outcomes . as icl - inducing chemotherapy directly causes dna damage , the dna repair capacity of cancer cells plays a major role in determining the effectiveness of dna - damaging drugs . for instance , a role for tls has been implicated in acquired drug resistance following chemotherapy . suppression of rev1 or pol not only sensitized cancer cells to platinum agents but also limited mutagenesis and acquired chemoresistance in mouse b - cell lymphoma and lung adenocarcinoma models ( doles et al . upregulation of rev3l mrna levels was observed in human glioma , and stable overexpression of rev3l attenuated cisplatin - induced toxicity , while downregulation enhanced its cytotoxicity ( wang et al . , 2009 ) . these results indicate that upregulation of tls represents one of the critical mechanisms that confer acquired chemoresistance on tumor cells , and targeting the enhanced tls pathway using specific tls polymerase inhibitors could be utilized as an adjuvant therapy for treating chemoresistant malignancy . selective inhibition of the active site of one of the many tls polymerases may be difficult to achieve , but recent structural data on protein - protein interactions that regulate tls polymerase activities may be helpful for identifying small molecules that specifically interfere with such interactions ( kikuchi et al . hyperdependency on compensatory dna repair due to defects in the fa pathway can be exploited for achieving synthetic lethality . the best example is development of poly - adp ribose polymerase ( parp ) inhibitors to treat breast and ovarian cancers harboring brca1/2 mutations ( bryant et al . , 2005 ; farmer et al . , 2005 ) . because dna lesions that can not be repaired by base excision repair ( ber ) due to the inhibition of parp enzymes should be repaired by hr during the s - phase , cancer cells that are defective in hr are selectively sensitive to parp inhibition owing to the concomitant loss of two dna repair pathways theoretically , tumors that share common hr defects ( i.e. , brcaness ) could be managed with parp inhibitors . indeed , impaired brca1 phosphorylation and subsequent defects in hr caused by cdk1 inhibition was shown to sensitize brca1-proficient tumors to parp inhibition , suggesting that functional disruption of hr signaling could expand the utility of parp inhibitors ( johnson et al . , 2011 ) . parp enzymes are also involved in the error - prone microhomology - mediated end joining ( mmej ) pathway . dna polymerase promotes this process , and a recent genetic study showed that , unlike single knockouts , fancd2polq double knockout mice die in utero ( ceccaldi et al . , 2015 ; mateos - gomez et al . , 2015 given that pol was found to be highly upregulated in epithelial ovarian cancers , this result indicates that the synthetic lethal relationship between the hr pathway and the pol -dependent mmej could be employed as a new therapeutic target for cancers with defective hr , or the fa pathway in general . although inhibition of the fa pathway can occur at multiple levels , fancd2 monoubiquitination has been a primary target for pharmacological interventions . a previous study demonstrated that proteasome inhibition by bortezomib or depletion of proteasome subunits leads to suppression of fancd2 monoubiquitination and foci formation ( jacquemont and taniguchi , 2007 ) . bortezomib also downregulates fancd2 gene expression by inhibiting nf-b signaling and enhances cytotoxicity of melphalan - resistant multiple myeloma cells ( yarde et al . , 2009 ) . bortezomib has been used as standard care for relapsed / refractory multiple myeloma and mantle cell lymphoma , and eventually , its use could be expanded to include a combination therapy , for instance with parp inhibition in hr - proficient tumors . the natural compound curcumin and its analogs such as ef24 and 4h - ttd have been shown to inhibit fancd2 activation and sensitize a variety of cancer cells to dna damage ( chirnomas et al . , 2006 ; landais et al . , in addition , mln4924 , an inhibitor of a nedd8 activating enzyme , was shown to impair fancd2 activation , rendering cancer cells hypersensitive to icl - inducing agents ( kee et al . , 2012 ) . the usp1-uaf1 deubiquitinating enzyme complex plays an essential role in the fa pathway by antagonizing the level of fancd2-ub . thus , disrupting the ubiquitin - deubiquitination cycle of fancd2 could lead to the inhibition of the fa pathway . indeed , several inhibitors of the usp1-uaf1 complex have been developed to target the fa pathway . small molecule inhibitors such as pimozide and gw7647 , as well as a more selective inhibitor ml323 , inhibit fancd2 deubiquitination and potentiate cisplatin cytotoxicity of chemoresistant cancer cells ( chen et al . inhibiting the usp1-uaf1 complex also compromises tls by increasing levels of monoubiquitinated pcna , another substrate of usp1 , suggesting that inhibition of the usp1-uaf1 complex can simultaneously target two major steps in the fa pathway ( liang et al . , 2014 ) . usp1 also prevents the inhibitor of dna - binding-1 ( id1 ) transcription factor from undergoing destruction and thus maintains the stemness of malignant cells ( williams et al . , 2011b ) . another usp1 inhibitor c527 was shown to promote id1 degradation and induce differentiation of leukemic cells ( mistry et al . , 2013 ) . an sirna - based synthetic lethal screening identified several genes including atm , parp1 , cdk1 , nbs1 , and plk1 that are required for the survival of cells deficient in fancg , indicating that these genes could be targeted in conjunction with an fa pathway inhibitor ( kennedy et al . , 2007 ) . as deficiency of atm signaling is found in several types of leukemia and triple - negative breast cancer , the fa pathway inhibitor could be used as a single agent in these cancers as well . in addition , chk1 inhibition was shown to be synthetically lethal with fanca deficiency following cisplatin treatment ( chen et al . , 2009 ) . these results suggest that targeting the fa pathway can become more effective by inhibiting dna damage signaling simultaneously , and appropriate combinations of ddr and dna repair inhibitors could be customized for increasing the efficacy of chemotherapy . since the discovery of fancd2 monoubiquitination in the early 2000s , the study of fa has integrated important topics in biology , including ubiquitin signaling , dna repair , and the pathogenesis of cancer . fancd2 activation acts as a surrogate marker for the dna icl repair process , and knowledge of how fancd2 is activated to regulate downstream repair steps has opened new therapeutic opportunities for cancer treatment we must increase our understanding of the regulatory mechanisms underlying fancd2 activation by the fa core complex . more specifically , a comprehensive understanding of the complex network of posttranslational modifications that regulate the fa core complex may lead to the identification of additional targets for therapeutic interventions . moreover , finding ways to inhibit the enzymatic steps of the fa pathway , including incisions and tls , may be useful . basic information on dna repair regulation could also have a broad translational impact . profiling dna repair in individuals with nonfunctional or upregulated dna repair capacity optimizing the selection of a chemotherapy approach requires the identification of reliable dna repair biomarkers . thus , development of fast and affordable ways to monitor individual dna repair activity is highly desirable . furthermore , synthetic lethality could be extended beyond dna repair pathways , to include aberrant signaling of activated oncogenes and growth factors , or enhanced anti - apoptotic signaling . overall , further deciphering of the complex regulatory network underlying the fa pathway will enable development of new strategies to exploit aberrant regulation of dna repair in cancer .

genome instability , primarily caused by faulty dna repair mechanisms , drives tumorigenesis . therapeutic interventions that exploit deregulated dna repair in cancer have made considerable progress by targeting tumor - specific alterations of dna repair factors , which either induces synthetic lethality or augments the efficacy of conventional chemotherapy and radiotherapy . the study of fanconi anemia ( fa ) , a rare inherited blood disorder and cancer predisposition syndrome , has been instrumental in understanding the extent to which dna repair defects contribute to tumorigenesis . the fa pathway functions to resolve blocked replication forks in response to dna interstrand cross - links ( icls ) , and accumulating knowledge of its activation by the ubiquitin - mediated signaling pathway has provided promising therapeutic opportunities for cancer treatment . here , we discuss recent advances in our understanding of fa pathway regulation and its potential application for designing tailored therapeutics that take advantage of deregulated dna icl repair in cancer .

MECHANISM OF DNA ICL REPAIR IN THE FA PATHWAY REGULATION OF FANCD2 ACTIVATION BY THE FA CORE COMPLEX Functional modules of the FA core complex and its regulation DNA damage response and FA pathway activation Fine-tuning FA pathway activation DNA REPAIR FACTORS AS THERAPEUTIC TARGETS IN CANCER THERAPEUTIC POTENTIAL OF FA PATHWAY INHIBITORS CONCLUSION

fanconi anemia ( fa ) is a chromosomal instability syndrome characterized by developmental abnormalities , progressive bone marrow failure , and increased cancer susceptibility ( dandrea , 2010 ) . thus , the study of fa has provided important insights into the pathogenesis of cancer and could lead to the development of anti - cancer therapeutics for the general population . fa is caused by germ - line mutations in one of the 17 currently known fa genes whose gene products participate in a common dna repair pathway called the fa pathway ( kim and dandrea , 2012 ) . the pathway functions to resolve dna interstrand cross - links ( icls ) , lethal dna lesions that block dna replication and transcription ( clauson et al . the fa pathway coordinates various steps in icl repair : recognition and unhooking of icls , translesion dna synthesis ( tls ) , and homologous recombination ( hr ) ( kottemann and smogorzewska , 2013 ) . central to the fa pathway is the conjugation of monoubiquitin to fancd2 at lys561 and , to a lesser extent , its binding partner fanci at lys523 in human ( garcia - higuera et al . this step is activated by the multi - subunit ubiquitin e3 ligase complex , also known as the fa core complex , which is composed of eight fanc proteins ( fanca / b / c / e / f / g / l / m ) along with the accessory proteins , faap100 , faap20 , and faap24 ( walden and deans , 2014 ) . the fa pathway senses an icl - mediated stalled replication fork , and the fancm - faap24 heterodimer in the complex recognizes dna damage caused by the dna icl and induces ataxia - telangiectasia and rad3-related ( atr)-dependent activation of the fa core complex ( collis et al . although not required for fancd2 monoubiquitination , atpase activity of fancm is essential for remodeling and stabilization of replication forks , and activation of the cell cycle checkpoint ( blackford et al . , 2008 ; schwab et al although the mechanisms of icl repair are not yet fully understood , the most detailed model derived from studies in xenopus egg extracts suggests that two dna replication forks converge on an icl to initiate repair ( fig . fancd2-monoubiquitin ( fancd2-ub ) has been implicated in the recruitment of several structure - specific nucleases , which are responsible for unhooking the dna icl ( fig . fancp / slx4 functions as a scaffold that targets structure - specific nucleases such as the ercc1-fancq / xpf heterodimer and slx1 to a lesion , utilizing its ubz4 ( ubiquitin - binding zinc finger 4 ) motif that recognizes fancd2-ub ( cybulski and howlett , 2011 ; yamamoto et al . recent biochemical and genetic studies indicated that nuclease activity of the ercc1-xpf complex plays a prominent role in carrying out unhooking incisions , which is controlled by the presence of slx4 ( hodskinson et al . interestingly , mutations in fa - q patients disrupt the function of xpf in icl repair , without altering its function in nucleotide excision repair ( ner ) , highlighting the multifunctional activity of the xpf endonuclease ( bogliolo et al . , 2015 ) , suggesting that the complex may process different intermediates in icl repair such as holliday junction ( hj ) resolution during double - strand break ( dsb ) repair ( chen et al . fan1 , another structure - specific nuclease implicated in icl repair , contains a ubz4 motif required for interaction with fancd2-ub ( smogorzewska et al . however , fan1 deficiency leads not to fa , but rather to a rare kidney disease called karyomegalic interstitial nephritis ( kin ) , suggesting that fan1 may play a redundant role with other nucleases in the fa pathway or deal with lesions independently of the fa pathway ( zhou et al . , slx4 also participates in diverse genome maintenance pathways including telomere maintenance and regulation of dna damage checkpoints ( kim , 2014 ) . tls , a dna damage tolerance mechanism mediated by specialized tls polymerases ( chang and cimprich , 2009 ) , allows a nascent leading strand to bypass the unhooked adduct and extend from primer - template termini to resume replication ( fig . the precise mechanism for the recruitment of tls polymerases to dna icl lesions remains elusive , but the fa core complex has been shown to facilitate this process ( budzowska et al . pol has been shown to execute the lesion bypass step of icl repair that occurs in xenopus egg extracts ( raschle et al . biallelic mutations in brca1 were recently found in a breast cancer patient with a fa - like disorder , and thus brca1 has been designated as a new fa gene , fancs ( sawyer et al . it is also required for unloading of the cdc45-mcm - gins ( cmg ) helicase complex from stalled forks and loading of fancd2-ub onto dna lesions , which functions independently of hr ( bunting et al . the usp1 knockout mouse exhibits fa phenotypes , and usp1 cells are hypersensitive to dna cross - linking agents , indicating that timely deubiquitination process is essential for the integrity of the fa pathway ( kim et al . fancd2 monoubiquitination by the fa core complex is a key regulatory step in the fa pathway as it connects an upstream dna damage response ( ddr ) to downstream enzymatic dna repair processes . notably , disruption of the interaction between fancl and its ubiquitin e2 conjugating enzyme ube2 t caused by ube2 t mutations in fa patients leads to compromised fancd2 monoubiquitination , suggesting that ube2 t ( fanct ) mutations define a new fa subtype ( hira et al . , recent biochemical and genetic experiments classified the fa core complex into three distinct modules , namely , the fancb - fancl - faap100 core catalytic unit , and the fanca - fancg - faap20 and fancc - fance - fancf auxiliary units ( huang et al . , 2012 ; deficiency in fanca also results in destabilization of its binding partners fancg and faap20 , and hypersensitivity to icl - inducing agents , highlighting its role as a scaffold to preserve the integrity of the complex . however , it remains unclear why the fa core complex consists of at least ten subunits that do not appear to have any homology or evolutionary connections , and what precise roles each module exerts . recognition of icl - stalled replication forks by the fancm - faap24 heterodimer , which recruits replication protein a ( rpa ) and the human homolog of the caenorhabditis elegans biological clock protein clk-2 ( hclk2 ) , initiates the activation of the atr - mediated checkpoint and the fa core complex ( collins et al . chk1-mediated phosphorylation of fance at thr346 and ser374 , and atr - dependent fanca phosphorylation at ser1449 are required for the functional integrity of the fa pathway ( collins et al . interestingly , the three - dimensional structure of the fancd2-fanci heterodimer revealed a unique fancd2 interacting domain in fanci , which undergoes a conformational change that stabilizes its interaction with fancd2 ( joo et al . the fancd2-fanci heterodimer is sumoylated following replication stress in an atr - dependent manner , and sumoylated fancd2 is a target for the sumo - targeted ubiquitin e3 ligase ( stubl ) rnf4 , which results in polyubiquitination and removal of fancd2 from damage sites via the dvc1-p97 ubiquitin segregase complex ( gibbs - seymour et al . improper clearance of fancd2 due to disruption of sumo signaling compromises cellular survival against replication stress , suggesting that timely inactivation of fancd2 is required for the functional integrity of the fa pathway . characterization of a breast cancer patient with a unique fanca mutation that disrupts the interaction with faap20 revealed that in the absence of faap20 interaction , a fanca sumoylation site becomes exposed , which initiates proteasome - dependent fanca degradation mediated by rnf4 ( xie et al . aberrant accumulation of fanca at sites of repair by the loss of rnf4 may prevent replication fork restarting and thus inhibit completion of dna repair . this protein group sumoylation may be relevant to the activation of the fa core complex as well ; pervasive sumoylation may stabilize the fa core complex assembly and promote its activation , which is followed by selective degradation of its components by a stubl such as rnf4 . , 2015 ) , and thus the role of sumoylation in regulating the fa pathway awaits further characterization . the muts complex , a damage sensor required for mismatch repair , has been shown to facilitate the recruitment of the fa core complex to chromatin , thereby playing a redundant role with the fancm complex ( huang et al . ubiquitin - like with phd and ring finger domains 1 ( uhrf1 ) , a key epigenetic regulator of chromatin modification at replication forks , was recently identified as a dna icl recognition factor that initiates icl repair ( liang et al . , 2015 uhrf1 is rapidly targeted to a dna icl , which promotes recruitment of fancd2 and structure - specific nucleases required for icl processing . it will be interesting to determine if these factors crosstalk with the fa core complex to fine - tune the steps of fancd2 activation . fancd2 monoubiquitination by the fa core complex is a key regulatory step in the fa pathway as it connects an upstream dna damage response ( ddr ) to downstream enzymatic dna repair processes . notably , disruption of the interaction between fancl and its ubiquitin e2 conjugating enzyme ube2 t caused by ube2 t mutations in fa patients leads to compromised fancd2 monoubiquitination , suggesting that ube2 t ( fanct ) mutations define a new fa subtype ( hira et al . , recent biochemical and genetic experiments classified the fa core complex into three distinct modules , namely , the fancb - fancl - faap100 core catalytic unit , and the fanca - fancg - faap20 and fancc - fance - fancf auxiliary units ( huang et al . , 2012 ; deficiency in fanca also results in destabilization of its binding partners fancg and faap20 , and hypersensitivity to icl - inducing agents , highlighting its role as a scaffold to preserve the integrity of the complex . however , it remains unclear why the fa core complex consists of at least ten subunits that do not appear to have any homology or evolutionary connections , and what precise roles each module exerts . recognition of icl - stalled replication forks by the fancm - faap24 heterodimer , which recruits replication protein a ( rpa ) and the human homolog of the caenorhabditis elegans biological clock protein clk-2 ( hclk2 ) , initiates the activation of the atr - mediated checkpoint and the fa core complex ( collins et al . chk1-mediated phosphorylation of fance at thr346 and ser374 , and atr - dependent fanca phosphorylation at ser1449 are required for the functional integrity of the fa pathway ( collins et al . the fancd2-fanci heterodimer is sumoylated following replication stress in an atr - dependent manner , and sumoylated fancd2 is a target for the sumo - targeted ubiquitin e3 ligase ( stubl ) rnf4 , which results in polyubiquitination and removal of fancd2 from damage sites via the dvc1-p97 ubiquitin segregase complex ( gibbs - seymour et al . improper clearance of fancd2 due to disruption of sumo signaling compromises cellular survival against replication stress , suggesting that timely inactivation of fancd2 is required for the functional integrity of the fa pathway characterization of a breast cancer patient with a unique fanca mutation that disrupts the interaction with faap20 revealed that in the absence of faap20 interaction , a fanca sumoylation site becomes exposed , which initiates proteasome - dependent fanca degradation mediated by rnf4 ( xie et al . aberrant accumulation of fanca at sites of repair by the loss of rnf4 may prevent replication fork restarting and thus inhibit completion of dna repair . this protein group sumoylation may be relevant to the activation of the fa core complex as well ; pervasive sumoylation may stabilize the fa core complex assembly and promote its activation , which is followed by selective degradation of its components by a stubl such as rnf4 . , 2015 ) , and thus the role of sumoylation in regulating the fa pathway awaits further characterization . the muts complex , a damage sensor required for mismatch repair , has been shown to facilitate the recruitment of the fa core complex to chromatin , thereby playing a redundant role with the fancm complex ( huang et al . ubiquitin - like with phd and ring finger domains 1 ( uhrf1 ) , a key epigenetic regulator of chromatin modification at replication forks , was recently identified as a dna icl recognition factor that initiates icl repair ( liang et al . , 2015 ) uhrf1 is rapidly targeted to a dna icl , which promotes recruitment of fancd2 and structure - specific nucleases required for icl processing . germ - line mutations or promoter hyper - methylation of dna repair genes confer increased risk for multiple cancers ( negrini et al . in addition , replication stress resulting from high levels of dna damage that interfere with dna replication and progression is augmented by faulty dna repair mechanisms caused by selection of somatic mutations that disrupt dna repair process ( gaillard et al . this is possible because cancer cells either become more susceptible to conventional chemotherapy that causes dna damage due to a decreased capacity to address genotoxicity , or become hyperdependent on another compensatory dna repair pathway , which provides a therapeutic window for specific killing of cancer cells . in - depth analysis of the fa signaling pathway has helped to understand the molecular mechanism of the chemotherapeutic effects of dna icl - inducing agents and allowed for targeted anti - cancer therapy . this effect could be achieved either by inhibiting the intact or upregulated fa pathway to chemosensitize cancer cells , or by exploiting the synthetic lethality of cancer cells that are defective in the fa pathway ( fig . as icl - inducing chemotherapy directly causes dna damage , the dna repair capacity of cancer cells plays a major role in determining the effectiveness of dna - damaging drugs . for instance , a role for tls has been implicated in acquired drug resistance following chemotherapy . these results indicate that upregulation of tls represents one of the critical mechanisms that confer acquired chemoresistance on tumor cells , and targeting the enhanced tls pathway using specific tls polymerase inhibitors could be utilized as an adjuvant therapy for treating chemoresistant malignancy . hyperdependency on compensatory dna repair due to defects in the fa pathway can be exploited for achieving synthetic lethality . , 2015 given that pol was found to be highly upregulated in epithelial ovarian cancers , this result indicates that the synthetic lethal relationship between the hr pathway and the pol -dependent mmej could be employed as a new therapeutic target for cancers with defective hr , or the fa pathway in general . although inhibition of the fa pathway can occur at multiple levels , fancd2 monoubiquitination has been a primary target for pharmacological interventions . bortezomib has been used as standard care for relapsed / refractory multiple myeloma and mantle cell lymphoma , and eventually , its use could be expanded to include a combination therapy , for instance with parp inhibition in hr - proficient tumors . the natural compound curcumin and its analogs such as ef24 and 4h - ttd have been shown to inhibit fancd2 activation and sensitize a variety of cancer cells to dna damage ( chirnomas et al . the usp1-uaf1 deubiquitinating enzyme complex plays an essential role in the fa pathway by antagonizing the level of fancd2-ub . thus , disrupting the ubiquitin - deubiquitination cycle of fancd2 could lead to the inhibition of the fa pathway . indeed , several inhibitors of the usp1-uaf1 complex have been developed to target the fa pathway . inhibiting the usp1-uaf1 complex also compromises tls by increasing levels of monoubiquitinated pcna , another substrate of usp1 , suggesting that inhibition of the usp1-uaf1 complex can simultaneously target two major steps in the fa pathway ( liang et al . an sirna - based synthetic lethal screening identified several genes including atm , parp1 , cdk1 , nbs1 , and plk1 that are required for the survival of cells deficient in fancg , indicating that these genes could be targeted in conjunction with an fa pathway inhibitor ( kennedy et al . as deficiency of atm signaling is found in several types of leukemia and triple - negative breast cancer , the fa pathway inhibitor could be used as a single agent in these cancers as well . these results suggest that targeting the fa pathway can become more effective by inhibiting dna damage signaling simultaneously , and appropriate combinations of ddr and dna repair inhibitors could be customized for increasing the efficacy of chemotherapy . since the discovery of fancd2 monoubiquitination in the early 2000s , the study of fa has integrated important topics in biology , including ubiquitin signaling , dna repair , and the pathogenesis of cancer . fancd2 activation acts as a surrogate marker for the dna icl repair process , and knowledge of how fancd2 is activated to regulate downstream repair steps has opened new therapeutic opportunities for cancer treatment we must increase our understanding of the regulatory mechanisms underlying fancd2 activation by the fa core complex . more specifically , a comprehensive understanding of the complex network of posttranslational modifications that regulate the fa core complex may lead to the identification of additional targets for therapeutic interventions . moreover , finding ways to inhibit the enzymatic steps of the fa pathway , including incisions and tls , may be useful . profiling dna repair in individuals with nonfunctional or upregulated dna repair capacity optimizing the selection of a chemotherapy approach requires the identification of reliable dna repair biomarkers . furthermore , synthetic lethality could be extended beyond dna repair pathways , to include aberrant signaling of activated oncogenes and growth factors , or enhanced anti - apoptotic signaling . overall , further deciphering of the complex regulatory network underlying the fa pathway will enable development of new strategies to exploit aberrant regulation of dna repair in cancer .

cholesterol is a ubiquitous molecule that plays a vital role in maintaining the overall integrity of eukaryotic cells by giving rise to the requisite structure , fluidity , and organization of the membrane . it is synthesized endogenously through an extensive multistep biosynthetic pathway , with the first sterol in the pathway being lanosterol . interestingly , of all the sterol intermediates between lanosterol and cholesterol , none possess cholesterol s ability to order and condense lipid membranes ; however , the details regarding cholesterol s structural interactions within the membrane are poorly understood . one of the more intriguing aspects of this planar tetracyclic molecule pertains to its two structurally distinct faces : its -face contains two protruding methyl groups that are conspicuously absent on its -face ( figure 1a ) . there has been much speculation that this facial asymmetry may be essential for cholesterol s unique membrane condensing abilities as well as being essential to the way in which it interacts with and stabilizes neighboring lipids and transmembrane proteins . to test this theory , models have been used to simulate the properties and interactions of a demethylated form of cholesterol ( 18,19-di - nor - cholesterol , which lacks the -face methyl groups ) . however , direct experimental evidence has not been obtained , as the molecule has never been previously synthesized . herein , we describe the first synthesis of a smoothened 18,19-di - nor - cholesterol , which we have completed as part of a collaborative effort , in hopes of providing a way to experimentally measure its effects on lipid membranes and other important transmembrane interactions . ( a ) structures of cholesterol and 18,19-di - nor - cholesterol ; the -face methyl groups of cholesterol ( shown in red ) are absent in 18,19-di - nor - cholesterol . blue numbers are methyl groups absent in 18,19-di - nor - cholesterol ; red letters label each ring . wedged bonds are referred to as having a -configuration , and dashed bonds , an -configuration . utilizing a nine - step sequence developed in our laboratory , commercially available 19-nor - testosterone was first transformed into perhydrochrysenone derivative a ( scheme 1 ) . in this transformation , the unwanted c18 methyl group was removed while still preserving the trans - c , d ring fusion . additionally , the cis - a , b ring fusion of compound a allows for the later introduction of the double bond . the -stereochemistry of the hydroxyl group at c3 in compound a is not important , as the 3-hydroxy group will be established during the double bond introduction ( see figure 1b for numbering ) . as depicted in our retrosynthesis ( scheme 1 ) , starting from a , we can essentially fragment our synthesis into three main parts : d - ring contraction , side - chain installation , and introduction of the double bond . installation of the double bond was chosen as the final step in our synthesis of 18,19-di - nor - cholesterol because of its high reactivity toward a variety of reaction conditions and because it could be obtained from 18,19-di - nor - epicoprostanol ( c ) via enone formation and double bond deconjugation . it was then envisaged that synthon c could be obtained through the addition of an isohexyl side chain to a steroid precursor containing a ch2coor group at c17 . synthon b fits this description and could be accessed from a by an oxidative d - ring - opening ( to yield a 1,4-diester ) , a dieckmann condensation to reclose the ring , the removal of the 16-ketone group , and a one - carbon homologation reaction at c17 to give synthon b. the success of this entire approach , however , is contingent upon whether ring closure via the dieckmann condensation ( preceding the c17 homologation reaction ) would yield the desired 17-carbomethoxy group as the major isomeric product ( scheme 2 , compound 4 ) . although four isomers are possible , extrapolating results from previous studies on the synthesis of a - nor - steroids and examining the steric interactions at play in our di - nor system led us to expect that this transformation would , in fact , yield our desired product ( see supporting information scheme s1 ) . this was therefore seen as our most critical , and yet most elegant , synthetic step , as this reaction would simultaneously contract the d - ring and selectively install a c17 functional group while preserving the trans - c , d ring fusion . to begin , we first protected perhydrochrysenone derivative a as the acetate ( 2 ) before carrying out a chromium(vi)-mediated , oxidative d - ring - opening and esterification to give 1,4-diester ( 3 ) . the key step in our synthetic approach was next . employing dieckmann condensation conditions , compound 3 was heated at 40 c for 45 min in basic meoh to yield a single -ketoester product . a crystal structure confirmed the stereochemistry to be that of the expected 17-methyl ester ( 4 ) ( see the supporting information ) . after removing the 16-keto group via formation of thioketal 5 followed by raney nickel dethioketalization to yield compound 6 having little success with the arndt eistert homologation reaction , we chose to proceed via the introduction of a carbonitrile group . we first protected compound 6 as a methoxymethyl ether ( 7 ) and then reduced the ester moiety using lialh4 to afford primary alcohol 8 . a carbonitrile was then introduced via a two - part procedure , wherein a methanesulfonyl ester was formed and then stirred at 65 c for 16 h with nacn in dmf to give carbonitrile 9 , which was subsequently hydrolyzed to carboxylic acid 10 ( scheme 2 ) . for steroids with a c18 methyl group , the introduction of a side - chain via an -substitution at c20 is known to favor the desired ( r)-diastereomer ; however , this selectivity is attributed to steric interferences caused by the c18 methyl group , which is absent in our 18,19-di - nor series . after confirming that alkylations at c20 are not stereoselective for 18-nor - steroids , we used evans chiral auxiliary [ ( r)-4-benzyl-2-oxazolidinone ] to ensure ( r)-stereochemistry at c21 . however , in a variety of subsequent alkylation attempts , we were unable to introduce the entire isohexyl group in satisfactory yield , presumably because of the added bulk of the chiral auxiliary . therefore , we instead installed the side chain in two pieces , first utilizing the highly reactive allyl iodide , to obtain compound 12 as the desired ( r)-diastereomer in 86% yield . next , we needed to remove the chiral auxiliary in order to generate the c21 methyl group ( currently positioned as the carbonyl carbon ) . although one - step reduction procedures failed to remove the chiral auxiliary , it was found to be easily removed by first hydrolyzing the benzyloxazolidinone and then reducing the resultant carboxylic acid with lialh4 to give primary alcohol 13 . at this point , with all critical stereocenters intact , a crystal structure was obtained ( see the supporting information ) . finally , deoxygenation was completed via a simple two - part protocol by first forming the methylsulfonate ester and then displacing this group using lialh4 to reveal the c21 methyl group and give compound 14 . transformation of the allyl group into the isohexyl side chain was the last step in completing the construction of synthon c. although the side chain was not introduced in one piece via our initial plan , transformation of the allyl group into an isohexyl chain was straightforward and higher yielding overall than the initial isohexyl introduction attempts . accordingly , the terminal olefin was oxidatively cleaved using o3/me2s to give the crude aldehyde intermediate , which was immediately treated with isobutyl(triphenyl)phosphonium bromide under wittig conditions to give the isohexenyl side chain of compound 15 in 85% yield as a single double bond isomer whose stereochemistry we did not determine . a subsequent hydrogenation proceeded near quantitatively , affording 18,19-di - nor - epicoprostanol ( 16 ) and completing the synthesis of synthon c ( scheme 3 ) . lastly , we needed to introduce the double bond , which is well - known in literature , through formation of a -3-ketone followed by deconjugation . therefore , we first synthesized the 3-keto - precursor ( 17 ) , which was easily obtained by first removing the methoxymethyl protecting group of compound 16 with anhydrous hcl and then oxidizing the resulting crude alcohol using jones reagent to give ketone 17 . however , subsequent attempts to introduce the -3-ketone proved to be difficult in the absence of the c19 methyl group . after having little success with a variety of steroid protocols , we ultimately adapted a general procedure for the formation of ,-unsaturated ketones , wherein bromination of a silyl enol ether first yields an -bromoketone that undergoes a dehydrobromination . after optimizing conditions , compound 17 was treated with trimethylsilyl triflate ( tmsotf ) and et3n to form the tms - enol ether in situ . upon cooling the reaction to 78 c and adding nahco3 and n - bromosuccinimide ( nbs ) , the intermediate bromoketone ( obtained as a mixture of bromides ; see supporting information scheme s2 ) was heated in the presence of li2co3 and libr in dmf at 120 c for 16 h to generate the desired -3-ketone ( 18 ) in a moderate 32% yield , with an unwanted -3-ketone byproduct obtained in 55% yield ( reflecting the ratio of the bromide intermediates ) . although the desired -3-ketone was the minor product , of the reaction protocols attempted this protocol gave the highest product yield and the least amount of byproducts . it was also far more economical than the other attempted procedures , in that we were consistently able to recover and reuse starting material 17 via hydrogenation of the unwanted -3-ketone . thus , after hydrogenation , using 10% pd / c , starting material 17 could be recovered in 54% yield . by running this reaction sequence three consecutive times , we were able to obtain our desired 18,19-di - nor - cholest-4-ene-3-one ( 18 ) in a respectable 60% yield , with 16% of our total starting material ( 17 ) recovered . deconjugation was the final step in our synthesis , which proved to be relatively straightforward using typical literature protocols . compound 18 was dissolved in ac2o and tmsi to form the dienol acetate , which was then reduced with nabh4 in etoh . this simultaneously deconjugated the enone and set our final stereocenter ( 3-oh ) , affording our target 18,19-di - nor - cholesterol ( 1 ) in 60% yield ( 3.6% overall from perhydrochrysenone a ) ( scheme 4 ) . the synthesis of the smoothened 18,19-di - nor - cholesterol was achieved starting from commercially available 19-nor - testosterone . the novel use of the dieckmann condensation was key to accessing our target molecule , allowing us to contract the d - ring to a 5-membered ring and to simultaneously introduce a modifiable functional group and set the stereochemistry at c17 while maintaining the trans - c , d ring fusion . furthermore , the completion of this molecule can now allow for the direct comparison of cholesterol and demethylated 18,19-di - nor - cholesterol in a variety of biological and biophysical studies , some of which are currently underway . chromatography was performed using flash chromatography grade silica gel ( 3263 m ; scientific adsorbents ) . nmr spectra were recorded in cdcl3 at 400 mhz ( h ) , 100 mhz ( c ) , or 400 mhz ( 2d cosy ) . chemical shifts ( ) are reported downfield from internal me4si ( : 0.00 ) . high resolution fab - ms determinations were made with a matrix m - nitrobenzyl alcohol , using nai as necessary , utilizing a double - focusing analyzer composed of a magnetic sector followed by a electrostatic sector . all other chemicals were used as purchased without further purification unless noted . to a stirred solution of a(13 ) ( 5.28 g , 19.1 mmol ) at 0 c under n2 in anhydrous pyridine ( 20 ml ) was added acetic anhydride ( 3.6 ml , 38 mmol ) dropwise followed by a catalytic amount of 4-dimethylaminopyridine ( 116 mg , 0.95 mmol ) . the reaction mixture was allowed to warm to 25 c and was stirred for 16 h. the pyridine was then evaporated in vacuo , and the crude residue was then redissolved in dcm ( 100 ml ) and washed with 1 m hcl ( 3 25 ml ) followed by h2o ( 2 25 ml ) . the residue was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to yield compound 2 as colorless crystals in 96% yield ( 5.83 g , 18.3 mmol ) . compound 2 : mp 169171 c ( etoac / hexanes ) ; [ ]d 6.7 ( c = 0.22 , chcl3 ) ; ir : 2931 , 2905 , 2858 , 1724 , 1243 , 1032 cm ; h nmr ( cdcl3 ) 4.75 ( m , 1h , choac ) , 2.252.43 ( m , 4h ) , 2.04 ( s , 3h , oac ) , 2.121.10 ( m , 20h ) , 0.750.92 ( m , 2h ) ; c nmr ( cdcl3 ) 212.1 , 170.8 , 74.4 , 48.8 , 47.3 , 46.3 , 43.4 , 41.5 , 40.3 , 37.4 , 35.2 , 34.2 , 32.3 , 31.4 , 30.5 , 29.5 , 26.0 , 25.7 , 25.3 , 21.7 ; hr - fab ms [ m + na ] calcd for c20h30o3na : 341.2093 , found 341.2099 . compound 3 was prepared according to a literature procedure . a stock solution of cro3/h2o / acoh ( 5.0 g/7.10 ml/8.35 ml ; w / v / v ) was first prepared . then , in a two - necked flask fitted with a dropping funnel and a reflux condenser , compound 2 ( 3.05 g , 9.58 mmol ) was dissolved into a solution of acoh / h2o / meoh 600:20:1 ( 30 ml ) at 60 c . the stock cro3/h2o / acoh solution ( 13.5 ml ) was then added via dropping funnel to compound 2 , taking care not to heat the reaction mixture above 65 c ( 1 drop/5 s ) . the reaction flask was allowed to stir for 2 h at 60 c , at which time starting material disappearance was observed by tlc . at this point , heated h2o was added ( 30 ml , 60 c ) , the stirring was stopped , and the reaction was gradually cooled to room temperature . the reaction was allowed to stand for 16 h , during which time the desired compound precipitated . cold h2o ( 10 ml ) was then added , and the precipitate was then filtered off and washed several times with cold h2o and dried in vacuo . after 3 h of drying , the compound was redissolved in meoh ( 100 ml ) and cooled to 0 c under n2 . acetyl chloride ( 10 ml ) was slowly added dropwise to the flask , and after 10 min , the reaction was brought to rt . after 16 h , the reaction was cooled to 0 c and neutralized with saturated aqueous nahco3 . the reaction was diluted with h2o ( 100 ml ) and extracted with dcm ( 4 50 ml ) . the organic layers were combined , dried over na2so4 , and concentrated in vacuo . the residue was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to yield compound 3 as a white amorphous solid in 44% yield ( 1.49 g , 4.21 mmol ) . compound 3 : mp 5254 c ( amorphous solid , etoac / hexanes ) ; [ ]d + 13.4 ( c = 0.19 , chcl3 ) ; ir : 3401 , 2928 , 2863 , 1737 , 1436 , 1270 , 1161 cm ; h nmr ( cdcl3 ) 3.64 ( br s , 7h , choh , 2 ome ) , 2.550.72 ( m , 24h ) ; c nmr ( cdcl3 ) 173.6 ( 2 ) , 71.6 , 51.5 , 51.5 , 47.0 , 43.7 , 40.1 , 39.3 , 38.7 , 37.3 , 36.1 , 35.1 , 35.0 , 32.4 , 31.5 , 29.7 , 28.9 , 25.5 , 25.5 ; hr - fab ms [ m + na ] calcd for c20h32o5na : 375.2147 , found 375.2150 . similar to the literature , in a flame - dried flask under n2 , na(s ) ( 0.60 g , 26 mmol ) was dissolved portionwise into anhydrous meoh ( 15 ml ) . the excess meoh was then evaporated off , leaving only a small amount ( 2.5 ml ) to prevent the generated naome from precipitating . anhydrous thf ( 50 ml ) was added to the flask followed by the dropwise addition of a solution of compound 3 ( 2.63 g , 7.46 mmol ) in anhydrous thf ( 100 ml ) . the reaction was refluxed at 100 c for 45 min and then promptly cooled to 0 c and neutralized with 1 m hcl . the reaction mixture was then diluted with h2o ( 150 ml ) and extracted with etoac ( 4 50 ml ) . the organic extracts were then combined , dried over na2so4 , and concentrated in vacuo . the residue was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to give compound 4 as colorless crystals in 70% yield ( 1.65 g , 5.22 mmol ) . compound 4 : mp 163164 c ( etoac / hexanes ) ; [ ]d 86.8 ( c = 0.19 , chcl3 ) ; ir : 3399 , 2924 , 2861 , 1756 , 1727 , 1440 , 1271 , 1051 cm ; h nmr ( cdcl3 ) 3.76 ( s , 3h , co2me ) , 3.66 ( br s , 1h , h-3 ) , 2.91 ( d , 1h , j = 12.8 hz ) , 2.51 ( dd , 1h , j = 18.0 hz , j = 6.8 hz ) , 1.182.11 ( m , 19h ) , 0.921.11 ( m , 2h ) ; c nmr ( cdcl3 ) 210.4 , 169.7 , 71.8 , 62.3 , 52.6 , 48.0 , 47.0 , 46.3 , 43.3 , 40.1 , 37.6 , 36.4 , 35.5 , 31.3 , 30.2 , 29.9 , 29.3 , 26.8 , 25.9 ; hr - fab ms [ m + h ] calcd for c19h29o4 : 321.2066 , found 321.2064 . following a literature procedure , compound 4 ( 1.58 g , 4.93 mmol ) was dissolved into acoh ( 12 ml ) and ethanedithiol ( 1.04 ml , 12.3 mmol ) . to prevent the reaction mixture from solidifying , the flask was cooled in an ice bath no longer than 2 min before adding bf3hoac ( 3.43 ml , 24.7 mmol ) dropwise . the reaction was then allowed to stir at 0 c for 10 min and then for another 30 min at rt . the flask was then cooled to 0 c and neutralized with saturated aqueous nahco3 . the reaction mixture was then diluted with h2o ( 25 ml ) and extracted with dcm ( 4 25 ml ) . the organic extracts were then combined , dried over na2so4 , and concentrated in vacuo . the residue was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to give compound 5 as colorless crystals in 95% yield ( 1.86 g , 4.68 mmol ) . compound 5 : mp 103105 c ( etoac / hexanes ) ; [ ]d 65.0 ( c = 0.14 , chcl3 ) ; ir : 3368 , 2923 , 2861 , 1735 , 1438 , 1210 , 1156 , 1056 , 1033 , 731 cm ; h nmr ( cdcl3 ) 3.74 ( s 3h , ome ) , 3.64 ( br s 1h , h-3 ) , 3.403.45 ( m , 1h ) , 3.303.34 ( m , 1h ) , 3.133.22 ( m , 2h ) , 2.86 ( d , 1h , j = 11.3 hz ) , 2.51 ( dd , 1h , j = 13.3 hz , j = 5.5 hz ) , 1.181.97 ( m , 18h ) , 1.021.13 ( m , 1h ) , 0.850.97 ( m , 2h ) ; c nmr ( cdcl3 ) 172.4 , 71.9 , 69.7 , 65.9 , 52.5 , 51.7 , 49.7 , 48.3 , 47.6 , 41.1 , 40.2 , 40.1 , 37.3 , 36.5 , 35.6 , 31.4 , 30.1 , 30.0 , 29.6 , 26.6 , 26.1 ; hr - fab ms [ m + na ] calcd for c21h32o3s2na : 419.1691 , found 419.1698 . grace and co. raney4200 , slurry in h2o , active catalyst ) was successively washed and decanted with deionized h2o ( 3 ) and etoh ( ) . once rinsed thoroughly , the catalyst was kept in a solution of etoh to keep it from drying . compound 5 ( 1.87 g , 4.71 mmol ) was dissolved in etoh ( 30 ml ) . a slurry of activated raney nickel ( 17 g ) in etoh ( 30 ml ) was added portionwise , and the reaction was refluxed at 80 c for 1.5 h. the reaction was then cooled to rt , and the crude reaction mixture was filtered through a short column of silica gel ( eluting several times with etoac / etoh 1:1 ) to remove the raney nickel and concentrated in vacuo . the residue was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to give compound 6 as colorless crystals in 95% yield ( 1.37 g , 4.47 mmol ) . compound 6 : mp 123125 c ( etoac / hexanes ) ; [ ]d + 43.0 ( c = 0.10 , chcl3 ) ; ir : 3350 , 2924 , 2864 , 1735 , 1596 , 1440 , 1208 , 1156 , 1050 cm ; h nmr ( cdcl3 ) 3.65 ( s 3h , ome ) , 3.64 ( br s 1h , h-3 ) , 2.25 ( m , 1h ) , 0.951.99 ( m , 22h ) , 0.720.90 ( m , 2h ) ; c nmr ( cdcl3 ) 177.1 , 71.8 , 52.0 , 51.6 , 50.2 , 49.2 , 48.5 , 40.1 , 37.5 , 36.4 , 35.7 , 31.5 , 30.3 , 29.9 , 29.8 , 28.6 , 27.4 , 26.6 , 26.2 ; hr - fab ms [ m + h ] calcd for c19h31o3 : 307.2278 , found 307.2273 . compound 6 ( 1.34 g , 4.37 mmol ) was dissolved in anhydrous dcm ( 200 ml ) and cooled to 0 c under n2 . n , n - diisopropylethylamine ( 3.05 ml , 17.5 mmol ) was added followed by the dropwise addition of chloromethyl methyl ether ( 0.66 ml , 8.8 mmol ) . the reaction was allowed to warm to rt and left to stir for 16 h. upon completion , the reaction was washed with h2o ( 50 ml ) , brine ( 50 ml ) , and h2o ( 50 ml ) , dried over na2so4 , and concentrated in vacuo . the residue was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to give compound 7 as colorless crystals in 98% yield ( 1.50 g , 4.28 mmol ) . compound 7 : mp 6669 c ( cold hexanes ) ; [ ]d + 43.8 ( c = 0.18 , chcl3 ) ; ir : 2929 , 2867 , 1733 , 1440 , 1144 , 1107 , 1041 cm ; h nmr ( cdcl3 ) 4.68 ( s 2h , och2och3 ) , 3.67 ( s 3h , co2me ) , 3.54 ( m , 1h , h-3 ) , 3.37 ( s 3h , och2och3 ) , 2.25 ( m , 1h ) , 0.971.99 ( m , 22h ) , 0.770.90 ( m , 2h ) ; c nmr ( cdcl3 ) 177.1 , 94.7 , 77.0 , 55.3 , 52.0 , 51.7 , 50.3 , 49.3 , 48.5 , 40.4 , 37.5 , 35.8 , 33.7 , 31.7 , 30.4 , 29.8 , 28.7 , 27.5 , 27.1 , 26.6 , 26.3 ; hr - fab ms [ m + h ] calcd for c21h35o4 : 351.2535 , found 351.2536 . compound 7 ( 2.89 g , 8.25 mmol ) was dissolved in anhydrous thf ( 300 ml ) and then cooled to 0 c under n2 . over 30 min , lialh4 ( 1 m in thf ; 8.25 ml , 16.5 mmol ) was added dropwise to the reaction mixture , and upon completion of lialh4 addition , the reaction was found to be complete . the reaction was quenched by the careful addition of h2o ( 0.63 ml ) followed by 15% aqueous naoh ( 0.63 ml ) , and the reaction was allowed to stir for 30 min at rt . another aliquot of h2o ( 1.90 ml ) was added , and within 5 min a white precipitate formed . the precipitate was filtered off , and the filtrate was dried over na2so4 and concentrated in vacuo . the residue was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to give compound 8 as colorless crystals in 91% yield ( 2.42 g , 7.50 mmol ) . compound 8 : mp 7678 c ( etoac / hexanes ) ; [ ]d + 41.4 ( c = 0.14 , chcl3 ) ; ir : 3401 , 2928 , 2865 , 1441 , 1144 , 1107 , 1041 cm ; h nmr ( cdcl3 ) 4.69 ( s 2h , och2och3 ) , 3.623.73 ( m 1h ) , 3.453.57 ( m , 2h , h-3 ) , 3.37 ( s 3h , och2och3 ) , 0.721.99 ( m , 25h ) ; c nmr ( cdcl3 ) 94.7 , 77.0 , 66.8 , 55.3 , 52.3 , 48.8 , 48.7 , 47.1 , 40.5 , 37.8 , 35.9 , 33.7 , 31.7 , 30.8 , 30.1 , 28.3 , 27.1 , 27.1 , 26.6 , 26.3 ; hr - fab ms [ m + na ] calcd for c20h34o3na : 345.2406 , found 345.2404 . to a solution of compound 8 ( 0.90 g , 2.8 mmol ) in anhydrous dcm ( 30 ml ) at 0 c under n2 was added et3n ( 1.17 ml , 8.37 mmol ) . mscl ( 0.43 ml , 5.6 mmol ) was dissolved in anhydrous dcm ( 0.6 ml ) and added dropwise to the reaction flask . after 30 min at 0 c , the reaction was quenched with ice and subsequently washed with cold saturated aqueous nahco3 ( 10 ml ) , cold brine ( 10 ml ) , and cold h2o ( 10 ml ) , dried over na2so4 , and concentrated in vacuo at rt . the reaction flask was allowed to dry for 2 to 3 h under vacuum , and the residue was dissolved in anhydrous dmf ( 60 ml ) , and the flask was fitted with a reflux condenser . nacn ( 0.68 g , 14 mmol ) was crushed into a powder and added to the reaction flask , and the mixture was stirred at 65 c for 16 h. the reaction was then cooled to 25 c , and ice was added to the reaction mixture , causing the product to precipitate . the product was then filtered , washed several times with h2o , and dried in vacuo . the crude compound was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to give compound 9 as colorless crystals in 85% yield ( 0.79 g , 2.4 mmol ) . compound 9 : mp 113115 c ( etoac / hexanes ) ; [ ]d + 46.1 ( c = 0.17 , chcl3 ) ; ir : 2934 , 2909 , 2867 , 2851 , 2244 , 1143 , 1106 , 1040 cm ; h nmr ( cdcl3 ) 4.67 ( s 2h , och2och3 ) , 3.473.58 ( m 1h , h-3 ) , 3.35 ( s , 3h , och2och3 ) , 2.44 ( dd , 1h , j = 16.8 hz , j = 5.5 hz ) , 2.32 ( dd , 1h , j = 16.3 hz , j = 6.7 hz ) , 1.881.99 ( m , 4h ) , 0.741.86 ( m , 21h ) ; c nmr ( cdcl3 ) 119.3 , 94.7 , 76.9 , 55.3 , 51.7 , 50.8 , 48.5 , 40.6 , 40.3 , 37.6 , 35.8 , 33.6 , 31.6 , 29.7 , 29.6 , 29.5 , 27.7 , 27.1 , 26.4 , 26.2 , 21.2 ; hr - fab ms [ m + na ] calcd for c21h33no2na : 354.2409 , found 354.2403 . in a flask fitted with a reflux condenser , compound 9 ( 1.98 g , 5.97 mmol ) was dissolved in ethylene glycol ( 150 ml ) at 120 c . to this was slowly added a solution of 50% aqueous naoh ( 20 ml ) , and the reaction was allowed to stir for 16 h at 120 c . the reaction was then cooled to 25 c and acidified to a ph 4 with 6 n hcl . the reaction mixture was then extracted several times with dcm ( 4 50 ml ) . the organic fractions were then combined , dried over na2so4 , and concentrated in vacuo . the crude compound was purified through a short column of silica gel ( meoh / dcm , 1% isocratic elution ) to give compound 10 as colorless crystals in 85% yield ( 1.78 g , 5.07 mmol ) . compound 10 : mp 164166 c ( dcm / hexanes ) ; [ ]d + 35.7 ( c = 0.12 , chcl3 ) ; ir : 3367 , 2914 , 2866 , 1701 , 1141,1109 , 1047 cm ; h nmr ( cdcl3 ) 4.69 ( s 2h , och2och3 ) , 3.493.59 ( m 1h , h-3 ) , 3.37 ( s , 3h , och2och3 ) , 2.52 ( dd , 1h , j = 14.9 hz , j = 4.7 hz ) , 2.14 ( dd , 1h , j = 15.3 hz , j = 9.4 hz ) , 1.452.03 ( m , 13h ) , 0.931.36 ( m , 10h ) , 0.710.85 ( m , 3h ) ; c nmr ( cdcl3 ) 179.7 , 94.6 , 77.1 , 55.3 , 51.7 , 51.6 , 48.8 , 41.0 , 40.5 , 39.1 , 37.7 , 35.9 , 33.7 , 31.7 , 30.0 , 30.0 , 29.8 , 27.9 27.1 , 26.5 , 26.3 ; hr - fab ms [ m + na ] calcd for c21h34o4na : 373.2355 , found 373.2349 . following literature methodology , to a solution of compound 10 ( 0.36 g , 1.0 mmol ) dissolved in anhydrous thf ( 15 ml ) under n2 at 0 c was added et3n ( 0.79 ml , 5.7 mmol ) . the reaction was cooled to 0 c , and pivaloyl chloride ( 0.19 ml , 1.5 mmol ) was added dropwise . the reaction was stirred at 0 c for 2 h. then , licl ( 0.22 g , 5.1 mmol ) and ( r)-4-benzyl-2-oxazolidinone ( 0.27 g , 1.5 mmol ) were added , and the reaction was allowed to slowly warm to 25 c and was then stirred for 16 h. the reaction was then cooled to 0 c , quenched with saturated aqueous nahco3 , and further diluted with h2o ( 30 ml ) . the organic extracts were combined , dried over na2so4 , and concentrated in vacuo . the crude compound was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to give compound 11 as colorless crystals in 96% yield ( 0.50 g , 0.99 mmol ) . compound 11 : mp 143145 c ( cold etoac ) ; [ ]d + 10.2 ( c = 0.25 , chcl3 ) ; ir : 3392 , 2928 , 2865 , 1783 , 1699 , 1386 , 1212 , 1106 , 1039 cm ; h nmr ( cdcl3 ) 7.117.29 ( m , 5h ) , 4.62 ( s 2h , och2och3 ) , 4.574.64 ( m , 1h , nch ) , ( overlapping doublets , 2h , och2ch ) , 3.433.51 ( m , 1h , h-3 ) , 3.30 ( s , 3h , och2och3 ) , 3.23 ( dd , 1h , j = 13.1 hz , j = 3.3 hz , chhph ) , 3.03 ( dd , 1h , j = 16.6 hz , j = 4.1 hz , chhc(o)n ) , 2.662.79 ( overlapping doublets , 2h , chhc(o)n , chhph ) , 0.652.03 ( m , 25 h ) ; c nmr ( cdcl3 ) 173.5 , 153.7 , 135.5 , 129.6 ( 2 ) , 129.1 ( 2 ) , 127.5 , 94.7 , 77.0 , 66.3 , 55.4 , 55.4 , 51.6 , 51.5 , 48.8 , 40.6 , 40.5 , 40.4 , 38.1 , 37.8 , 35.9 , 33.7 , 31.7 , 30.4 , 30.0 , 29.9 , 28.0 , 27.1 , 26.6 , 26.3 ; hr - fab ms [ m + na ] calcd for c31h43no5na : 532.3039 , found 532.3043 . in a flame - dried flask under n2 , compound 11 ( 1.32 g , 2.59 mmol ) was dissolved in anhydrous thf ( 50 ml ) , and the reaction mixture was cooled to 78 c . nahmds ( 1 m in thf ; 4.32 ml , 4.53 mmol ) was added dropwise , and the reaction was allowed to stir for 1 h at 78 c . allyl iodide ( 0.47 ml , 5.2 mmol ) was then added dropwise , and the reaction was stirred at 78 c for another 1 h. after 1 h , the reaction was quenched with saturated aqueous nh4cl , h2o was added ( 50 ml ) , and the reaction was extracted several times with etoac ( 4 50 ml ) . the organic fractions were then combined , dried over na2so4 , and concentrated in vacuo . the crude compound was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to give compound 12 as a white foamy solid in 86% yield ( 1.22 g , 2.23 mmol ) , with recovery of unreacted starting material 11 in 7% . compound 12 : [ ]d 28.2 ( c = 0.13 , chcl3 ) ; ir : 3369 , 2927 , 2865 , 1782 , 1697 , 1640 , 1384 , 1208 , 1105 , 1040 cm ; h nmr ( cdcl3 ) 7.157.29 ( m , 5h ) , 5.705.81 ( m , 1h , ch = ch2 ) , 4.935.03 ( overlapping doublets , 2h , ch = ch2 ) , 4.61 ( s , 2h , och2och3 ) , 4.544.62 ( m , 1h , nch ) , 4.014.06 ( overlapping doublets , 2h , och2ch ) , 3.923.96 ( m , 1h , chc = o ) , 3.413.49 ( m , 1h , h-3 ) , 3.29 ( s , 3h , och2och3 ) , 3.24 ( dd , 1h , j = 13.2 hz , j = 3.2 hz , chhph ) , 2.56 ( dd , 1h , j = 13.2 hz , j = 2.8 hz , chhph ) , 2.382.48 ( m , 1h , chhch = ch2 ) , 2.242.32 ( m , 1h , chhch = ch2 ) , 1.381.85 ( m , 14h ) , 0.851.21 ( m , 9h ) , 0.600.78 ( m , 2h ) ; c nmr ( cdcl3 ) 176.1 , 163.4 , 135.8 , 135.7 , 129.6 ( 2 ) , 129.1 ( 2 ) , 127.4 , 117.0 , 94.6 , 76.9 , 65.9 , 56.0 , 55.3 , 52.0 , 49.6 , 48.7 , 46.4 , 45.2 , 40.3 , 38.2 , 37.6 , 35.8 , 34.8 , 33.7 , 31.6 , 30.7 , 30.1 , 27.6 , 27.2 , 27.0 , 26.4 , 26.2 ; hr - fab ms [ m + h ] calcd for c34h48no5 : 550.3533 , found 550.3539 . following a reported procedure , a solution of compound 12 ( 1.65 g , 3.00 mmol ) was dissolved in thf / h2o 5:1 ( 40 ml ) at 0 c followed by dropwise addition of 30% aqueous h2o2 ( 3 ml ) . this was followed by the portionwise addition of lioh ( 0.25 g , 6.0 mmol ) over 30 min . the solution was allowed to slowly come to rt . after stirring for 16 h , the reaction was acidified to ph 1 with 1 m hcl and was immediately extracted with etoac ( 4 20 ml ) . the organic extracts were combined , dried over na2so4 , and concentrated in vacuo for 3 h. the crude intermediate carboxylic acid was then redissolved in anhydrous thf ( 30 ml ) at 0 c under n2 . lialh4 ( 2 m in thf ; 3.75 ml , 7.50 mmol ) was added dropwise , and the reaction was brought to 25 c and stirred for 16 h. upon reaction completion , the reaction was cooled to 0 c and quenched carefully with 1 m hcl . the reaction was then diluted with h2o ( 30 ml ) and extracted with etoac ( 4 20 ml ) . the organic fractions were then combined , dried over na2so4 , and concentrated in vacuo . the crude compound was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to give compound 13 as colorless crystals in 91% yield ( 1.02 g , 2.73 mmol ) . compound 13 : mp 8991 c ( etoac / hexanes ) ; [ ]d + 37.2 ( c = 0.13 , chcl3 ) ; ir : 3401 , 2928 , 2866 , 1639 , 1440 , 1144 , 1107 , 1040 cm ; h nmr ( cdcl3 ) 5.755.86 ( m , 1h , ch = ch2 ) , 4.955.04 ( overlapping doublets , 2h , ch = ch2 ) , 4.66 ( s , 2h , och2och3 ) , 3.463.62 ( m 3h , h-3 , ch2oh ) , 3.34 ( s , 3h , och2och3 ) , 2.102.19 ( m , 1h , chhch = ch2 ) , 0.902.12 ( m , 25h ) , 0.700.85 ( m , 2h ) ; c nmr ( cdcl3 ) 138.5 , 115.8 , 94.6 , 77.0 , 65.2 , 55.2 , 52.0 , 48.8 , 48.3 , 44.9 , 42.6 , 40.4 , 37.8 , 35.8 , 33.6 , 32.7 , 31.7 , 30.8 , 30.1 , 28.2 , 27.1 , 26.5 , 26.2 , 25.7 ; hr - fab ms [ m + na ] calcd for c24h40o3na : 399.2875 , found 399.2883 . to a solution of compound 13 ( 1.04 g , 2.76 mmol ) in anhydrous dcm ( 80 ml ) at 0 c under n2 was added et3n ( 1.54 ml , 11.0 mmol ) . mscl ( 0.43 ml , 5.5 mmol ) was dissolved in anhydrous dcm ( 30 ml ) , and this solution was added dropwise to the reaction flask . after 30 min at 0 c , the reaction was quenched with ice and subsequently washed with cold saturated aqueous nahco3 ( 25 ml ) , cold brine ( 25 ml ) , and cold h2o ( 25 ml ) , dried over na2so4 , and condensed in vacuo at rt . the reaction flask was allowed to dry for 2 to 3 h under vacuum , the residue was then dissolved in anhydrous thf ( 40 ml ) , and the flask was fitted with a reflux condenser . lialh4 ( 2 m in thf , 13.8 ml , 27.6 mmol ) was added dropwise to the reaction flask , and the mixture was stirred at 50 c for 1 h. the reaction was then cooled to 0 c , quenched carefully with 1 m hcl , subsequently diluted with h2o ( 30 ml ) , and extracted with etoac ( 4 20 ml ) . the organic fractions were then combined , dried over na2so4 , and concentrated in vacuo . the crude compound was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to give compound 14 as colorless crystals in 94% yield ( 0.93 g , 2.6 mmol ) . compound 14 : mp 5557 c ( cold hexanes ) ; [ ]d + 41.9 ( c = 0.16 , chcl3 ) ; ir : 2933 , 2910 , 2861 , 1437 , 1147 , 1105 , 1046 , 913 cm ; h nmr ( cdcl3 ) 5.705.85 ( m , 1h ) , 4.954.99 ( overlapping doublets , 2h ) , 4.68 ( s , 2h , och2och3 ) , 3.493.59 ( m , 1h , h-3 ) , 3.37 ( s , 3h , och2och3 ) , 2.112.19 ( m , 1h ) , 0.751.98 ( m , 27h ) , 0.87 ( d , 3h , j = 6.7 hz ) ; c nmr ( cdcl3 ) 138.7 , 115.3 , 94.7 , 77.1 , 55.3 , 52.6 , 49.5 , 48.8 , 48.4 , 40.5 , 38.5 , 37.8 , 35.9 , 35.9 , 33.7 , 31.8 , 31.6 , 30.3 , 28.3 , 27.1 , 26.9 , 26.5 , 26.3 , 18.1 ; hr - fab ms [ m + na ] calcd for c24h40o2na : 383.2926 , found 383.2925 . combining two reported procedures , ozone gas was bubbled into a solution of compound 14 ( 0.62 g , 1.7 mmol ) and dissolved in dcm ( 50 ml ) at 78 c until the reaction turned light blue in color ( ca . , the ozone generator was turned off , and oxygen was allowed to bubble into the flask until the blue ozone color disappeared ( ca . 5 min ) . acetic acid ( 10 ml ) was added to the flask , and subsequently the dcm was evaporated under reduced pressure . dcm ( 10 ml ) was then added back to the flask containing the acetic acid and crude ozonide intermediate . c under n2 , and dimethyl sulfide ( 25 ml ) was added dropwise . wanting to avoid possible byproduct formation , the reaction was carefully monitored . after stirring at 25 c for 5 h , it was then placed in a freezer ( 5 c ) for 16 h to slow reaction progress . the reaction was then stirred an additional 8 h at rt followed by another 16 h at 5 c , after which the starting alkene was found to be completely converted into an aldehyde intermediate . the reaction was diluted with dcm ( 50 ml ) , washed with h2o , saturated aqueous nahco3 , and brine , dried over mg2so4 , concentrated , and dried under high vacuum for 2 h. during this time , in a separate flask , isobutyltriphenylphosphonium bromide ( 2.39 g , 3.5 mmol ) was dissolved into anhydrous thf ( 50 ml ) under n2 and cooled to 0 c . nahmds ( 1 m in thf ; 3.08 ml , 3.08 mmol ) was added dropwise to the reaction flask , and the mixture was stirred for 30 min at 0 c , becoming bright orange in color . tris[2-(2-methoxyethoxy)ethyl]amine ( tda-1 ) ( 68 l ) was then added to the bright orange ylide followed by the dropwise addition of the crude aldehyde dissolved in anhydrous thf ( 50 ml ) . , the remaining ylide was quenched by the addition of a few drops of saturated aqueous nh4cl . the thf was evaporated under reduced pressure , etoac ( 50 ml ) was added , and the mixture was sequentially washed with h2o , saturated aqueous nahco3 , and brine , dried over na2so4 , and concentrated in vacuo . the crude compound was purified by column chromatography on silica gel using dcm / hexanes gradient elution to remove excess triphenylphosphine and transitioning to a etoac / hexanes gradient elution to give compound 15 as colorless crystals in 85% yield ( 0.58 g , 1.5 mmol ) . compound 15 : mp 7476 c ( cold hexanes ) ; [ ]d + 44.0 ( c = 0.32 , chcl3 ) ; ir : 2929 , 2866 , 1456 , 1375 , 1145 , 1106 , 1046 , 917 cm ; h nmr ( cdcl3 ) 5.165.26 ( m , 2h ) , 4.70 ( s , 2h , och2och3 ) , 3.503.59 ( m , 1h , h-3 ) , 3.38 ( s , 3h , och2och3 ) , 2.532.62 ( m , 1h ) , 1.082.11 ( m , 28h ) , 0.95 ( d , 3h , j = 1.2 hz ) , 0.94 ( d , 3h , j = 1.6 hz ) , 0.87 ( d , 3h , j = 6.8 hz ) ; c nmr ( cdcl3 ) 138.06 , 127.0 , 94.7 , 77.1 , 55.3 , 52.6 , 49.8 , 48.9 , 48.5 , 40.6 , 37.9 , 36.5 , 35.9 , 33.7 , 31.8 , 31.5 , 31.4 , 30.3 , 28.3 , 27.2 , 26.9 , 26.7 , 26.6 , 26.3 , 23.4 , 23.3 , 18.4 ; hr - fab ms [ m + na ] calcd for c27h46o2na : 425.3396 , found 425.3389 . compound 15 ( 0.56 g , 1.4 mmol ) and 10% pd / c ( 200 mg ) were added to a borosilicate sealable glass vessel containing etoac ( 50 ml ) . the reaction vessel was attached to the parr hydrogenator ( shaker - type ) , and the air was evacuated and replaced with h2 at 60 psi . the reaction was allowed to shake for 3 h , at which time the h2 was evacuated under vacuum for 30 min . the vessel was repressurized and removed from the hydrogenator , and the reaction mixture was filtered through celite ( taking care to wash the pd / c several times with etoac ) . the eluent was concentrated and recrystallized from cold hexanes to give compound 16 as colorless crystals in 99% yield ( 0.56 g , 1.4 mmol ) . compound 16 : mp 6465 c ( cold hexanes ) ; [ ]d + 40.2 ( c = 0.17 , chcl3 ) ; ir : 2928 , 2867 , 1456 , 1375 , 1145 , 1107 , 1045 , 916 cm ; h nmr ( cdcl3 ) 4.70 ( s , 2h , och2och3 ) , 3.493.59 ( m , 1h , h-3 ) , 3.38 ( s , 3h , och2och3 ) , 0.731.99 ( m , 33h ) , 0.860.87 ( 3 , d , 9h ) ; c nmr ( cdcl3 ) 94.7 , 77.1 , 55.3 , 52.6 , 50.0 , 48.9 , 48.1 , 40.6 , 39.6 , 37.9 , 36.0 , 35.5 , 33.8 , 33.7 , 31.8 , 31.6 , 30.3 , 28.4 , 28.2 , 27.2 , 26.7 , 26.6 , 26.4 , 25.7 , 23.0 , 22.8 , 18.4 ; hr - fab ms [ m + na ] calcd for c27h48o2na : 427.3552 , found 427.3556 . to a flask containing compound 16 ( 306 mg , 0.76 mmol ) in meoh ( 15 ml ) after stirring 16 h at rt , the reaction was neutralized with saturated aqueous nahco3 , further diluted with h2o ( 30 ml ) , and extracted with dcm ( 4 15 ml ) . the organic extracts were combined and evaporated to give the crude alcohol intermediate , which was immediately redissolved in acetone ( 30 ml ) . jones reagent ( 0.5 ml ; 30% cro330% h2so440% h2o ) was added dropwise until the solution remained a light brown color . at this time , a few drops of isopropyl alcohol were added to quench the remaining jones reagent , and the reaction solution was filtered through a short silica column and eluted several times with etoac to remove chromium byproducts . the eluent was collected and concentrated and then purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to give compound 17 as colorless crystals in 97% yield ( 263 mg , 0.73 mmol ) . compound 17 : mp 5962 c ( amorphous solid , etoac / hexanes ) ; [ ]d = + 40.9 ( c = 0.17 , chcl3 ) ; ir : 2951 , 2927 , 2911 , 2861 , 1712 , 1463 cm ; h nmr ( cdcl3 ) 2.60 ( t , 1h , j = 14.0 hz ) , 0.862.32 ( m , 32h ) , 0.860.88 ( 3 , d , 9h ) ; c nmr ( cdcl3 ) 213.2 , 52.4 , 50.0 , 48.4 , 48.0 , 43.1 , 40.2 , 39.6 , 38.5 , 37.8 , 36.7 , 35.4 , 33.7 , 31.4 , 30.8 , 30.4 , 28.3 , 28.1 , 28.0 , 26.6 , 25.8 , 25.7 , 23.0 , 22.7 , 18.4 ; hr - fab ms [ m + na ] calcd for c25h42ona : 381.3133 , found 381.3132 . compound 17 ( 225 mg , 0.63 mmol ) was added to a flask containing anhydrous dcm ( 4 ml ) and et3n ( 0.22 ml , 1.6 mmol ) and cooled to 0 c under n2 . trimethylsilyl trifluoromethanesulfonate ( tmsotf ) ( 0.17 ml , 0.94 mmol ) was added dropwise , and the reaction was allowed to stir for 30 min at 0 c . after formation of the silyl enol ether ( vide tlc ) , the reaction was cooled to 78 c , and following a general literature procedure , nahco3(s ) ( 77 mg , 1.3 mmol ) and nbs ( 168 mg , 0.94 mmol ) were then added sequentially . the reaction was allowed to warm to 40 c over a period of 30 min . upon formation of the intermediate bromide , the reaction was quenched by the addition of saturated aqueous na2s2o3 ( 1 ml ) , removed from the dry ice bath , and allowed to stir at 25 c for 5 min . the reaction was further diluted with dcm ( 4 ml ) and washed with h2o ( 2 5 ml ) . the organic phase was then dried over na2so4 , concentrated in vacuo , and dried under vacuum for 30 min . according to standard protocol , the crude bromide compound was then redissolved in anhydrous dmf ( 7.5 ml ) , libr ( 162 mg , 1.88 mmol ) and li2co3 ( 324 mg , 4.39 mmol ) were added , and the reaction was heated to 120 c for 16 h. upon the formation of two new products ( vide tlc ) , the dmf was removed , and the crude mixture was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to yield the undesired -3-ketone in 55% ( 123 mg , 0.34 mmol ) yield and the desired -3-ketone ( 18 ) in 32% ( 71 mg , 0.20 mmol ) yield . the undesired -3-ketone was then hydrogenated using the conditions in the synthesis of compound 16 , regenerating reuseable starting material ( 17 , 122 mg , 0.34 mmol ) . in repeating this sequence of protocols two more times , the desired enone ( 18 ) was obtained as colorless crystals in 60% yield ( 135 mg , 0.38 mmol ) , with starting material ( 17 ) recovered in 16% yield ( 36 mg , 0.10 mmol ) . compound 18 : mp 6567 c ( cold hexanes ) ; [ ]d + 64.3 ( c = 0.10 , chcl3 ) ; ir : 2951 , 2930 , 2862 , 1678 , 1619 , 1466 , 1257 cm ; h nmr ( cdcl3 ) 5.81 ( s , 1h ) 2.332.47 ( m , 2h ) , 2.182.33 ( m , 3h ) , 1.882.06 ( m , 4h ) , 0.811.78 ( m , 21h ) , 0.840.86 ( 3 , d , 9h ) ; c nmr ( cdcl3 ) 200.1 , 167.2 , 124.5 , 52.1 , 49.7 , 49.1 , 47.8 , 47.3 , 42.9 , 39.5 , 36.7 , 35.8 , 35.5 , 33.7 , 31.5 , 31.1 , 31.0 , 28.2 , 28.1 , 26.7 , 26.6 , 25.6 , 23.0 , 22.7 , 18.3 ; hr - fab ms [ m + h ] calcd for c25h41o : 357.3157 , found 357.3151 . similar to a reported literature method , to a flask containing ac2o ( 6 ml ) and nai ( 140 mg , 0.93 mmol ) at 0 c under n2 was added trimethylsilyl chloride ( 0.11 ml , 0.89 mmol ) dropwise . the reagents were stirred for 30 min at 0 c , after which compound 18 ( 57 mg , 0.18 mmol ) was added . the reaction was allowed to slowly warm to rt , and stirring was continued another 16 h. upon formation of the 18,19-di - nor - cholesta-3,5-dien-3-ol acetate intermediate , the reaction was neutralized with a 1:1 mix of et3n / diethyl ether , water ( 5 ml ) was added , and the reaction was then extracted with hexanes ( 4 2.5 ml ) , dried over na2so4 , and concentrated in vacuo . the residue was immediately purified by column chromatography ( etoac / hexanes gradient elution ; adding 0.5% et3n to each gradient ) on a silica gel column packed with hexanes / et3n ( 49:1 ) . the dienol acetate intermediate was collected , the solvents were removed , and the compound was redissolved in etoh and cooled to 0 c . nabh4 ( 60 mg ) was added portionwise over 15 min , the reaction was allowed to come to rt , and stirring was then continued for another 1 h. the reaction mixture was then quenched with a few drops of saturated aqueous nh4cl , and the solvents were evaporated . the crude product was redissolved in dcm ( 20 ml ) and washed with brine ( 2 20 ml ) . purification by column chromatography on silica gel ( etoac / hexanes , gradient elution ) gave 18,19-di - nor - cholesterol ( 1 ) as colorless crystals in 60% yield ( 39 mg , 0.11 mmol ) . compound 1 : mp 124126 c ( dcm / hexanes ) ; [ ]d + 23.4 ( c = 0.19 , chcl3 ) ; ir : 3373 , 2926 , 2853 , 1449 , 1375 , 1056 cm ; h nmr ( cdcl3 ) 4.60 ( dd , 1h , j = 5.6 hz , h-6 ) , 3.513.58 ( m , 1h , h-3 ) , 2.50 ( m , 1h ) , 0.802.12 ( m , 29h ) , 0.860.88 ( 3 , d , 9h ) ; c nmr ( cdcl3 ) 137.3 , 121.8 , 71.4 , 53.0 , 49.9 , 47.7 , 45.2 , 45.2 , 43.1 , 43.0 , 39.6 , 35.7 , 35.6 , 33.9 , 32.0 , 31.3 , 31.1 , 30.6 , 28.3 , 28.2 , 26.7 , 25.7 , 23.0 , 22.8 , 18.4 ; hr - fab ms [ m + na ] calcd for c25h42ona : 381.3133 , found 381.3136 .

herein , we report the first synthesis of a demethylated form of cholesterol ( 18,19-di - nor - cholesterol ) , in which the c18 and c19 methyl groups of the -face were eliminated . recent molecular simulations modeling 18,19-di - nor - cholesterol have suggested that cholesterol s opposing rough -face and smooth -face play necessary roles in cholesterol s membrane condensing abilities and , additionally , that specific facial preferences are displayed as cholesterol interacts with different neighboring lipids and transmembrane proteins . inspired by these poorly characterized biochemical interactions , an extensive 18-step synthesis was completed as part of a collaborative effort , wherein synthesizing a smoothened cholesterol analogue would provide a direct way to experimentally measure the significance of the -face methyl groups . starting from known perhydrochrysenone a , the synthesis of 18,19-di - nor - cholesterol was accomplished with an excellent overall yield of 3.5% . the use of the highly stereoselective dieckmann condensation and the employment of evans chiral auxiliary were both key to ensuring the success of this synthesis .

Introduction Discussion and Results Conclusions Experimental Section

it is synthesized endogenously through an extensive multistep biosynthetic pathway , with the first sterol in the pathway being lanosterol . interestingly , of all the sterol intermediates between lanosterol and cholesterol , none possess cholesterol s ability to order and condense lipid membranes ; however , the details regarding cholesterol s structural interactions within the membrane are poorly understood . one of the more intriguing aspects of this planar tetracyclic molecule pertains to its two structurally distinct faces : its -face contains two protruding methyl groups that are conspicuously absent on its -face ( figure 1a ) . there has been much speculation that this facial asymmetry may be essential for cholesterol s unique membrane condensing abilities as well as being essential to the way in which it interacts with and stabilizes neighboring lipids and transmembrane proteins . to test this theory , models have been used to simulate the properties and interactions of a demethylated form of cholesterol ( 18,19-di - nor - cholesterol , which lacks the -face methyl groups ) . herein , we describe the first synthesis of a smoothened 18,19-di - nor - cholesterol , which we have completed as part of a collaborative effort , in hopes of providing a way to experimentally measure its effects on lipid membranes and other important transmembrane interactions . ( a ) structures of cholesterol and 18,19-di - nor - cholesterol ; the -face methyl groups of cholesterol ( shown in red ) are absent in 18,19-di - nor - cholesterol . blue numbers are methyl groups absent in 18,19-di - nor - cholesterol ; red letters label each ring . additionally , the cis - a , b ring fusion of compound a allows for the later introduction of the double bond . as depicted in our retrosynthesis ( scheme 1 ) , starting from a , we can essentially fragment our synthesis into three main parts : d - ring contraction , side - chain installation , and introduction of the double bond . installation of the double bond was chosen as the final step in our synthesis of 18,19-di - nor - cholesterol because of its high reactivity toward a variety of reaction conditions and because it could be obtained from 18,19-di - nor - epicoprostanol ( c ) via enone formation and double bond deconjugation . synthon b fits this description and could be accessed from a by an oxidative d - ring - opening ( to yield a 1,4-diester ) , a dieckmann condensation to reclose the ring , the removal of the 16-ketone group , and a one - carbon homologation reaction at c17 to give synthon b. the success of this entire approach , however , is contingent upon whether ring closure via the dieckmann condensation ( preceding the c17 homologation reaction ) would yield the desired 17-carbomethoxy group as the major isomeric product ( scheme 2 , compound 4 ) . although four isomers are possible , extrapolating results from previous studies on the synthesis of a - nor - steroids and examining the steric interactions at play in our di - nor system led us to expect that this transformation would , in fact , yield our desired product ( see supporting information scheme s1 ) . after removing the 16-keto group via formation of thioketal 5 followed by raney nickel dethioketalization to yield compound 6 having little success with the arndt eistert homologation reaction , we chose to proceed via the introduction of a carbonitrile group . for steroids with a c18 methyl group , the introduction of a side - chain via an -substitution at c20 is known to favor the desired ( r)-diastereomer ; however , this selectivity is attributed to steric interferences caused by the c18 methyl group , which is absent in our 18,19-di - nor series . after confirming that alkylations at c20 are not stereoselective for 18-nor - steroids , we used evans chiral auxiliary [ ( r)-4-benzyl-2-oxazolidinone ] to ensure ( r)-stereochemistry at c21 . however , in a variety of subsequent alkylation attempts , we were unable to introduce the entire isohexyl group in satisfactory yield , presumably because of the added bulk of the chiral auxiliary . therefore , we instead installed the side chain in two pieces , first utilizing the highly reactive allyl iodide , to obtain compound 12 as the desired ( r)-diastereomer in 86% yield . next , we needed to remove the chiral auxiliary in order to generate the c21 methyl group ( currently positioned as the carbonyl carbon ) . a subsequent hydrogenation proceeded near quantitatively , affording 18,19-di - nor - epicoprostanol ( 16 ) and completing the synthesis of synthon c ( scheme 3 ) . lastly , we needed to introduce the double bond , which is well - known in literature , through formation of a -3-ketone followed by deconjugation . therefore , we first synthesized the 3-keto - precursor ( 17 ) , which was easily obtained by first removing the methoxymethyl protecting group of compound 16 with anhydrous hcl and then oxidizing the resulting crude alcohol using jones reagent to give ketone 17 . however , subsequent attempts to introduce the -3-ketone proved to be difficult in the absence of the c19 methyl group . after having little success with a variety of steroid protocols , we ultimately adapted a general procedure for the formation of ,-unsaturated ketones , wherein bromination of a silyl enol ether first yields an -bromoketone that undergoes a dehydrobromination . upon cooling the reaction to 78 c and adding nahco3 and n - bromosuccinimide ( nbs ) , the intermediate bromoketone ( obtained as a mixture of bromides ; see supporting information scheme s2 ) was heated in the presence of li2co3 and libr in dmf at 120 c for 16 h to generate the desired -3-ketone ( 18 ) in a moderate 32% yield , with an unwanted -3-ketone byproduct obtained in 55% yield ( reflecting the ratio of the bromide intermediates ) . although the desired -3-ketone was the minor product , of the reaction protocols attempted this protocol gave the highest product yield and the least amount of byproducts . it was also far more economical than the other attempted procedures , in that we were consistently able to recover and reuse starting material 17 via hydrogenation of the unwanted -3-ketone . by running this reaction sequence three consecutive times , we were able to obtain our desired 18,19-di - nor - cholest-4-ene-3-one ( 18 ) in a respectable 60% yield , with 16% of our total starting material ( 17 ) recovered . this simultaneously deconjugated the enone and set our final stereocenter ( 3-oh ) , affording our target 18,19-di - nor - cholesterol ( 1 ) in 60% yield ( 3.6% overall from perhydrochrysenone a ) ( scheme 4 ) . the synthesis of the smoothened 18,19-di - nor - cholesterol was achieved starting from commercially available 19-nor - testosterone . the novel use of the dieckmann condensation was key to accessing our target molecule , allowing us to contract the d - ring to a 5-membered ring and to simultaneously introduce a modifiable functional group and set the stereochemistry at c17 while maintaining the trans - c , d ring fusion . furthermore , the completion of this molecule can now allow for the direct comparison of cholesterol and demethylated 18,19-di - nor - cholesterol in a variety of biological and biophysical studies , some of which are currently underway . high resolution fab - ms determinations were made with a matrix m - nitrobenzyl alcohol , using nai as necessary , utilizing a double - focusing analyzer composed of a magnetic sector followed by a electrostatic sector . at this point , heated h2o was added ( 30 ml , 60 c ) , the stirring was stopped , and the reaction was gradually cooled to room temperature . the reaction was allowed to warm to rt and left to stir for 16 h. upon completion , the reaction was washed with h2o ( 50 ml ) , brine ( 50 ml ) , and h2o ( 50 ml ) , dried over na2so4 , and concentrated in vacuo . over 30 min , lialh4 ( 1 m in thf ; 8.25 ml , 16.5 mmol ) was added dropwise to the reaction mixture , and upon completion of lialh4 addition , the reaction was found to be complete . the reaction was quenched by the careful addition of h2o ( 0.63 ml ) followed by 15% aqueous naoh ( 0.63 ml ) , and the reaction was allowed to stir for 30 min at rt . after 30 min at 0 c , the reaction was quenched with ice and subsequently washed with cold saturated aqueous nahco3 ( 10 ml ) , cold brine ( 10 ml ) , and cold h2o ( 10 ml ) , dried over na2so4 , and concentrated in vacuo at rt . the reaction flask was allowed to dry for 2 to 3 h under vacuum , and the residue was dissolved in anhydrous dmf ( 60 ml ) , and the flask was fitted with a reflux condenser . nacn ( 0.68 g , 14 mmol ) was crushed into a powder and added to the reaction flask , and the mixture was stirred at 65 c for 16 h. the reaction was then cooled to 25 c , and ice was added to the reaction mixture , causing the product to precipitate . to this was slowly added a solution of 50% aqueous naoh ( 20 ml ) , and the reaction was allowed to stir for 16 h at 120 c . in a flame - dried flask under n2 , compound 11 ( 1.32 g , 2.59 mmol ) was dissolved in anhydrous thf ( 50 ml ) , and the reaction mixture was cooled to 78 c . allyl iodide ( 0.47 ml , 5.2 mmol ) was then added dropwise , and the reaction was stirred at 78 c for another 1 h. after 1 h , the reaction was quenched with saturated aqueous nh4cl , h2o was added ( 50 ml ) , and the reaction was extracted several times with etoac ( 4 50 ml ) . lialh4 ( 2 m in thf ; 3.75 ml , 7.50 mmol ) was added dropwise , and the reaction was brought to 25 c and stirred for 16 h. upon reaction completion , the reaction was cooled to 0 c and quenched carefully with 1 m hcl . compound 13 : mp 8991 c ( etoac / hexanes ) ; [ ]d + 37.2 ( c = 0.13 , chcl3 ) ; ir : 3401 , 2928 , 2866 , 1639 , 1440 , 1144 , 1107 , 1040 cm ; h nmr ( cdcl3 ) 5.755.86 ( m , 1h , ch = ch2 ) , 4.955.04 ( overlapping doublets , 2h , ch = ch2 ) , 4.66 ( s , 2h , och2och3 ) , 3.463.62 ( m 3h , h-3 , ch2oh ) , 3.34 ( s , 3h , och2och3 ) , 2.102.19 ( m , 1h , chhch = ch2 ) , 0.902.12 ( m , 25h ) , 0.700.85 ( m , 2h ) ; c nmr ( cdcl3 ) 138.5 , 115.8 , 94.6 , 77.0 , 65.2 , 55.2 , 52.0 , 48.8 , 48.3 , 44.9 , 42.6 , 40.4 , 37.8 , 35.8 , 33.6 , 32.7 , 31.7 , 30.8 , 30.1 , 28.2 , 27.1 , 26.5 , 26.2 , 25.7 ; hr - fab ms [ m + na ] calcd for c24h40o3na : 399.2875 , found 399.2883 . after 30 min at 0 c , the reaction was quenched with ice and subsequently washed with cold saturated aqueous nahco3 ( 25 ml ) , cold brine ( 25 ml ) , and cold h2o ( 25 ml ) , dried over na2so4 , and condensed in vacuo at rt . the reaction flask was allowed to dry for 2 to 3 h under vacuum , the residue was then dissolved in anhydrous thf ( 40 ml ) , and the flask was fitted with a reflux condenser . lialh4 ( 2 m in thf , 13.8 ml , 27.6 mmol ) was added dropwise to the reaction flask , and the mixture was stirred at 50 c for 1 h. the reaction was then cooled to 0 c , quenched carefully with 1 m hcl , subsequently diluted with h2o ( 30 ml ) , and extracted with etoac ( 4 20 ml ) . , the ozone generator was turned off , and oxygen was allowed to bubble into the flask until the blue ozone color disappeared ( ca . the reaction was diluted with dcm ( 50 ml ) , washed with h2o , saturated aqueous nahco3 , and brine , dried over mg2so4 , concentrated , and dried under high vacuum for 2 h. during this time , in a separate flask , isobutyltriphenylphosphonium bromide ( 2.39 g , 3.5 mmol ) was dissolved into anhydrous thf ( 50 ml ) under n2 and cooled to 0 c . , the remaining ylide was quenched by the addition of a few drops of saturated aqueous nh4cl . the reaction vessel was attached to the parr hydrogenator ( shaker - type ) , and the air was evacuated and replaced with h2 at 60 psi . compound 16 : mp 6465 c ( cold hexanes ) ; [ ]d + 40.2 ( c = 0.17 , chcl3 ) ; ir : 2928 , 2867 , 1456 , 1375 , 1145 , 1107 , 1045 , 916 cm ; h nmr ( cdcl3 ) 4.70 ( s , 2h , och2och3 ) , 3.493.59 ( m , 1h , h-3 ) , 3.38 ( s , 3h , och2och3 ) , 0.731.99 ( m , 33h ) , 0.860.87 ( 3 , d , 9h ) ; c nmr ( cdcl3 ) 94.7 , 77.1 , 55.3 , 52.6 , 50.0 , 48.9 , 48.1 , 40.6 , 39.6 , 37.9 , 36.0 , 35.5 , 33.8 , 33.7 , 31.8 , 31.6 , 30.3 , 28.4 , 28.2 , 27.2 , 26.7 , 26.6 , 26.4 , 25.7 , 23.0 , 22.8 , 18.4 ; hr - fab ms [ m + na ] calcd for c27h48o2na : 427.3552 , found 427.3556 . to a flask containing compound 16 ( 306 mg , 0.76 mmol ) in meoh ( 15 ml ) after stirring 16 h at rt , the reaction was neutralized with saturated aqueous nahco3 , further diluted with h2o ( 30 ml ) , and extracted with dcm ( 4 15 ml ) . after formation of the silyl enol ether ( vide tlc ) , the reaction was cooled to 78 c , and following a general literature procedure , nahco3(s ) ( 77 mg , 1.3 mmol ) and nbs ( 168 mg , 0.94 mmol ) were then added sequentially . upon formation of the intermediate bromide , the reaction was quenched by the addition of saturated aqueous na2s2o3 ( 1 ml ) , removed from the dry ice bath , and allowed to stir at 25 c for 5 min . according to standard protocol , the crude bromide compound was then redissolved in anhydrous dmf ( 7.5 ml ) , libr ( 162 mg , 1.88 mmol ) and li2co3 ( 324 mg , 4.39 mmol ) were added , and the reaction was heated to 120 c for 16 h. upon the formation of two new products ( vide tlc ) , the dmf was removed , and the crude mixture was purified by column chromatography on silica gel ( etoac / hexanes , gradient elution ) to yield the undesired -3-ketone in 55% ( 123 mg , 0.34 mmol ) yield and the desired -3-ketone ( 18 ) in 32% ( 71 mg , 0.20 mmol ) yield . the undesired -3-ketone was then hydrogenated using the conditions in the synthesis of compound 16 , regenerating reuseable starting material ( 17 , 122 mg , 0.34 mmol ) . in repeating this sequence of protocols two more times , the desired enone ( 18 ) was obtained as colorless crystals in 60% yield ( 135 mg , 0.38 mmol ) , with starting material ( 17 ) recovered in 16% yield ( 36 mg , 0.10 mmol ) . the reaction was allowed to slowly warm to rt , and stirring was continued another 16 h. upon formation of the 18,19-di - nor - cholesta-3,5-dien-3-ol acetate intermediate , the reaction was neutralized with a 1:1 mix of et3n / diethyl ether , water ( 5 ml ) was added , and the reaction was then extracted with hexanes ( 4 2.5 ml ) , dried over na2so4 , and concentrated in vacuo . the dienol acetate intermediate was collected , the solvents were removed , and the compound was redissolved in etoh and cooled to 0 c . nabh4 ( 60 mg ) was added portionwise over 15 min , the reaction was allowed to come to rt , and stirring was then continued for another 1 h. the reaction mixture was then quenched with a few drops of saturated aqueous nh4cl , and the solvents were evaporated . purification by column chromatography on silica gel ( etoac / hexanes , gradient elution ) gave 18,19-di - nor - cholesterol ( 1 ) as colorless crystals in 60% yield ( 39 mg , 0.11 mmol ) .

myosin binding protein - c ( mybp - c ) is a regulatory and structural protein associated with the a - bands ( thickfilaments ) of vertebrate striated muscle sarcomeres . regulatory effects are mediated in part by the n - terminus of cardiac cmybp - c , which is made up of modular immunoglobulin-(ig- ) like domains , termed c0 at the n - terminus , followed by domains c1 through c10 ( figure 1(a ) ) . between the c0 and c1 domains there is a sequence of ~50 amino acids that contains a high percentage of proline and alanine residues ( referred to as the pro - ala rich region ( p / a ) ) . between the c1 and c2 domains there is a stretch of ~100 highly conserved amino acids referred to as mybp - c motif or m - domain . the m - domain is phosphorylated by -adrenergic agonists and phosphorylation increases the rate of cross - bridge cycling , thereby contributing to increased inotropic responses of the heart . phosphorylation reduces binding of the m - domain to actin and myosin s2 in vitro , but the precise mechanism(s ) by which phosphorylation accelerates actomyosin interactions is not well understood . in efforts to uncover mechanisms by which the n - terminus of cmybp - c affects cross - bridge cycling several groups have investigated the effects of recombinant proteins containing n - terminal cmybp - c domains on actomyosin interactions . for instance , razumova et al . investigated effects of n - terminal domains in in vitro motility assays and found that recombinant proteins containing the c1 through c2 domains of mouse cmybp - c ( referred to as c1c2 , inclusive of the m - domain ) could activate thin filament motility even in the absence of ca . incubation of permeabilized rat trabeculae with c1c2 also increased ca sensitivity of force and increased the rate of tension redevelopment ( ktr ) . the activating effects of c1c2 were attributed to the combined effects of the c1 and m - domains because these domains together were necessary and sufficient to increase ca sensitivity of force , whereas other domains including the c0 , p / a , and c1 domains had little , if any , effect on force activation . however , in apparent contrast to these results , herron et al . found that a recombinant protein comprised of the human c0 , p / a , and c1 domains ( i.e. , c0c1 ) activated tension and increased ktr in permeabilized myocytes from human myocardium , whereas c1c2 ( which lacked the p / a region ) had no effect . these authors concluded that the p / a rich region was required to activate tension and increase ktr . a potential explanation for the different conclusions reached in the two studies is that species - specific differences are responsible for the different functional effects observed for the mouse versus human proteins . consistent with this idea , the full - length cmybp - c protein is quite similar between mouse and human isoforms ( 89% identity , table 1 ) and the c1 , m , and c2 domains are highly conserved ( > 90% identity ) , but domains near the n - terminus show greater sequence divergence with 81% and 46% identity in the c0 domain and p / a regions , respectively ( figure 1(b ) ) . the large difference in sequence homology in the p / a region is primarily due to differences in the content of proline and alanine residues : in the human p / a region , proline and alanine account for 51% of the sequence , while the mouse isoform contains only 28% proline and alanine . interestingly , a similar trend was noted for a p / a rich sequence in fast skeletal myosin essential light chains ( elc1 ) where bicer and reiser found that p / a content scaled with mammalian size . the purpose of the present study was to determine whether species - specific differences in the p / a rich regions of human and mouse cmybp - c can account for functional differences in the ability of recombinant proteins from the two species to activate actomyosin interactions . results show that both the human p / a rich region and the c1 domains can activate actomyosin interactions , whereas the mouse p / a and c1 domains are less effective . these results thus reconcile apparently disparate experimental results and raise the possibility that species - specific variations in cmybp - c regulatory domains contribute to cross - species variations in cardiac function . the recombinant mouse protein mc0c1 ( containing the c0 , p / a , and c1 domains , figure 1(c ) ) was subcloned from a full - length mouse cdna ( genbank gi:3747133 ) , expressed in m15 cells , and purified using ni - nta affinity chromatography as previously described . recombinant human hc0c1 was cloned from a human cdna obtained by rt - pcr from whole heart human total rna ( stratagene , la jolla , ca ) using a one - step rt - pcr kit ( invitrogen , carlsbad , ca ) . a pcr product encoding hc0c1 was generated using gene specific primers flanking the desired domains ( forward primer 5-ggcccatatgcctgagccggggaagaag-3 , reverse primer 5-ggccaagctttcatccggtgcccatggcctcgtg-3 ) and cloned into the pqe-2 expression vector ( qiagen , valencia , ca ) using ndei and hindiii restriction sites on the upstream and downstream primers , respectively . protein concentrations were determined by measuring light absorbance at 280 nm ( corrected for turbidity at 310 nm ) using extinction coefficients calculated from swiss institute for bioinformatics software . the in - fusion pcr cloning system ( clontech , mountain view , ca ) was used according to manufacturer 's instructions and the protocol by zhu et al . to create seamless chimeric c0c1 proteins consisting of various combinations of mouse and human c0 , p / a , and c1 domains . boundaries of the c0 , p / a , and c1 domains are shown in figure 1(b ) and were as described by gautel et al . and as listed for human and mouse cmybp - c on the universal protein resources databank ( uniprot ) . pcr products encoding the mouse and human domains were amplified from mouse and human cmybp - c cdna with additional 15 bp flanking sequences at the 5 and 3 ends that overlap with the adjacent segment of the construct ( table 2 , figure 1(c ) ) . the pqe-2 expression vector ( qiagen ) was digested with ndei and hindiii and gel - purified . pcr products and the digested pqe-2 vector were mixed at a 1 : 2 : 2 : 2 molar ratio with the in - fusion enzyme according to manufacturer 's instructions and transformed into top10 cells ( invitrogen ) . correctly ligated plasmids were chosen after selection with carbenicillin and sequences were verified by dna sequencing at the uc davis dna sequencing facility . heavy meromyosin ( hmm ) and s1 were prepared from rabbit psoas skeletal myosin via -chymotryptic digest as described . bovine cardiac f - actin , tropomyosin , and troponin were purified from ether powder as described [ 1416 ] . thin filaments , comprised of f - actin and regulatory proteins , were reconstituted in ab buffer ( in mmol / l : 25 kcl , 25 imidazole ( ph 7.4 ) , 4 mgcl2 , 1 egta , and 1 dtt ) by combining at an actin : tropomyosin : troponin ratio of 4 : 1 : 1 for in vitro motility assays and at 7 : 2 : 2 for atpase assays . the thin filament mix was labeled with rhodamine - phalloidin according to homsher et al . and used in in vitro motility assays . treatment of all animals was in strict accordance with guidelines and protocols established by the university of california institutional animal care and use committee . male sprague - dawley rats ( 200250 g ) were euthanized by intraperitoneal injection of sodium pentobarbital . hearts were then rapidly excised and right ventricles were dissected in a ringer 's solution at ph 7.4 ( in mmol / l : 100 nacl , 24 nahco3 , 2.5 kcl , 1 mgso4 , 1 na2hpo4 , and 1 cacl2 ) . trabeculae were permeabilized in situ by incubation of splayed ventricles overnight in a relaxing solution containing 50% glycerol and 1% individual trabeculae were dissected free from ventricle walls , pinned to the bottom of a sylgar - coated petri dish , and stored for up to one week in glycerinated relaxing solution at 20c . steady state force and rate of tension redevelopment ( ktr ) measurements were performed as previously described . briefly , permeabilized trabeculae were mounted between a force transducer ( model 403a , aurora scientific inc . ) and a torque motor ( model 312-cl , aurora scientific inc ) . sarcomere length was adjusted to ~2.3 m in relaxing solution and monitored throughout the course of the experiment using an inverted microscope ( olympus ix-71 ) fitted with a 12-mega pixel digital camera ( olympus dp70 ) . relaxing , preactivating , and ca - activating solutions were prepared as previously described using a custom software package [ 18 , 19 ] . solutions were maintained at 0.18 m ionic strength and ph 7.0 at 15c ( in mmol / l : 15 phosphocreatine , 15 egta , at least 40 mops , 1 free mg , 135 na + k , 1 dtt , 250 units ml creatine kinase ( ck ) , and 5 atp ) . ca concentration ( reported as pca = log[ca ] ) was established by varying amounts of cacl2 . recombinant proteins were added to relaxing and preactivating solutions by buffer exchange using desalting spin columns ( pierce , rockford , il ) . in vitro motility assays briefly , hmm was applied to a nitrocellulose - coated coverslip followed by incubation with bovine serum albumin ( bsa ) to prevent nonspecific adsorption of thin filaments or recombinant proteins to the motility surface . shredded actin filaments followed by atp were then added to block nonfunctional myosin heads ( dead - heads ) . next , rhodamine - phalloidin labeled bovine cardiac thin filaments ( 4 : 1 : 1 actin : tropomyosin : troponin ) were added , followed by a motility buffer containing ab buffer , 2 mm atp , and an oxygen - scavenging system to limit photo - bleaching . recombinant cmybp - c proteins were dialyzed into ab buffer and added to the slide surface in the motility buffer . motility was viewed using an olympus ix-71 microscope with an hg - arc lamp , tritc filter , and a 100/1.4 na oil - immersion objective . video files were recorded using a q - imaging retiga exi digital camera and imageproplus software . filament motility was analyzed using custom software developed in labview and ni vision development ( national instruments , austin , tx ) kindly provided by dr . atpase assays were performed by mixing 0.2 m myosin s1 and 3.5 m reconstituted bovine cardiac thin filaments ( mixed at a 7 : 2 : 2 actin : tropomyosin : troponin ratio ) in atpase buffer ( in mmol / l : 10 imidazole ( ph 7.4 ) , 2 mgcl2 , 1 egta , and 1 dtt ) with or without 1 m recombinant cmybp - c proteins . the addition of 1 mm atp started the reaction and reactions were quenched at three different time points with stop solution ( 3.3% sds , 0.12 m na - edta , ph 7.4 ) . effects of recombinant mouse and human c0c1 proteins ( inclusive of the c0 , p / a , and c1 domains ) were first assessed in permeabilized rat cardiac trabeculae . figure 2(a ) shows effects of human ( h)c0c1 and mouse ( m)c0c1 on force generation at pca 9.0 and at maximal ca activation ( pca 4.5 ) . neither hc0c1 nor mc0c1 affected resting force in the absence of ca ( pca 9.0 ) or maximal force at pca 4.5 even at concentrations up to 80 m , the highest concentration used by herron et al . . this result is consistent with observations by razumova et al . who found that preincubation of rat trabeculae with 30 m mc0c1 did not affect force , but it differs somewhat from herron et al . who found that 30 m hc0c1 activated force generation even in the absence of ca at pca 9.0 . because measurements by herron et al . were conducted primarily using permeabilized myocytes from human myocardium , species - specific differences ( rat versus human ) could potentially contribute to the different effects observed here . however , as shown in figures 2(b ) and 2(c ) , hc0c1 was effective at increasing force and ktr at submaximal ca ( pca 5.3 ) near the half - maximal [ ca ] ( pca50 ) required for maximal force generation . 50 m mc0c1 also increased ca sensitivity of tension , albeit to a lesser extent than hc0c1 , but had no effect on ktr . because results from permeabilized rat trabeculae suggested that species - specific differences contribute to differences in effects of hc0c1 and mc0c1 , we sought to compare effects of hc0c1 versus mc0c1 in a defined system that minimizes variability in cross - species isoform expression . we therefore compared effects of hc0c1 and mc0c1 using in vitro motility assays with reconstituted cardiac thin filaments and skeletal hmm . control values for average filament velocities in the absence ( pca 9 ) and presence ( pca 5 ) of ca were 0.3 0.1 m / s ( n = 12 ) and 3.8 0.5 m / s ( n = 9 ) , respectively , demonstrating that reconstituted thin filaments were well regulated by ca under control conditions in the absence of added recombinant proteins . figure 3(a ) compares filament sliding speeds at pca 9.0 in the absence or presence of hc0c1 or mc0c1 . under control conditions at low ca , filament sliding speed was low and the vast majority of filaments were stopped ( fraction moving was 4% ) . addition of 1 m mc0c1 to motility buffers did not activate motility , whereas addition of hc0c1 significantly increased motility . these results are similar to those obtained in rat trabeculae ( figure 2 ) where hc0c1 increased ca sensitivity of tension and ktr to a greater extent than mc0c1 . they are also consistent with herron et al . who found that hc0c1 could activate tension development in myocytes in the absence of ca . to determine whether the p / a region contributes to the activating properties of hc0c1 , chimeric c0c1 proteins were created that substituted human and mouse p / a regions . figure 3(b ) shows effects on filament motility of exchanging the human and mouse p / a domains in chimeric proteins . insertion of the human p / a region into mouse c0c1 to create the chimeric protein mhmc0c1 ( mouse c0 , human p / a , and mouse c1 ) increased filament motility at pca 9 . this result demonstrates that the human p / a region but not the mouse p / a region is sufficient to confer activating effects on c0c1 proteins . however , activation was not complete since sliding speeds were still somewhat less than in the presence of hc0c1 comprised of all human sequences . this suggests that domains outside of the p / a region must also contribute to the activating effects of hc0c1 . consistent with this idea , when the mouse p / a region was exchanged into hc0c1 , that is , in hmhc0c1 ( human c0 , mouse p / a , and human c1 ) , filament sliding speeds were increased relative to control but were less than in the presence of hc0c1 . collectively , these results demonstrate that the human p / a rich region is sufficient to activate motility in the absence of ca , but that the c0 or c1 domains must also contribute to the ability of hc0c1 to activate filament motility . to determine whether human c1 or c0 domains also contribute to the activating effects of hc0c1 , we created additional chimeric proteins that substituted human and mouse c0 and c1 domains . to assess if the c1 domain is required for activating effects , 1 m mmhc0c1 or hhmc0c1 proteins were added to in vitro motility assays . as shown in figure 3(c ) , adding the human c1 to mouse c0 and p / a domains , ( mmhc0c1 ) activated thin filament motility at pca 9 , demonstrating that the human c1 domain can also confer activating effects . however , filament velocity was reduced compared to hc0c1 ( figure 3(a ) ) , indicating that other domains ( e.g. , p / a ; figure 3(b ) ) contribute to the activating effects of hc0c1 . conversely , when the mouse c1 domain was added to human c0 and p / a domains ( hhmc0c1 ) activation occurred but to a lesser extent than hc0c1 ( figure 3(a ) ) . these results show that mouse c1 can not substitute for human c1 and can not restore full activating effects when expressed with the human c0 and p / a domains . to assess whether the human c0 domain also contributes to activating effects of hc0c1 , hmmc0c1 and mhhc0c1 proteins were created and their effects in motility assays were assessed at pca 9 . as shown in figure 3(d ) , when the human c0 domain was added to mouse p / a and c1 domains ( hmmc0c1 ) , activation of filament motility did not occur and effects of hmmc0c1 were not different from the parent mc0c1 . similarly , when the mouse c0 domain was expressed with the human p / a and c1 domains ( mhhc0c1 ) , filament motility was the same as in the presence of hc0c1 ( figure 3(a ) ) . these results show that c0 does not contribute to the activating effects of hc0c1 in motility assays , and that the human p / a and c1 domains together are sufficient to account for the full activating properties of hc0c1 on actomyosin interactions in the absence of ca . when expressed in rat neonatal cardiomyocytes , hc0c1 localized to sarcomere a - bands , suggesting that interactions of hc0c1 with myosin or other thick filament proteins are required for the observed activating effects . consistent with this idea , the c0 domain was reported to bind to myosin s1 and c1 can bind to myosin s2 . therefore to test whether interactions with myosin s2 are required for the activating effects of the human p / a and c1 domains , effects of chimeric c0c1 proteins were assessed in atpase assays using myosin s1 ( without s2 ) and regulated thin filaments . as shown in figure 4 , under control conditions in the absence of recombinant proteins , atpase rates were low ( 0.2 0.2 sec , n = 17 ) at low ca ( pca 10 ) and increased ( 3.3 1.0 sec , n = 16 ) in the presence of maximal ca ( pca 3 ) . similar to the results obtained in the in vitro motility assays , addition of 1 m hc0c1 activated atpase activity at pca 10 , whereas mc0c1 did not significantly affect atpase rates . addition of the human p / a domain or the c1 domains to the mouse c0c1 backbone ( mhmc0c1 or mmhc0c1 ) also activated atpase activity in the absence of ca , but not to the full extent of hc0c1 . substituting the mouse p / a and c1 domains in the human c0c1 backbone ( hmhc0c1 and hhmc0c1 ) reduced atpase activity when compared to hc0c1 . similar to motility assays ( figure 3(d ) ) , exchanging the c0 domains ( hmmc0c1 and mhhc0c1 ) had no effect on the atpase rates when compared to mc0c1 and hc0c1 controls ( figure 4 ) . taken together , these results confirm conclusions from the in vitro motility assays that either the human p / a or c1 domains can confer activating properties to the hc0c1 protein , but that both domains are required for full effects . however , because all activating effects occurred in the absence of myosin s2 , results from the atpase assays demonstrate that activation does not require interactions of hc0c1 with myosin s2 . the major result from this study is that species - specific differences between mouse and human cmybp - c contribute to functional differences in the activities of recombinant cmybp - c proteins . in particular , we found significant sequence divergence ( 46% identity ) in a proline - alanine ( p / a ) rich region near the n - terminus of the molecule . the human p / a rich region but not the mouse p / a region promoted actomyosin interactions as shown by the ability of proteins containing the human p / a region to increase thin filament motility and actomyosin atpase activity even in the absence of ca ( figure 3 ) . interestingly , the human c1 domain but not mouse c1 also conferred activating effects even though the sequences of the c1 domains are much more similar ( 90% identity ) . taken together , these results suggest that even modest sequence variations in conserved domains can lead to significant functional differences between homologous cmybp - c proteins . results from this study partially reconcile disparate results from two previously published studies . using human cmybp - c recombinant proteins , herron et al . attributed activating effects to the p / a rich region , whereas razumova et al . , using mouse recombinant proteins , found that the c1 and m domains together were required to activate force . in the present study we performed a side - by - side comparison of the effects of mouse and human c0c1 proteins and found in good agreement with herron et al . that the human p / a region can confer activating effects on actomyosin interactions , whereas also in good agreement with razumova et al . thus , species - specific differences can account in part for the different conclusions reached in the two studies . the precise mechanism(s ) by which the p / a and c1 domains promote actomyosin interactions is not known , but interactions with either thick filaments or actin are possible . in support of the former , hc0c1 localizes to sarcomere a - bands however , the present results exclude interactions with thick filaments because myosin s2 was not required for hc0c1 to increase acto - s1 atpase activity ( figure 4 ) . furthermore , while c0 was reported to bind to myosin s1 , c0 made little or no contribution to the activating effects of hc0c1 ( figures 3 and 4 ) . thus , interactions with myosin do not appear important for the activating effects of hc0c1 , although interactions with other thick filament proteins ( e.g. , myosin light chains ) can not be excluded . alternatively , the p / a region could interact with thin filaments to promote activation . in support of this idea squire et al . proposed that the p / a region of human cmybp - c binds actin based on sequence similarity to a p / a rich segment found in essential myosin light chains ( mlc ) . although in a previous study we found little evidence that the mouse p / a region binds to actin because mc0c1 bound weakly if at all to f - actin in cosedimentation assays , kulikovskya et al . reported that human c0c1 can bind actin . side - by - side comparisons of binding affinity are needed , but these data are thus in the right direction for the pro - ala sequence to contribute to functional effects in human ( e.g. , because of greater actin binding affinity ) but exert less effects in mouse because of reduced interactions with actin [ 6 , 7 ] . the functional significance of the pro - ala rich sequence of cmybp - c in vivo is not known . however , the pro - ala rich sequences have been identified in other thick filament regulatory proteins including cardiac and skeletal isoforms of myosin essential light chains ( mlc ) . in these proteins the pro - ala sequences modulate cross - bridge cycling rates and shortening velocity ( vmax ) by binding to actin . the proline - alanine rich regions of different mlc isoforms slow cross - bridge kinetics either by binding directly to actin or by functioning as a rigid spacer arm that extends an actin binding site located near the n - terminus of the mlc out toward the thin filament . in either case , interactions with actin are thought to create a drag that limits filament sliding and slows cross - bridge cycling and shortening velocity . consistent with this idea , atrial myocytes that express an atrial mlc isoform with reduced affinity for actin have nearly twice the vmax and maximal power output than ventricular myocytes expressing an mlc that binds to actin with greater affinity . by analogy with mlc , it is possible that the pro - ala rich region of cmybp - c performs a similar role and contributes to the ability of cmybp - c to limit myocyte shortening velocity , cross - bridge cycling , and power output . if so , then the species - specific differences described here between the pro - ala regions of mouse and human cmybp - c could serve to fine - tune shortening velocity to optimize power output ( the product of force and velocity ) such that contractile efficiency is maximized in hearts that contract under different hemodynamic loads and at different speeds . consistent with this idea , we found that the percentage of proline and alanine residues in the pro - ala region varies inversely with heart rate in mammals such that larger mammals have a greater proportion of pro - ala residues , shaffer and harris . thus , decreased pro - ala content of cmybp - c in small mammals could accelerate cross - bridge cycling kinetics , whereas increased pro - ala content in larger mammals could slow cycling rates . species - specific changes in cmybp - c could thereby fine - tune larger shifts in cross - bridge cycling kinetics that occur due to differences in isoform expression of other contractile proteins such as in myosin heavy chain that shifts from fast -mhc ( high atpase activity and cross - bridge cycling ) expressed in small mammals to slow -mhc expressed in larger mammals [ 3336 ] . such systematic changes in cross - bridge cycling kinetics could also tune cardiac relaxation rates , for instance , to ensure adequate diastolic filling times even at high heart rates in small mammals . in summary , results presented here demonstrate significant species - specific differences in the ability of the p / a rich region and c1 domains of mouse versus human cmybp - c to activate actomyosin interactions . these differences suggest that the function of cmybp - c varies in different species and raises the intriguing possibility that cmybp - c - fine - tunes cardiac contraction in different animals to better match contractile speed to hemodynamic load .

the n - terminus of cmybp - c can activate actomyosin interactions in the absence of ca2 + , but it is unclear which domains are necessary . prior studies suggested that the pro - ala rich region of human cmybp - c activated force in permeabilized human cardiomyocytes , whereas the c1 and m - domains of mouse cmybp - c activated force in permeabilized rat cardiac trabeculae . because the amino acid sequence of the p / a region differs between human and mouse cmybp - c isoforms ( 46% identity ) , we investigated whether species - specific differences in the p / a region could account for differences in activating effects . using chimeric fusion proteins containing combinations of human and mouse c0 , pro - ala , and c1 domains , we demonstrate here that the human p / a and c1 domains activate actomyosin interactions , whereas the same regions of mouse cmybp - c are less effective . these results suggest that species - specific differences between homologous cmybp - c isoforms confer differential effects that could fine - tune cmybp - c function in hearts of different species .

1. Introduction 2. Methods 3. Results 4. Discussion

regulatory effects are mediated in part by the n - terminus of cardiac cmybp - c , which is made up of modular immunoglobulin-(ig- ) like domains , termed c0 at the n - terminus , followed by domains c1 through c10 ( figure 1(a ) ) . between the c0 and c1 domains there is a sequence of ~50 amino acids that contains a high percentage of proline and alanine residues ( referred to as the pro - ala rich region ( p / a ) ) . between the c1 and c2 domains there is a stretch of ~100 highly conserved amino acids referred to as mybp - c motif or m - domain . the m - domain is phosphorylated by -adrenergic agonists and phosphorylation increases the rate of cross - bridge cycling , thereby contributing to increased inotropic responses of the heart . phosphorylation reduces binding of the m - domain to actin and myosin s2 in vitro , but the precise mechanism(s ) by which phosphorylation accelerates actomyosin interactions is not well understood . in efforts to uncover mechanisms by which the n - terminus of cmybp - c affects cross - bridge cycling several groups have investigated the effects of recombinant proteins containing n - terminal cmybp - c domains on actomyosin interactions . investigated effects of n - terminal domains in in vitro motility assays and found that recombinant proteins containing the c1 through c2 domains of mouse cmybp - c ( referred to as c1c2 , inclusive of the m - domain ) could activate thin filament motility even in the absence of ca . the activating effects of c1c2 were attributed to the combined effects of the c1 and m - domains because these domains together were necessary and sufficient to increase ca sensitivity of force , whereas other domains including the c0 , p / a , and c1 domains had little , if any , effect on force activation . found that a recombinant protein comprised of the human c0 , p / a , and c1 domains ( i.e. , c0c1 ) activated tension and increased ktr in permeabilized myocytes from human myocardium , whereas c1c2 ( which lacked the p / a region ) had no effect . these authors concluded that the p / a rich region was required to activate tension and increase ktr . a potential explanation for the different conclusions reached in the two studies is that species - specific differences are responsible for the different functional effects observed for the mouse versus human proteins . consistent with this idea , the full - length cmybp - c protein is quite similar between mouse and human isoforms ( 89% identity , table 1 ) and the c1 , m , and c2 domains are highly conserved ( > 90% identity ) , but domains near the n - terminus show greater sequence divergence with 81% and 46% identity in the c0 domain and p / a regions , respectively ( figure 1(b ) ) . the large difference in sequence homology in the p / a region is primarily due to differences in the content of proline and alanine residues : in the human p / a region , proline and alanine account for 51% of the sequence , while the mouse isoform contains only 28% proline and alanine . interestingly , a similar trend was noted for a p / a rich sequence in fast skeletal myosin essential light chains ( elc1 ) where bicer and reiser found that p / a content scaled with mammalian size . the purpose of the present study was to determine whether species - specific differences in the p / a rich regions of human and mouse cmybp - c can account for functional differences in the ability of recombinant proteins from the two species to activate actomyosin interactions . results show that both the human p / a rich region and the c1 domains can activate actomyosin interactions , whereas the mouse p / a and c1 domains are less effective . these results thus reconcile apparently disparate experimental results and raise the possibility that species - specific variations in cmybp - c regulatory domains contribute to cross - species variations in cardiac function . the recombinant mouse protein mc0c1 ( containing the c0 , p / a , and c1 domains , figure 1(c ) ) was subcloned from a full - length mouse cdna ( genbank gi:3747133 ) , expressed in m15 cells , and purified using ni - nta affinity chromatography as previously described . to create seamless chimeric c0c1 proteins consisting of various combinations of mouse and human c0 , p / a , and c1 domains . boundaries of the c0 , p / a , and c1 domains are shown in figure 1(b ) and were as described by gautel et al . and as listed for human and mouse cmybp - c on the universal protein resources databank ( uniprot ) . pcr products encoding the mouse and human domains were amplified from mouse and human cmybp - c cdna with additional 15 bp flanking sequences at the 5 and 3 ends that overlap with the adjacent segment of the construct ( table 2 , figure 1(c ) ) . thin filaments , comprised of f - actin and regulatory proteins , were reconstituted in ab buffer ( in mmol / l : 25 kcl , 25 imidazole ( ph 7.4 ) , 4 mgcl2 , 1 egta , and 1 dtt ) by combining at an actin : tropomyosin : troponin ratio of 4 : 1 : 1 for in vitro motility assays and at 7 : 2 : 2 for atpase assays . solutions were maintained at 0.18 m ionic strength and ph 7.0 at 15c ( in mmol / l : 15 phosphocreatine , 15 egta , at least 40 mops , 1 free mg , 135 na + k , 1 dtt , 250 units ml creatine kinase ( ck ) , and 5 atp ) . recombinant cmybp - c proteins were dialyzed into ab buffer and added to the slide surface in the motility buffer . atpase assays were performed by mixing 0.2 m myosin s1 and 3.5 m reconstituted bovine cardiac thin filaments ( mixed at a 7 : 2 : 2 actin : tropomyosin : troponin ratio ) in atpase buffer ( in mmol / l : 10 imidazole ( ph 7.4 ) , 2 mgcl2 , 1 egta , and 1 dtt ) with or without 1 m recombinant cmybp - c proteins . effects of recombinant mouse and human c0c1 proteins ( inclusive of the c0 , p / a , and c1 domains ) were first assessed in permeabilized rat cardiac trabeculae . figure 2(a ) shows effects of human ( h)c0c1 and mouse ( m)c0c1 on force generation at pca 9.0 and at maximal ca activation ( pca 4.5 ) . neither hc0c1 nor mc0c1 affected resting force in the absence of ca ( pca 9.0 ) or maximal force at pca 4.5 even at concentrations up to 80 m , the highest concentration used by herron et al . who found that preincubation of rat trabeculae with 30 m mc0c1 did not affect force , but it differs somewhat from herron et al . who found that 30 m hc0c1 activated force generation even in the absence of ca at pca 9.0 . were conducted primarily using permeabilized myocytes from human myocardium , species - specific differences ( rat versus human ) could potentially contribute to the different effects observed here . because results from permeabilized rat trabeculae suggested that species - specific differences contribute to differences in effects of hc0c1 and mc0c1 , we sought to compare effects of hc0c1 versus mc0c1 in a defined system that minimizes variability in cross - species isoform expression . control values for average filament velocities in the absence ( pca 9 ) and presence ( pca 5 ) of ca were 0.3 0.1 m / s ( n = 12 ) and 3.8 0.5 m / s ( n = 9 ) , respectively , demonstrating that reconstituted thin filaments were well regulated by ca under control conditions in the absence of added recombinant proteins . figure 3(a ) compares filament sliding speeds at pca 9.0 in the absence or presence of hc0c1 or mc0c1 . who found that hc0c1 could activate tension development in myocytes in the absence of ca . to determine whether the p / a region contributes to the activating properties of hc0c1 , chimeric c0c1 proteins were created that substituted human and mouse p / a regions . figure 3(b ) shows effects on filament motility of exchanging the human and mouse p / a domains in chimeric proteins . insertion of the human p / a region into mouse c0c1 to create the chimeric protein mhmc0c1 ( mouse c0 , human p / a , and mouse c1 ) increased filament motility at pca 9 . this result demonstrates that the human p / a region but not the mouse p / a region is sufficient to confer activating effects on c0c1 proteins . this suggests that domains outside of the p / a region must also contribute to the activating effects of hc0c1 . consistent with this idea , when the mouse p / a region was exchanged into hc0c1 , that is , in hmhc0c1 ( human c0 , mouse p / a , and human c1 ) , filament sliding speeds were increased relative to control but were less than in the presence of hc0c1 . collectively , these results demonstrate that the human p / a rich region is sufficient to activate motility in the absence of ca , but that the c0 or c1 domains must also contribute to the ability of hc0c1 to activate filament motility . to determine whether human c1 or c0 domains also contribute to the activating effects of hc0c1 , we created additional chimeric proteins that substituted human and mouse c0 and c1 domains . to assess if the c1 domain is required for activating effects , 1 m mmhc0c1 or hhmc0c1 proteins were added to in vitro motility assays . as shown in figure 3(c ) , adding the human c1 to mouse c0 and p / a domains , ( mmhc0c1 ) activated thin filament motility at pca 9 , demonstrating that the human c1 domain can also confer activating effects . , p / a ; figure 3(b ) ) contribute to the activating effects of hc0c1 . these results show that mouse c1 can not substitute for human c1 and can not restore full activating effects when expressed with the human c0 and p / a domains . to assess whether the human c0 domain also contributes to activating effects of hc0c1 , hmmc0c1 and mhhc0c1 proteins were created and their effects in motility assays were assessed at pca 9 . as shown in figure 3(d ) , when the human c0 domain was added to mouse p / a and c1 domains ( hmmc0c1 ) , activation of filament motility did not occur and effects of hmmc0c1 were not different from the parent mc0c1 . similarly , when the mouse c0 domain was expressed with the human p / a and c1 domains ( mhhc0c1 ) , filament motility was the same as in the presence of hc0c1 ( figure 3(a ) ) . these results show that c0 does not contribute to the activating effects of hc0c1 in motility assays , and that the human p / a and c1 domains together are sufficient to account for the full activating properties of hc0c1 on actomyosin interactions in the absence of ca . when expressed in rat neonatal cardiomyocytes , hc0c1 localized to sarcomere a - bands , suggesting that interactions of hc0c1 with myosin or other thick filament proteins are required for the observed activating effects . therefore to test whether interactions with myosin s2 are required for the activating effects of the human p / a and c1 domains , effects of chimeric c0c1 proteins were assessed in atpase assays using myosin s1 ( without s2 ) and regulated thin filaments . as shown in figure 4 , under control conditions in the absence of recombinant proteins , atpase rates were low ( 0.2 0.2 sec , n = 17 ) at low ca ( pca 10 ) and increased ( 3.3 1.0 sec , n = 16 ) in the presence of maximal ca ( pca 3 ) . similar to the results obtained in the in vitro motility assays , addition of 1 m hc0c1 activated atpase activity at pca 10 , whereas mc0c1 did not significantly affect atpase rates . addition of the human p / a domain or the c1 domains to the mouse c0c1 backbone ( mhmc0c1 or mmhc0c1 ) also activated atpase activity in the absence of ca , but not to the full extent of hc0c1 . substituting the mouse p / a and c1 domains in the human c0c1 backbone ( hmhc0c1 and hhmc0c1 ) reduced atpase activity when compared to hc0c1 . taken together , these results confirm conclusions from the in vitro motility assays that either the human p / a or c1 domains can confer activating properties to the hc0c1 protein , but that both domains are required for full effects . however , because all activating effects occurred in the absence of myosin s2 , results from the atpase assays demonstrate that activation does not require interactions of hc0c1 with myosin s2 . the major result from this study is that species - specific differences between mouse and human cmybp - c contribute to functional differences in the activities of recombinant cmybp - c proteins . in particular , we found significant sequence divergence ( 46% identity ) in a proline - alanine ( p / a ) rich region near the n - terminus of the molecule . the human p / a rich region but not the mouse p / a region promoted actomyosin interactions as shown by the ability of proteins containing the human p / a region to increase thin filament motility and actomyosin atpase activity even in the absence of ca ( figure 3 ) . interestingly , the human c1 domain but not mouse c1 also conferred activating effects even though the sequences of the c1 domains are much more similar ( 90% identity ) . taken together , these results suggest that even modest sequence variations in conserved domains can lead to significant functional differences between homologous cmybp - c proteins . using human cmybp - c recombinant proteins , herron et al . attributed activating effects to the p / a rich region , whereas razumova et al . , using mouse recombinant proteins , found that the c1 and m domains together were required to activate force . in the present study we performed a side - by - side comparison of the effects of mouse and human c0c1 proteins and found in good agreement with herron et al . that the human p / a region can confer activating effects on actomyosin interactions , whereas also in good agreement with razumova et al . thus , species - specific differences can account in part for the different conclusions reached in the two studies . the precise mechanism(s ) by which the p / a and c1 domains promote actomyosin interactions is not known , but interactions with either thick filaments or actin are possible . alternatively , the p / a region could interact with thin filaments to promote activation . proposed that the p / a region of human cmybp - c binds actin based on sequence similarity to a p / a rich segment found in essential myosin light chains ( mlc ) . although in a previous study we found little evidence that the mouse p / a region binds to actin because mc0c1 bound weakly if at all to f - actin in cosedimentation assays , kulikovskya et al . side - by - side comparisons of binding affinity are needed , but these data are thus in the right direction for the pro - ala sequence to contribute to functional effects in human ( e.g. the functional significance of the pro - ala rich sequence of cmybp - c in vivo is not known . however , the pro - ala rich sequences have been identified in other thick filament regulatory proteins including cardiac and skeletal isoforms of myosin essential light chains ( mlc ) . in these proteins the pro - ala sequences modulate cross - bridge cycling rates and shortening velocity ( vmax ) by binding to actin . the proline - alanine rich regions of different mlc isoforms slow cross - bridge kinetics either by binding directly to actin or by functioning as a rigid spacer arm that extends an actin binding site located near the n - terminus of the mlc out toward the thin filament . by analogy with mlc , it is possible that the pro - ala rich region of cmybp - c performs a similar role and contributes to the ability of cmybp - c to limit myocyte shortening velocity , cross - bridge cycling , and power output . if so , then the species - specific differences described here between the pro - ala regions of mouse and human cmybp - c could serve to fine - tune shortening velocity to optimize power output ( the product of force and velocity ) such that contractile efficiency is maximized in hearts that contract under different hemodynamic loads and at different speeds . consistent with this idea , we found that the percentage of proline and alanine residues in the pro - ala region varies inversely with heart rate in mammals such that larger mammals have a greater proportion of pro - ala residues , shaffer and harris . thus , decreased pro - ala content of cmybp - c in small mammals could accelerate cross - bridge cycling kinetics , whereas increased pro - ala content in larger mammals could slow cycling rates . species - specific changes in cmybp - c could thereby fine - tune larger shifts in cross - bridge cycling kinetics that occur due to differences in isoform expression of other contractile proteins such as in myosin heavy chain that shifts from fast -mhc ( high atpase activity and cross - bridge cycling ) expressed in small mammals to slow -mhc expressed in larger mammals [ 3336 ] . in summary , results presented here demonstrate significant species - specific differences in the ability of the p / a rich region and c1 domains of mouse versus human cmybp - c to activate actomyosin interactions . these differences suggest that the function of cmybp - c varies in different species and raises the intriguing possibility that cmybp - c - fine - tunes cardiac contraction in different animals to better match contractile speed to hemodynamic load .

major depression ( md ) is the main cause of disability and the fourth - leading contributor to the global burden of disease . by the year 2020 , md is projected to reach second place in the ranking of disability - adjusted life years . trials of available antidepressant medications alone or combined with psychotherapies are effective for 60%80% of those affected with md.1 conversely , up to 40% of patients with md do not show satisfactory improvement attributable to multiple biopsychosocial factors . at its worst , md can lead to suicide , and as a consequence about 850,000 lives are lost every year.2 treatment - resistant depression ( trd ) evades universal definition ; however , a poor response to two adequate ( optimal dosage and 612 weeks duration ) trials of two different classes of antidepressants has been proposed as its operational characterization.3 researchers have categorized trd in accordance to antidepressant trials : stage 0 , has not had a single adequate trial of medication ; stage 1 , failure of an adequate trial of one class of an antidepressant that is monotherapy ; stage 2 , failure of adequate trials of two distinctly different classes that is , selective serotonin reuptake inhibitors ( ssris ) and tricyclic antidepressants of antidepressant , involving two monotherapy trials ; stage 3 , stage 2 plus failure to respond to one augmentation strategy of lithium or thyroid augmentation of one of the monotherapies ; stage 4 , stage 3 plus a failure to a second augmentation strategy in terms of monoamine oxidase inhibitors ; and stage 5 , stage 4 plus failure of an adequate course of ect.4 there are other staging methods of trd.5 these staging methods help researchers and clinicians to understand trd patients and accordingly plan interventions for enhancing the response , remission rate , and quality of life . however , trd continues to challenge mental health care providers despite the understanding of psychosocial and biological markers and psychopharmacology of mood disorders and also the availability of multiple therapeutic options including optimization , switching , and combination of antidepressants . notably , currently there is an increasing interest in the utilization of several neuromodulation therapies ( nts ) in the management of patients with trd.6 this is because psychopharmacological therapy exposes the entire body to a potentially therapeutic substance in order to treat a relatively small region of the brain , whereas nts are designed to target specific brain circuits that are important in the pathogenesis of md . additionally , nts are not systemic and , therefore , the side - effect profile is limited and different from medications , and there are minimal , if any , drug interactions.7 furthermore , evidence - based data has been emerging continuously about fda - approved and yet - to - be - approved nts in the trd population over the past decade . multiple computer searches were conducted using pubmed , google scholar , quertle(r ) , and medline databases for the years 20002012 . a number of keywords were used : treatment - resistant depression , treatment - refractory depression , partial - response depression , nonresponse depression , neuromodulation techniques , neurostimulation approaches , and somatic therapies . these words were combined with modified electroconvulsive therapy ( mect ) , repetitive transcranial magnetic stimulation ( rtms ) , vagus nerve stimulation ( vns ) , magnetic seizure therapy ( mst ) , deep brain stimulation ( dbs ) , transcranial direct current stimulation , cranial electric stimulation ( ces ) , epidural cortical stimulation ( ecs ) , focused ultrasound ( fus ) , near - infrared light therapy ( nir ) , low - field magnetic stimulation ( lfms ) , and optogenetic stimulation ( os ) for a second round of computer searches . a third round of searches included words such as mechanisms , brain areas involved , and outcomes combined with aforesaid therapies . as a corollary , relevant articles published in english - language peer - reviewed journals were retrieved . only clinical trials , systematic reviews , and meta - analyses that addressed trd and nts were retained for extensive review and inclusion in this study . some exceptions were made with regard to some unique case reports , open and controlled studies , and small and large case series describing usefulness of nts in patients with trd and md . similarly , studies focusing on neurosurgical ablation approaches in trd populations were not considered for inclusion . references of selected articles were also reviewed for identifying relevant trd trials , which were also included in this review . nts for neuropsychiatric disorders including md are categorized into the following : ( 1 ) seizure therapies , including mect and mst , ( 2 ) noninvasive therapies , including rtms , tdcs , and ces , ( 3 ) neurosurgical approaches , including vns , ecs , and dbs , and ( 4 ) new approaches on the horizon , including fus , nir , lfms , and os.8 another category represents neurosurgical ablation therapies , including cingulotomy and limbic leucotomy used in trd . such technical details as invasiveness , anesthesia needed , seizures induced , target related to deep brain structures , contactness , stimulation being focal or generalized and form of stimulation of each neuromodulation therapy are presented in table 1 . there is an increasing focus on exploring biomarkers underlying the pathogenesis of mood disorders9 that help in the development of new drugs and nts . in several related studies , overactive subcallosal cingulate gyrus ( scg ) glucose metabolism has been reported in md that is reduced with successful antidepressant therapies.10 interestingly , dbs is reported to modulate neural pathways linked with scg in relieving md.8,11,12 according to some studies , antidepressant effects were also found when dbs targeted ventral capsule / ventral striatum ( vc / vs ) in patients with severe obsessive - compulsive disorder ( ocd ) and md.13,14 in a study of single patients with dystonia suffering from depression , dbs of globus pallidus internus ( gpi ) showed improvement in dystonia but also showed antidepressant effects through modulation of mesolimbic dopamine pathways.15 in another study , also of single patients with tardive dyskinesia ( td ) and md , dbs brought about improvement in depressive mood.16 other studies have also reported improvement in both depression and td after dbs of the inferior thalamic peduncle ( itp ) , which modulates orbitofrontal cortex hyperactivity.17,18 bewernick and colleagues reported that dbs of the nucleus accumbens ( nac ) was associated with decreased ratings of depression and anxiety in trd patients.19 rush and colleagues20 noticed antidepressant effects when vns was used for epilepsy . vns modulates neural pathways associated with mood regulation : the nucleus tractus solitaries , raphe nucleus , and locus ceruleus.21 in fact , the vns device stimulates left cervical vagus nerve containing afferent neurons tracking through the brain stem to cortical and subcortical networks.2023 furthermore , some neurobiological studies reported disruptions in right and left dorsolateral prefrontal cortex ( r / ldlpfc ) in mood disorders . also , rtms of r / ldlpfc results in antidepressive effects coupled with increasing cerebral blood supply to this brain areas.2325 certainly , nts target more specific , localized regions in the brain , which are somehow dysfunctional in md . it remains uncertain how the depression is relieved ; this is yet to be understood well , and hence basic neurobiological studies are needed . similarly with regard to ect modified ect has been used extensively in psychotic depression , schizophrenia , mania , and other mental disorders . it requires light anesthesia and is a recognized mode of treatment for trd.31,32 it remains the most effective therapy in trd patients with a response rate of 50%70% , though the strength of recommendation of ect is c.33,34 it targets nonspecific , broad regions of the cortex , and its mechanism of action is elusive . notably , high post - ect relapse rate and safety profile are of great concern for trd patients and health providers as well . in a study of patients with nonpsychotic md that tested whether pre - ect medication resistance is associated with post - ect relapse rates , it was observed that 34.6% of nonmedication - resistant patients who were not exposed to at least one antidepressant medication trial relapsed , while 50.0% of medication - resistant patients relapsed , a difference that was not statistically significant but clinically relevant.35 furthermore , in the first week after acute remission , 9.8% of patients not having at least one antidepressant medication trial met relapse criteria , while 31.4% of medication - resistant patients met relapse criteria , a difference that was statistically significant . it was concluded that md patients who have had at least one adequate antidepressant medication trial or no such trial before ect may be especially prone to early relapse after successful acute remission with mect.35 research is needed to develop strategies in order to prevent relapse following successful ect in md , which may be maintenance ect and a combination of pharmacotherapy and mect . furthermore , it is also important to identify the predictors of nonresponse to mect . in a large sample of patients with trd , mect was effective in 66% of patients . mect nonresponse was associated with bipolar subtype , mixed features , slightly less severe depressive symptoms , and longer duration of the depressive episode.36 in another study that aimed to investigate whether the clinical course of trd patients following a course of mect might be associated with changes of plasma brain - derived neurotrophic factor ( bdnf ) concentrations , it was shown that at baseline , plasma bdnf levels of patients were significantly lower than those of control subjects , and those after ect were significantly increased in parallel with the decrease of the hamilton depression rating scale ( hdrs ) total score . only remitter patients who showed higher baseline bdnf levels than nonremitters reached normalized bdnf levels after mect . these findings suggested the potential usefulness of baseline plasma bdnf levels as predictors of response to mect in trd patients.37 in an earlier study of 18 patients with trd , levels of bdnf and 3-methoxy-4-hydroxyphenylglycol but not homovanillic acid were increased following mect in responders , which suggested that dopamine and bdnf might be involved in the mechanism of action of mect.38 in a recent study of adolescents with trd , both continuation and maintenance of mect were useful and safe for selected adolescents with severe trd , and symptom remission was achieved without experiencing cognitive impairment;39 the latter is a surprising finding and needs replication studies . interestingly , in another development , data support the use of ketamine as anesthetic agent prior to ect for increasing its antidepressant effect as compared to propofol . in a related study , 31 inpatients with the hdrs was used to evaluate these patients before ect and after the completion of the second , fourth , sixth , and eighth ect sessions . the hdrs scores improved earlier in the ketamine group , with decreases in hdrs scores that were significantly greater in the ketamine group . the implication of this finding is that the symptoms of md might be alleviated rapidly if ketamine anesthesia is used in trd patients during ect.40 a retrospective evaluation of 5482 ect treatments in 455 patients with trd found therapeutic advantages in combination therapies versus ect . a total of 18.2% of treatments were ect monotherapy , 8.87% were done with one antidepressant . results revealed that seizure duration was unaffected by most antidepressants , but ssri caused a lengthened seizure activity . postictal suppression was lower in mirtazapine and higher in ssri and snri - treated patients . a significant enhancement of therapeutic effectiveness was seen in the patient group receiving tricyclics , ssri , or mirtazapine , with no serious adverse events . baghai and colleagues suggested that controlled studies are necessary to investigate further the possible advantages of ect and pharmacotherapy combinations , especially the use of modern dual - acting antidepressants , which also have proven their efficacy in trd.41 although mect is effective in trd , it significantly produces transient confusion , anterograde amnesia , and retrograde amnesia . therefore , scientists have focused attention on technological refinements in ect and also developing techniques that do not cause cognitive impairment and at the same time remain effective in md and trd.4246 the fda has approved rtms for the treatment of md and trd in adolescents and adults . its other indications include chronic pain , movement disorders , stroke , epilepsy , tinnitus , and other psychiatric disorders . notably , rtms is safer on long - term use and acts more selectively than mect on brain areas implicated in the pathogenesis of md.47,48 the rtms has two forms : high - frequency rapid ( hfr ) ( > 1 hz ) and low - frequency slow ( lfs ) ( 1 hz ) . furthermore , hfr rtms is preferred over lfs sychronized tms , as the former was associated with more antidepressant effects in depressed patients as reflected by significant increases in blood supply to prefrontal cortical and limbic regions.23 a sequential bilateral rtms ( lf right [ lfr ] then hf left [ hfl ] ) is also effective in trd patients but not more effective than unilateral hfl rtms.49,50 in an open - label study , 21 patients who failed two antidepressant trials were given rtms ( hf , 10 hz and intensity of 110% ) for 4 weeks , keeping the dose of preexisting antidepressants unchanged . the majority of patients ( n = 19 ) completed the 4-week study and were assessed . in intention - to - treat analysis , the mean hdrs scores were reduced from 30.80 5.00 to 19.00 6.37 . no patient discontinued rtms due to adverse effects , including headache , which was reported by 16% of patients . the study indicated the potential utility of rtms as an augmenting agent in trd.51 like lfr then hfl sequential bilateral rtms , hfl and lfr unilateral rtms are also efficacious in trd . in a 6-week double - blind , randomized , sham - controlled trial in 50 patients with trd , three trains of lf rtms to the right prefrontal cortex of 140 seconds duration at 1 hz were applied daily , followed immediately by 15 trains of 5 seconds duration of hfl rtms at 10 hz . according to this study , there was a significantly greater response to active than sham stimulation at 2 weeks and across the full duration of the study . a significant proportion of the study group receiving active treatment met response ( 44% ) or remission ( 36% ) criteria by study end compared to the sham stimulation group ( 8% ) , and none remitted ( 0% ) . it was noted that sequentially applying both hfl rtms and lfr rtms to the right prefrontal cortex resulted in substantial improvement in patients with trd . furthermore , the treatment response accumulated to a clinically meaningful level over 46 weeks of active treatment.23 in another controlled investigation , patients with trd were randomized to receive 15 sessions of active or sham rtms delivered to the ldlpfc at 110% the estimated prefrontal cortex threshold . the results showed response rate ( 50% decrease in hdrs score ) for the rtms group was 30.6% , significantly greater than the 6.1% rate in the sham group . the remission rate ( an hdrs score < 8) for the rtms group was 20% , significantly greater than the 3% rate in the sham group . the authors concluded that rtms to ldlpfc can produce statistically and clinically significant antidepressant effects in patients with trd.25 in another study , subjects between the ages of 18 and 85 years were recruited from a tertiary care university hospital . seventy - four subjects with trd and an hdrs score > 21 were randomized to receive unilateral , bilateral , or sham rtms . according to this study , the remission rate was significantly higher in the bilateral group than the sham group , but the remission rate in the unilateral group did not differ from either group . these findings warrant larger controlled studies that compare the efficacy of sequential bilateral rtms and hfl / lfr rtms in md and trd.50,52 from a safety perspective , rtms can rarely induce accidental seizures , especially among patients with brain insult and on medications that reduce seizure threshold . however , this major side effect could be curtailed if expert guidelines are followed.53,54 over the past 10 years , a number of meta - analyses of rtms efficacy studies were conducted and the summary of these studies is as follows : a minimum of five to a maximum of 33 studies included ; almost all included studies except one focused on depression rather than trd ; rtms was more effective than sham rtms ; quality of studies improved successively ; and rtms designs also improved and effect size of rtms was comparable to antidepressant drugs.5560 finally , moreines and colleagues61 have reviewed the neuropsychological effects of somatic therapies including rtms that were associated with reversible mild reductions in sustained attention , spatial planning , and verbal retention . the fda approved the use of vns in patients with md and trd in 2005.62,63 vns principally stimulates the left cervical vagus nerve with a programmable neurostimulator . observations of mood elevation during vns for resistant epilepsy have suggested its potential role in trd.21,22,64 vns targets the nucleus tractus solitarius , frontolimbic network , the locus ceruleus , and dorsal raphe nucleus , which regulate mood . notably , initial studies on vns reported inconsistent findings regarding reduced metabolism and blood flow in targeted brain networks with no putative antidepressant mechanism.21,63,65 similarly , a multicenter study on vns found no significant reductions in depression scores for the experimental group as a whole , but antidepressant responses were observed among 40% of 30 recruited patients with trd.20 however , subsequent studies on vns reported positive results . in a naturalistic , 1-year , follow - up study of 30 trd patients who received vns , the results were as follows : response rate of 40%46% was sustained and the remission rate significantly increased , from 17% to 29% with an additional 9 months of long - term vns . it was concluded that long - term vns was associated with sustained benefit linked with good functional status.66 another naturalistic study with 2 years follow - up of 74 european patients with trd showed a significant reduction at all the three time points , ie , 3 , 12 , and 24 months of vns in the hdrs scores . after 2 years , 53.1% of the patients responded well , and 38.9% fulfilled the remission criteria . the proportion of patients with remission remained constant as the duration of vns increased , with no concomitant antidepressant medication significant impact . this 2-year open - label trial of vns suggested a clinical response and a benign adverse - effect profile among patients with trd.67 in a recent study of 15 consecutive outpatients with trd , vns significantly decreased beck depression inventory ( bdi ) scores compared to baseline at 6 and 12 months , from a mean of 37.8 7.8 before vns activation to a mean of 24.6 11.4 at 12 months . by 1 year , 28.6% of patients responded to vns and 7.1% remitted . hdrs showed similar improvement at 1 year , with a 43% response rate and 14.3% remission rate . reported side effects of vns in decreasing frequency were hoarseness , dyspnea , nausea , pain , and anxiety , and no patient terminated treatment due to side effects . according to this study , a substantial minority of patients with trd benefited from vns.68 vns but it has no adverse neuropsychological effects.61 in a study of single patients , vns produced good results , with cost savings over mect.69 according to a systematic review , vns examined in four clinical trials with 355 patients demonstrated steadily increasing improvement with full benefit after 612 months , sustained up to 2 years . but the primary results of the only controlled trial were negative and attributed to small sample size . further controlled studies with large sample size are warranted to establish its efficacy and tolerability in future.62,70 the issue of predictors of response to vns is addressed sparsely . in an open - label study of trd , the predictors of response to vns were history of resistant depression , mild to moderate resistant depression , not - severe resistant depression , and no history of use of ect.71 trials of vns in combination with pharmacotherapy are also needed in trd populations . transcranial direct current stimulation , a noninvasive technique with no fda approval , has been used in patients with md with mixed results . tdcs of the prefrontal cortex has been proposed as a therapeutic intervention in md.72,73 in a parallel - group , double - blind clinical trial , 40 patients with md who were medication - free were randomized into three groups . they were assessed by a blind rater using hdrs and bdi after ten sessions of tdcs during a 2-week period . according to this investigation , significantly larger reductions in depression scores after dlpfc tdcs moreover , the beneficial effects of tdcs in the dlpfc group persisted for 1 month after the end of treatment . the authors suggested further investigation on the effects of tdcs for the treatment of md.72 another double - blind , randomized study tested tdcs in 40 depressed participants and used the following parameters : 1-ma current strength , five treatment sessions , active or sham , and given on alternate days . anodal stimulation was centered over the left dlpfc , with the cathode placed on the lateral aspect of the contralateral orbit . overall , depression scores improved significantly over ten tdcs treatments , but there was no between - group difference in the five - session , sham - controlled phase . according to this study,73 tdcs was found to be safe , with no adverse effects on a variety of assessed neuropsychological functions.61 it was recommended that the efficacy of tdcs in md be further evaluated over a longer treatment period , using enhanced stimulation parameters.73 in another study , 22 patients with trd were randomly assigned to a crossover protocol comparing tdcs and placebo stimulation add - on to a stable antidepressant medication . the parameters of active tdcs were 1 or 2 ma for 20 minutes / day , anode over the left dlpfc , and cathode over the contralateral supraorbital region . the results showed that there was no significant difference in depression scores after 2 weeks of real compared with 2 weeks of sham tdcs . in contrast , subjective mood ratings showed an increase in positive emotions after real tdcs compared with sham tdcs . anodal tdcs , applied for 2 weeks , was not superior to placebo stimulation in patients with trd . the authors suggested that modified and improved tdcs protocols should be carried out in controlled trials to develop tdcs with better efficacy in trd.74 all aforementioned studies except one74 addressed the usefulness of tdcs in md , and hence more controlled trials are needed in trd patients . deep brain stimulation , yet to be approved by the fda , is a reversible invasive technique that involves stereotactical implantation of electrodes powered by a pulse generator into the specific dysfunctional brain regions implicated in mood disorders , parkinson s disease , alzheimer s disease , movement disorders , and other neuropsychiatric disorders . high frequency dbs of motor , mood , and cognitive neuronal circuits is reported to improve these conditions.75 dbs therapy , dose- and site - dependent , is a less invasive and less extreme alternative to ablative psychosurgeries.76 research data supports dbs that targets cortico - striatal - pallido - thalamocortical loop , the vc / vs , and other neuronal networks in patients with md , trd , ocd , and tourette s syndrome.7781 additionally , nac that contains dopamine , a reward system and involved in the pathogenesis of md , is a promising target for dbs . in a study , twelve months later , five patients reached 50% reduction of the hdrs score , with significantly increased pleasure activities . furthermore , the [ f]-2-fluoro-2-deoxy - d - glucose positron emission tomography data revealed that dbs decreased metabolism in the scg , orbital prefrontal cortex , and amygdala . this study supported antidepressant and antianhedonic effects of dbs in patients with trd . however , the small sample size limits the interpretation of results , and further research recruiting larger samples is needed.82 in a multicenter study of 21 trd patients who received dbs , it was found that patients treated with scg dbs had variable response with time : 57% at 1 month , 48% at 6 months , and 29% at 12 months . the response rate after 12 months of dbs increased to 62% when redefined as a reduction in the baseline hrsd of 40% or more . additionally , reductions in depressive symptoms were associated with amelioration in disease severity in patients who responded to surgery . overall , this study corroborated the results of other research that the outcome of scg dbs may be replicated across multiple centers.83 in two influential review articles , researchers have provided greater details of somatic treatments in terms of target structures , motivation , response rates , mechanism of action , and technical issues.8,9 accordingly , somatic therapies targeted scg , vc / vs , left cervical vagus nerve , r / l dlpfc , gpi , lateral habenula , and itp in md and trd patients , and improvement reported ranged from 30.6% to 66.7%.8,10,12,14 ( table 2 ) . furthermore , an improvement of 100% was reported in two dbs studies that included one patient with dystonia and trd and another patient with md and tardive dyskinesia.16,17 on a long - term basis ( 6 years ) , dbs is safe and effective in patients with trd , as substantiated by recent data.8789 according to these studies,8789 chronic dbs scg was effective in trd and bipolar patients and well tolerated with minor hemorrhagic events,86,90,91 but no neurocognitive impairment was reported61 ( table 3 ) . as a mechanism of action , overactive scg glucose metabolism seen in md is reduced with antidepressant therapies and dbs.10,11 magnetic seizure therapy , also known as magnetic convulsion therapy and yet to be approved by the fda , has antidepressant effects . studies conducted in humans and primates suggest that cognitive side effects of mst are more benign than those of mect . notably , postictal orientation recovery time is short and rapid with mst.61,92,93 furthermore , several studies have corroborated improved cognitive outcomes with mst as compared to mect . however , neither therapy causes structural changes , ie , volume , total number , or numerical density in neurons or glia in the frontal cortex , hippocampus , and their subregions in human and nonhuman brain.9496 overall , magnetic seizures with benign side - effect profile are therapeutically better than mect seizures . other than adverse neurocognitive effects , ect is also associated with reversible bradycardia and tachycardia immediate post - ect and ictal and postictal stages , respectively . in nonhuman studies of mst , these effects were minimal , reflecting a more superficial cortical site of action with less impact on deep brain structures , which are implicated in sympathetic and parasympathetic nervous system control , relative to ect.97 both antidepressant activity and cognitive side - effect profile of mst were further addressed in an open - label study , which tested whether it is associated with clinically significant antidepressant effects in trd as an add - on therapy to controlled pharmacotherapy.85 twenty patients with trd were randomly assigned to receive either mst or ect for more than 2 years . the primary outcome measure was antidepressant response assessed by madrs , and secondary outcome measures included hdrs , hamilton anxiety scale , bdi , and 90-item symptom checklist . antidepressant response as defined by 50% improvement in madrs ratings was statistically significant and of similar size in both treatment groups with no cognitive side effects . characteristics in mst- and ect - induced seizures were comparable , especially regarding ictal activity and postictal suppression . kayser and colleagues suggested that mst may be a potential alternative to ect if efficacy and safety are validated in larger clinical trials.85 mst is reported to result in minimal retrograde and anterograde amnesia.61 in summary , more studies are needed to further substantiate the efficacy of mst in mood disorder , including trd patients . notably , there is converging evidence that nts have a lower risk of neurocognitive side effects compared to mect , which are benign.61 ( table 4 ) . by and large , short- and long - term research is needed to establish the efficacy , safety , and cost - effectiveness of neurostimulation therapies.98 in addition , these therapies in general need proper selection of patients in line with tailored treatment guidelines.99 also , treatment teams should strictly follow ethical guidelines , especially those concerning autonomy , voluntary consent , beneficence , and nonmaleficence prior to using nts in individual patients.48,80,100 there are other nts , including ces and ecs , used uncommonly for a variety of disorders , such as anxiety , headaches , pain , stroke recovery , movement disorders , insomnia , and depression , but the data are largely limited in trd patients.101,102 in a systematic review , rosa and lisanby have described the technical details of all nts , including indications , safety , and effectiveness of ecs and ces.8 at the neurophysiological level , ces is quite different from tdcs.103 in one study , with ecs that used prefrontal cortical modulation , an average 55% improvement in depression scores was demonstrated.104 ces is associated with headache and nausea followed by skin irritation.105 unlike dbs , epidural cortical stimulation has fewer side effects.8 there are other neuromodulation therapies on the horizon , which include fus , lfms , and nir.106109 the data about these approaches are limited and need further research , especially concerning their role in mood disorders , including trd populations . with regard to os , microbial light - sensitive proteins called opsins are introduced into neurons and function as ion channels that open or close according to light exposure . channelrhodopsin-2 is one that allows na+ ions to enter the cell following exposure to ~470 nm blue light.110 according to rosa and lisanby,8 the advent of this technique has multiple implications : targeting specific fiber tracts that overlap in space ; selectively activating or inactivating specific projection neurons to the same target ; being a contactless form of stimulation relying on photoactivation ; and its potential use in treating mood disorders . like dbs , os will also require surgical implantation of the light - emitting electrode ; however , os certainly has other advantages over dbs.8 in one nonhuman study , antidepressant effects of os of medial prefrontal cortex have already been reported in a chronic social defeat stress model in rodents.111 more studies on newer nts are needed in human subjects with md and trd . this is a qualitative review of literature on somatic therapies used in the management of md and refractory depression . about 30% of patients with trd not responding to several intervention approaches , including optimization , augmentation and a combination of antidepressant drugs , are the principle candidates for nts.59 among these therapies , mect is most extensively and effectively used in severe depression and trd but associated with serious neurocognitive adverse effects because of nonspecific , broad excitation of cortical and deeper structures of the brain , and its mechanism of action is continuingly debatable.2633 other noninvasive somatic treatments such as rtms , tdcs , mst , and ces target more specific neuronal networks in the brain that are dysfunctional in md , trd , and other neuropsychiatric disorders , and reported to have fairly good safety and clinical profiles with more benign neuropsychological side effects.8,9,2325,4861,7274,85 invasive nts , ie , vns , dbs , and ecs with nonserious adverse effect profile , are also reported to be effective in patients with md and trd.810,1222,61,104 new nts on the horizon are also promising in patients with md and trd . although short- and long - term evidence - based comparative - effectiveness data on the role of nts in adult patients trd is emerging at a rapid pace,112 further research on their technical optimization , mechanisms of action , efficacy , side effect profile , and cost - effectiveness in larger populations of trd patients are warranted in future . there is converging evidence that up to 40% patients with md fail to respond to an initial antidepressant therapy . modified ect has a definite place in the management of patients with trd ; however , it carries well - known potential for neurocognitive impairment . like ect , mst also has neuropsychological adverse effects but of a milder nature . the role of other neuromodulation methods , including vns , rtms , dbs , and tdcs , in trd patients is expanding with greater efficacy and fewer side effects . these treatment modalities could be used alone or in combination with antidepressant therapy and/or psychotherapy . besides their therapeutic utility , neuromodulation techniques can further open windows into the biological basis of disordered neurocircuits related to md and trd . most studies on somatic therapies are of small sample size and hence reflect less reliable and valid results . therefore , collaborative , multisite and/or multicountry studies that use the same protocols and also recruit larger samples with trd are urgently needed . this methodological dilemma could be circumvented by determining a hypothesis a priori and others as exploratory . most importantly , trd evades a universally accepted definition , and hence tools to measure refractoriness of depression and strict eligibility criteria need to be developed . evidently , poor results of recent md and trd trials indicate the heterogeneous nature of depression and trd as well . therefore , treatment trials of somatic therapies should target more specific subpopulations together with the detection of endophenotypes to predict their response another challenge is blinding , which is vulnerable , and both the use of external raters and avoiding contact between subjects will solve this problem . additionally , open - label studies , especially of vns and dbs , tend to produce weak results , and therefore alternative designs including partial crossover and comparison against waiting list are needed . there is a relative lack of follow - up studies on somatic therapies , and hence more naturalistic studies are required in future . it is observed that the optimal parameters of somatic therapies are not defined , which could be managed by the use of adaptive designs and collaborative networks . finally , unlike nonpharmacologic research in adults with trd,112 there is a relative lack of direct comparison with antidepressant drugs , and hence comparative research is needed . most of these recommendations were constructed closely matching the challenges reported in the literature on nts , md , and trd populations.8,72,113

backgroundpatients with treatment - resistant depression ( trd ) who showed partial response to pharmacological and psychotherapeutic interventions need a trial of neuromodulation therapies ( nts).objectivethis paper aims to review evidence - based data on the use of nts in trd.methodusing keywords and combined - word strategy , multiple computer searches of pubmed , google scholar , quertle(r ) , and medline were conducted for retrieving relevant articles published in english - language peer - reviewed journals ( 20002012 ) . those papers that addressed nts in trd were retained for extensive review.resultsdespite methodological challenges , a range of 30%93% of trd patients showed substantial improvement to one of the nts . one hundred percent improvement was reported in two single - case studies on deep brain stimulation . some studies reported no benefits from transcranial direct current stimulation . nts were reported to have good clinical efficacy , better safety margin , and benign side - effect profile . data are limited regarding randomized clinical trials , long - term efficacy , and cost - effectiveness of these approaches . both modified electroconvulsive therapy and magnetic seizure therapy were associated with reversible but disturbing neurocognitive adverse effects . besides clinical utility , nts including approaches on the horizon may unlock the biological basis underlying mood disorders including trd.conclusionnts are promising in patients with trd , as the majority of them show good clinical response measured by standardized depression scales . nts need further technological refinements and optimization together with continuing well - designed studies that recruit larger numbers of participants with trd .

Introduction Search method Categorization of NTs Mechanisms of action of NTs Electroconvulsive therapy Repetitive transcranial magnetic stimulation Vagus nerve stimulation Transcranial direct current stimulation Deep brain stimulation Magnetic seizure therapy Newer neurostimulation therapies Discussion Conclusion Recommendations

at its worst , md can lead to suicide , and as a consequence about 850,000 lives are lost every year.2 treatment - resistant depression ( trd ) evades universal definition ; however , a poor response to two adequate ( optimal dosage and 612 weeks duration ) trials of two different classes of antidepressants has been proposed as its operational characterization.3 researchers have categorized trd in accordance to antidepressant trials : stage 0 , has not had a single adequate trial of medication ; stage 1 , failure of an adequate trial of one class of an antidepressant that is monotherapy ; stage 2 , failure of adequate trials of two distinctly different classes that is , selective serotonin reuptake inhibitors ( ssris ) and tricyclic antidepressants of antidepressant , involving two monotherapy trials ; stage 3 , stage 2 plus failure to respond to one augmentation strategy of lithium or thyroid augmentation of one of the monotherapies ; stage 4 , stage 3 plus a failure to a second augmentation strategy in terms of monoamine oxidase inhibitors ; and stage 5 , stage 4 plus failure of an adequate course of ect.4 there are other staging methods of trd.5 these staging methods help researchers and clinicians to understand trd patients and accordingly plan interventions for enhancing the response , remission rate , and quality of life . notably , currently there is an increasing interest in the utilization of several neuromodulation therapies ( nts ) in the management of patients with trd.6 this is because psychopharmacological therapy exposes the entire body to a potentially therapeutic substance in order to treat a relatively small region of the brain , whereas nts are designed to target specific brain circuits that are important in the pathogenesis of md . additionally , nts are not systemic and , therefore , the side - effect profile is limited and different from medications , and there are minimal , if any , drug interactions.7 furthermore , evidence - based data has been emerging continuously about fda - approved and yet - to - be - approved nts in the trd population over the past decade . multiple computer searches were conducted using pubmed , google scholar , quertle(r ) , and medline databases for the years 20002012 . a number of keywords were used : treatment - resistant depression , treatment - refractory depression , partial - response depression , nonresponse depression , neuromodulation techniques , neurostimulation approaches , and somatic therapies . these words were combined with modified electroconvulsive therapy ( mect ) , repetitive transcranial magnetic stimulation ( rtms ) , vagus nerve stimulation ( vns ) , magnetic seizure therapy ( mst ) , deep brain stimulation ( dbs ) , transcranial direct current stimulation , cranial electric stimulation ( ces ) , epidural cortical stimulation ( ecs ) , focused ultrasound ( fus ) , near - infrared light therapy ( nir ) , low - field magnetic stimulation ( lfms ) , and optogenetic stimulation ( os ) for a second round of computer searches . as a corollary , relevant articles published in english - language peer - reviewed journals were retrieved . only clinical trials , systematic reviews , and meta - analyses that addressed trd and nts were retained for extensive review and inclusion in this study . some exceptions were made with regard to some unique case reports , open and controlled studies , and small and large case series describing usefulness of nts in patients with trd and md . nts for neuropsychiatric disorders including md are categorized into the following : ( 1 ) seizure therapies , including mect and mst , ( 2 ) noninvasive therapies , including rtms , tdcs , and ces , ( 3 ) neurosurgical approaches , including vns , ecs , and dbs , and ( 4 ) new approaches on the horizon , including fus , nir , lfms , and os.8 another category represents neurosurgical ablation therapies , including cingulotomy and limbic leucotomy used in trd . in several related studies , overactive subcallosal cingulate gyrus ( scg ) glucose metabolism has been reported in md that is reduced with successful antidepressant therapies.10 interestingly , dbs is reported to modulate neural pathways linked with scg in relieving md.8,11,12 according to some studies , antidepressant effects were also found when dbs targeted ventral capsule / ventral striatum ( vc / vs ) in patients with severe obsessive - compulsive disorder ( ocd ) and md.13,14 in a study of single patients with dystonia suffering from depression , dbs of globus pallidus internus ( gpi ) showed improvement in dystonia but also showed antidepressant effects through modulation of mesolimbic dopamine pathways.15 in another study , also of single patients with tardive dyskinesia ( td ) and md , dbs brought about improvement in depressive mood.16 other studies have also reported improvement in both depression and td after dbs of the inferior thalamic peduncle ( itp ) , which modulates orbitofrontal cortex hyperactivity.17,18 bewernick and colleagues reported that dbs of the nucleus accumbens ( nac ) was associated with decreased ratings of depression and anxiety in trd patients.19 rush and colleagues20 noticed antidepressant effects when vns was used for epilepsy . vns modulates neural pathways associated with mood regulation : the nucleus tractus solitaries , raphe nucleus , and locus ceruleus.21 in fact , the vns device stimulates left cervical vagus nerve containing afferent neurons tracking through the brain stem to cortical and subcortical networks.2023 furthermore , some neurobiological studies reported disruptions in right and left dorsolateral prefrontal cortex ( r / ldlpfc ) in mood disorders . it requires light anesthesia and is a recognized mode of treatment for trd.31,32 it remains the most effective therapy in trd patients with a response rate of 50%70% , though the strength of recommendation of ect is c.33,34 it targets nonspecific , broad regions of the cortex , and its mechanism of action is elusive . in a study of patients with nonpsychotic md that tested whether pre - ect medication resistance is associated with post - ect relapse rates , it was observed that 34.6% of nonmedication - resistant patients who were not exposed to at least one antidepressant medication trial relapsed , while 50.0% of medication - resistant patients relapsed , a difference that was not statistically significant but clinically relevant.35 furthermore , in the first week after acute remission , 9.8% of patients not having at least one antidepressant medication trial met relapse criteria , while 31.4% of medication - resistant patients met relapse criteria , a difference that was statistically significant . in a large sample of patients with trd , mect was effective in 66% of patients . mect nonresponse was associated with bipolar subtype , mixed features , slightly less severe depressive symptoms , and longer duration of the depressive episode.36 in another study that aimed to investigate whether the clinical course of trd patients following a course of mect might be associated with changes of plasma brain - derived neurotrophic factor ( bdnf ) concentrations , it was shown that at baseline , plasma bdnf levels of patients were significantly lower than those of control subjects , and those after ect were significantly increased in parallel with the decrease of the hamilton depression rating scale ( hdrs ) total score . these findings suggested the potential usefulness of baseline plasma bdnf levels as predictors of response to mect in trd patients.37 in an earlier study of 18 patients with trd , levels of bdnf and 3-methoxy-4-hydroxyphenylglycol but not homovanillic acid were increased following mect in responders , which suggested that dopamine and bdnf might be involved in the mechanism of action of mect.38 in a recent study of adolescents with trd , both continuation and maintenance of mect were useful and safe for selected adolescents with severe trd , and symptom remission was achieved without experiencing cognitive impairment;39 the latter is a surprising finding and needs replication studies . the implication of this finding is that the symptoms of md might be alleviated rapidly if ketamine anesthesia is used in trd patients during ect.40 a retrospective evaluation of 5482 ect treatments in 455 patients with trd found therapeutic advantages in combination therapies versus ect . baghai and colleagues suggested that controlled studies are necessary to investigate further the possible advantages of ect and pharmacotherapy combinations , especially the use of modern dual - acting antidepressants , which also have proven their efficacy in trd.41 although mect is effective in trd , it significantly produces transient confusion , anterograde amnesia , and retrograde amnesia . furthermore , hfr rtms is preferred over lfs sychronized tms , as the former was associated with more antidepressant effects in depressed patients as reflected by significant increases in blood supply to prefrontal cortical and limbic regions.23 a sequential bilateral rtms ( lf right [ lfr ] then hf left [ hfl ] ) is also effective in trd patients but not more effective than unilateral hfl rtms.49,50 in an open - label study , 21 patients who failed two antidepressant trials were given rtms ( hf , 10 hz and intensity of 110% ) for 4 weeks , keeping the dose of preexisting antidepressants unchanged . it was noted that sequentially applying both hfl rtms and lfr rtms to the right prefrontal cortex resulted in substantial improvement in patients with trd . the authors concluded that rtms to ldlpfc can produce statistically and clinically significant antidepressant effects in patients with trd.25 in another study , subjects between the ages of 18 and 85 years were recruited from a tertiary care university hospital . however , this major side effect could be curtailed if expert guidelines are followed.53,54 over the past 10 years , a number of meta - analyses of rtms efficacy studies were conducted and the summary of these studies is as follows : a minimum of five to a maximum of 33 studies included ; almost all included studies except one focused on depression rather than trd ; rtms was more effective than sham rtms ; quality of studies improved successively ; and rtms designs also improved and effect size of rtms was comparable to antidepressant drugs.5560 finally , moreines and colleagues61 have reviewed the neuropsychological effects of somatic therapies including rtms that were associated with reversible mild reductions in sustained attention , spatial planning , and verbal retention . the fda approved the use of vns in patients with md and trd in 2005.62,63 vns principally stimulates the left cervical vagus nerve with a programmable neurostimulator . notably , initial studies on vns reported inconsistent findings regarding reduced metabolism and blood flow in targeted brain networks with no putative antidepressant mechanism.21,63,65 similarly , a multicenter study on vns found no significant reductions in depression scores for the experimental group as a whole , but antidepressant responses were observed among 40% of 30 recruited patients with trd.20 however , subsequent studies on vns reported positive results . it was concluded that long - term vns was associated with sustained benefit linked with good functional status.66 another naturalistic study with 2 years follow - up of 74 european patients with trd showed a significant reduction at all the three time points , ie , 3 , 12 , and 24 months of vns in the hdrs scores . this 2-year open - label trial of vns suggested a clinical response and a benign adverse - effect profile among patients with trd.67 in a recent study of 15 consecutive outpatients with trd , vns significantly decreased beck depression inventory ( bdi ) scores compared to baseline at 6 and 12 months , from a mean of 37.8 7.8 before vns activation to a mean of 24.6 11.4 at 12 months . according to this study , a substantial minority of patients with trd benefited from vns.68 vns but it has no adverse neuropsychological effects.61 in a study of single patients , vns produced good results , with cost savings over mect.69 according to a systematic review , vns examined in four clinical trials with 355 patients demonstrated steadily increasing improvement with full benefit after 612 months , sustained up to 2 years . in an open - label study of trd , the predictors of response to vns were history of resistant depression , mild to moderate resistant depression , not - severe resistant depression , and no history of use of ect.71 trials of vns in combination with pharmacotherapy are also needed in trd populations . transcranial direct current stimulation , a noninvasive technique with no fda approval , has been used in patients with md with mixed results . according to this study,73 tdcs was found to be safe , with no adverse effects on a variety of assessed neuropsychological functions.61 it was recommended that the efficacy of tdcs in md be further evaluated over a longer treatment period , using enhanced stimulation parameters.73 in another study , 22 patients with trd were randomly assigned to a crossover protocol comparing tdcs and placebo stimulation add - on to a stable antidepressant medication . deep brain stimulation , yet to be approved by the fda , is a reversible invasive technique that involves stereotactical implantation of electrodes powered by a pulse generator into the specific dysfunctional brain regions implicated in mood disorders , parkinson s disease , alzheimer s disease , movement disorders , and other neuropsychiatric disorders . high frequency dbs of motor , mood , and cognitive neuronal circuits is reported to improve these conditions.75 dbs therapy , dose- and site - dependent , is a less invasive and less extreme alternative to ablative psychosurgeries.76 research data supports dbs that targets cortico - striatal - pallido - thalamocortical loop , the vc / vs , and other neuronal networks in patients with md , trd , ocd , and tourette s syndrome.7781 additionally , nac that contains dopamine , a reward system and involved in the pathogenesis of md , is a promising target for dbs . overall , this study corroborated the results of other research that the outcome of scg dbs may be replicated across multiple centers.83 in two influential review articles , researchers have provided greater details of somatic treatments in terms of target structures , motivation , response rates , mechanism of action , and technical issues.8,9 accordingly , somatic therapies targeted scg , vc / vs , left cervical vagus nerve , r / l dlpfc , gpi , lateral habenula , and itp in md and trd patients , and improvement reported ranged from 30.6% to 66.7%.8,10,12,14 ( table 2 ) . furthermore , an improvement of 100% was reported in two dbs studies that included one patient with dystonia and trd and another patient with md and tardive dyskinesia.16,17 on a long - term basis ( 6 years ) , dbs is safe and effective in patients with trd , as substantiated by recent data.8789 according to these studies,8789 chronic dbs scg was effective in trd and bipolar patients and well tolerated with minor hemorrhagic events,86,90,91 but no neurocognitive impairment was reported61 ( table 3 ) . however , neither therapy causes structural changes , ie , volume , total number , or numerical density in neurons or glia in the frontal cortex , hippocampus , and their subregions in human and nonhuman brain.9496 overall , magnetic seizures with benign side - effect profile are therapeutically better than mect seizures . in nonhuman studies of mst , these effects were minimal , reflecting a more superficial cortical site of action with less impact on deep brain structures , which are implicated in sympathetic and parasympathetic nervous system control , relative to ect.97 both antidepressant activity and cognitive side - effect profile of mst were further addressed in an open - label study , which tested whether it is associated with clinically significant antidepressant effects in trd as an add - on therapy to controlled pharmacotherapy.85 twenty patients with trd were randomly assigned to receive either mst or ect for more than 2 years . kayser and colleagues suggested that mst may be a potential alternative to ect if efficacy and safety are validated in larger clinical trials.85 mst is reported to result in minimal retrograde and anterograde amnesia.61 in summary , more studies are needed to further substantiate the efficacy of mst in mood disorder , including trd patients . by and large , short- and long - term research is needed to establish the efficacy , safety , and cost - effectiveness of neurostimulation therapies.98 in addition , these therapies in general need proper selection of patients in line with tailored treatment guidelines.99 also , treatment teams should strictly follow ethical guidelines , especially those concerning autonomy , voluntary consent , beneficence , and nonmaleficence prior to using nts in individual patients.48,80,100 there are other nts , including ces and ecs , used uncommonly for a variety of disorders , such as anxiety , headaches , pain , stroke recovery , movement disorders , insomnia , and depression , but the data are largely limited in trd patients.101,102 in a systematic review , rosa and lisanby have described the technical details of all nts , including indications , safety , and effectiveness of ecs and ces.8 at the neurophysiological level , ces is quite different from tdcs.103 in one study , with ecs that used prefrontal cortical modulation , an average 55% improvement in depression scores was demonstrated.104 ces is associated with headache and nausea followed by skin irritation.105 unlike dbs , epidural cortical stimulation has fewer side effects.8 there are other neuromodulation therapies on the horizon , which include fus , lfms , and nir.106109 the data about these approaches are limited and need further research , especially concerning their role in mood disorders , including trd populations . about 30% of patients with trd not responding to several intervention approaches , including optimization , augmentation and a combination of antidepressant drugs , are the principle candidates for nts.59 among these therapies , mect is most extensively and effectively used in severe depression and trd but associated with serious neurocognitive adverse effects because of nonspecific , broad excitation of cortical and deeper structures of the brain , and its mechanism of action is continuingly debatable.2633 other noninvasive somatic treatments such as rtms , tdcs , mst , and ces target more specific neuronal networks in the brain that are dysfunctional in md , trd , and other neuropsychiatric disorders , and reported to have fairly good safety and clinical profiles with more benign neuropsychological side effects.8,9,2325,4861,7274,85 invasive nts , ie , vns , dbs , and ecs with nonserious adverse effect profile , are also reported to be effective in patients with md and trd.810,1222,61,104 new nts on the horizon are also promising in patients with md and trd . although short- and long - term evidence - based comparative - effectiveness data on the role of nts in adult patients trd is emerging at a rapid pace,112 further research on their technical optimization , mechanisms of action , efficacy , side effect profile , and cost - effectiveness in larger populations of trd patients are warranted in future . therefore , treatment trials of somatic therapies should target more specific subpopulations together with the detection of endophenotypes to predict their response another challenge is blinding , which is vulnerable , and both the use of external raters and avoiding contact between subjects will solve this problem .

several recent studies have demonstrated that some genes or groups of genes can show variation in copy number , and that this variation can have important functional consequences ( 16 ) . for example , the genes ccl3l1 , ccl4l1 and tbc1d3 are present on a segmental duplication that can vary between 0 and 10 copies per person ( 7 ) ; this variation appears to be a determinant of individual susceptibility to , and progression of , infection with hiv-1 ( 8) . similarly , a group of beta - defensin genes including defb4 commonly varies between two and seven copies per person , with occasional extremely expanded alleles containing 811 repeats visible as these beta - defensin genes , as well as the independently variable alpha - defensins defa1a3 ( 1012 ) , are candidate genes for variation in susceptibility to infectious disease , as well as autoimmune and inflammatory disorders ( 10 ) , and low copy number of the defb4 segmental duplication has been associated with crohn 's disease of the colon ( 13 ) . where frequent copy number variation encompasses the 03 copy number range , many established technologies can be used to provide rapid , cheap and accurate measurement of dna copy number . in contrast , where the copy number range is higher , such as for ccl3l1 or the defb4 cluster , accurately distinguishing a count of five copies from six requires a precision not available from easily implemented methods . for example , combining maph with determination of restriction enzyme digest variant ratios ( maph / redvr ) has been capable of high reproducibility in determining copy number at defb4 ( 10 ) and defa1a3 ( 11 ) , but uses large amounts of genomic dna ( > 1 g ) and is labour - intensive . real - time pcr is gaining in popularity as a method of determining copy number ( 8,13 ) , but requires careful set - up and does not easily provide the highest throughput required for large association studies . an ideal high - throughput method for measuring copy number in large - scale association studies would be accurate , inexpensive , robust and use only small amounts of genomic dna , and the pressing need for methods with these properties is increasingly recognized ( 14 ) . in assessing pathological deletions or duplications of single - copy genes , relatively simple multiplex fluorescent pcr methods ( 1517 ) multiplex fluorescent pcr methods compare the amount of pcr product made from a test amplicon with the yield from a reference locus in the same multiplex reaction . the experimental variability of multiplex pcr is presumably due to the different amplification properties of the test and reference loci , and the differential sensitivity of the yield of each amplification reaction to the precise conditions . to obtain reliable results in multiplex pcr , great care needs to be taken with a number of experimental factors , including dna quality and ( as far as possible ) matching the amplification properties of test and reference amplicons . some of this experimental variation has been reduced by the design of short amplicons , combined with careful attention to primer design and pcr conditions , in the qmpsf technology ( 15,16 ) . in this study we have adapted the quantitative multiplex pcr approach by using primers designed to amplify from repeated dna elements . the primers are precisely designed to amplify from a copy of the element within the variable repeat unit , plus exactly one other unlinked reference locus . we have applied this method to the copy - variable defb4 repeat unit , and compare the results from this approach with results on the same samples from three independent alternative methods . this new prt method is comparable in accuracy to these alternative methods , and its simple format , and requirement for only small ( 1020 ng ) amounts of genomic dna , should allow accurate , inexpensive and rapid copy number typing of large cohorts of samples in association studies . genomic dna from dutch and hapmap samples was used at concentrations of 510 ng/l , and for most experiments samples were arrayed in 96-well microtitre plates and processed in batches of 96 . mlpa was carried out as described ( 19 ) using 250 ng genomic dna and the salsa mlpa kit p139 defensin from mrc - holland , and data were analyzed as described ( 20 ) . details on methods used to collect the data , downloading from the website and clones used are also available on the site . pcr was carried out using 5 ng input genomic dna , 0.5 m primer hspd5.8f ( ccagatgagaccagtgtcc ) and 0.5 m fam- or hex - labelled primer hspd5.8r ( ttttaagttcagcaattacagc ) ( figure 1 ) , in a buffer [ modified from ( 21 ) ] containing final concentrations of 50 mm tris hcl ( ph 8.8 ) , 12 mm ammonium sulphate , 5 mm magnesium chloride , 125 g / ml bsa ( non - acetylated , ambion inc ) , 7.4 mm 2-mercaptoethanol and 1.1 mm each dntp ( sodium salts ) , with 0.5u taq dna polymerase in a total volume of 10 l . products were amplified using 30 cycles of 95c for 30 s , 53c for 30 s and 70c for 30 s , followed by a single chase phase of 53c for 1 min/70c for 20 min to enhance complete extension in the final round and hence reduce levels of single - stranded dna products . the cycle number of 30 was chosen after empirical tests to determine conditions that yielded quantifiable ( i.e. not saturating ) amounts of product ; the annealing temperature was also chosen after empirical comparisons , and the choice of 53c is near the maximum annealing temperature that can be used , probably associated with a reduction in the efficiency of amplification . the top line shows the general structure of the repeat unit containing defb4 ( which has two inverted rather than tandem repeats in the march 2006 assembly ) . defb4 and defb103107 , together with the locations of probes or amplicons used in maph , redvr , mlpa and prt , and the extent of the clone ( 10c3 ) used in array - cgh ( ' wt whole genome acgh ' ) . in the detailed display at the bottom , the primers amplify products from the hspdp3 pseudogene upstream of defb4 on chromosome 8 , and from a reference copy on chromosome 5 , but have multiple mismatches ( bold ) with other copies of the element . in this way a single primer pair can be used to amplify two very similar products , one from near defb4 , the other from chromosome 5 . two amplifications were carried out for each sample , one with a fluorescent fam label , the other with a fluorescent hex label ; 1 l of each pcr product was added , without further purification , to a 10 l digestion containing 1 react 2 buffer [ 50 mm tris hcl ( ph8.0 ) , 10 mm mgcl2 , 50 mm nacl ] ( invitrogen ) and 5u haeiii ( new england biolabs ) . after incubation at 37c for 416 h , 2 l was added to 10 l hidi formamide with rox-500 marker ( applied biosystems ) , and analyzed by electrophoresis on an abi3100 36 cm capillary using pop4 polymer and an injection time of 30 s. peak areas corresponding to the 302 bp haeiii fragment from near defb4 and the 315 bp fragment from chromosome 5 were recorded for both fam- and hex - labelled products using genescan and genotyper software ( applied biosystems ) . the ratio 302/315 bp was compared between fam- and hex - labelled products , and results were accepted if the difference between the ratios was < 15% of their mean ; this criterion led to the rejection of 10% of tests ( figure 2 ) . comparison of ratios ( chromosome 8 : chromosome 5 ) from fam- and hex - labelled products in a single experiment . pairs of ratios ( triangles ) meeting the quality - control criteria ( 73 samples ) cluster around groups corresponding to copy numbers of 2 , 3 , 4 , 5 and 6 . crosses show results rejected for having too great a difference between fam and hex ratios ( 10 samples ) . if accepted , the mean of the ratios of the fam- and hex - labelled products was used in further analysis . although nave inference of a copy number equal to double the mean ratio would lead to reasonably accurate results , there were small but definite shifts between experiments in the relative amplification of the test and reference products . mean ratios were therefore used in conjunction with reference standards to calibrate each experiment , and the resulting ( least - squares ) linear regression used to infer the copy numbers for unknown samples . in most experiments maph / redvr copy numbers were used to calibrate prt assays ; subsequently , dna samples giving reproducible results from several prt assays have also been successfully used as calibration standards . genomic dna from dutch and hapmap samples was used at concentrations of 510 ng/l , and for most experiments samples were arrayed in 96-well microtitre plates and processed in batches of 96 . mlpa was carried out as described ( 19 ) using 250 ng genomic dna and the salsa mlpa kit p139 defensin from mrc - holland , and data were analyzed as described ( 20 ) . details on methods used to collect the data , downloading from the website and clones used are also available on the site . pcr was carried out using 5 ng input genomic dna , 0.5 m primer hspd5.8f ( ccagatgagaccagtgtcc ) and 0.5 m fam- or hex - labelled primer hspd5.8r ( ttttaagttcagcaattacagc ) ( figure 1 ) , in a buffer [ modified from ( 21 ) ] containing final concentrations of 50 mm tris hcl ( ph 8.8 ) , 12 mm ammonium sulphate , 5 mm magnesium chloride , 125 g / ml bsa ( non - acetylated , ambion inc ) , 7.4 mm 2-mercaptoethanol and 1.1 mm each dntp ( sodium salts ) , with 0.5u taq dna polymerase in a total volume of 10 l . products were amplified using 30 cycles of 95c for 30 s , 53c for 30 s and 70c for 30 s , followed by a single chase phase of 53c for 1 min/70c for 20 min to enhance complete extension in the final round and hence reduce levels of single - stranded dna products . the cycle number of 30 was chosen after empirical tests to determine conditions that yielded quantifiable ( i.e. not saturating ) amounts of product ; the annealing temperature was also chosen after empirical comparisons , and the choice of 53c is near the maximum annealing temperature that can be used , probably associated with a reduction in the efficiency of amplification . the top line shows the general structure of the repeat unit containing defb4 ( which has two inverted rather than tandem repeats in the march 2006 assembly ) . defb4 and defb103107 , together with the locations of probes or amplicons used in maph , redvr , mlpa and prt , and the extent of the clone ( 10c3 ) used in array - cgh ( ' wt whole genome acgh ' ) . in the detailed display at the bottom , the primers amplify products from the hspdp3 pseudogene upstream of defb4 on chromosome 8 , and from a reference copy on chromosome 5 , but have multiple mismatches ( bold ) with other copies of the element . in this way a single primer pair can be used to amplify two very similar products , one from near defb4 , the other from chromosome 5 . two amplifications were carried out for each sample , one with a fluorescent fam label , the other with a fluorescent hex label ; 1 l of each pcr product was added , without further purification , to a 10 l digestion containing 1 react 2 buffer [ 50 mm tris hcl ( ph8.0 ) , 10 mm mgcl2 , 50 mm nacl ] ( invitrogen ) and 5u haeiii ( new england biolabs ) . after incubation at 37c for 416 h , 2 l was added to 10 l hidi formamide with rox-500 marker ( applied biosystems ) , and analyzed by electrophoresis on an abi3100 36 cm capillary using pop4 polymer and an injection time of 30 s. peak areas corresponding to the 302 bp haeiii fragment from near defb4 and the 315 bp fragment from chromosome 5 were recorded for both fam- and hex - labelled products using genescan and genotyper software ( applied biosystems ) . the ratio 302/315 bp was compared between fam- and hex - labelled products , and results were accepted if the difference between the ratios was < 15% of their mean ; this criterion led to the rejection of 10% of tests ( figure 2 ) . comparison of ratios ( chromosome 8 : chromosome 5 ) from fam- and hex - labelled products in a single experiment . pairs of ratios ( triangles ) meeting the quality - control criteria ( 73 samples ) cluster around groups corresponding to copy numbers of 2 , 3 , 4 , 5 and 6 . crosses show results rejected for having too great a difference between fam and hex ratios ( 10 samples ) . if accepted , the mean of the ratios of the fam- and hex - labelled products was used in further analysis . although nave inference of a copy number equal to double the mean ratio would lead to reasonably accurate results , there were small but definite shifts between experiments in the relative amplification of the test and reference products . mean ratios were therefore used in conjunction with reference standards to calibrate each experiment , and the resulting ( least - squares ) linear regression used to infer the copy numbers for unknown samples . in most experiments maph / redvr copy numbers were used to calibrate prt assays ; subsequently , dna samples giving reproducible results from several prt assays have also been successfully used as calibration standards . we reasoned that many of the problems of accuracy and reproducibility associated with multiplex pcr might be avoided if the test and reference amplicons were as similar as possible . this principle has recently been successfully exploited in an innovative approach to the diagnosis of trisomy ; because some sequences present on chromosome 21 ( for example ) have nearly identical paralogues at another site in the genome , a single pair of primers can be used to amplify both test and reference loci , distinguishing them via minor differences of internal sequence ( 22 ) . although copy - variable loci are very unlikely to contain extensive regions with nearly identical counterparts at other locations , we were able to exploit the same advantages of amplifying both test and reference loci with a single primer pair by designing precisely placed primers in a diverged ( low copy number ) repetitive sequence , thereby allowing a paralogue ratio test ( prt ) . a heat - shock protein pseudogene of 2 kb ( hspdp3 ) is found 2 kb upstream of the defb4 gene ( figure 1 ) , and at 10 locations elsewhere in the genome . we were able to design primers that matched the copy near defb4 and one other copy on chromosome 5 exactly , but which had multiple mismatches to copies at other chromosomal locations . the ( test ) chromosome 8 ( defb4 ) and ( reference ) chromosome 5 copies give pcr products too close in size ( 443 and 447 bp respectively ) to separate reliably by capillary electrophoresis , but they could be easily distinguished and quantified after digestion with the restriction enzyme haeiii to give products of 302 and 315 bp . because products from other copies of the pseudogene were predicted to have characteristic alternative fragment lengths following haeiii digestion , the absence of detectable fragments of these lengths confirmed that under the conditions used , the products detected came exclusively from the chromosome 5 and chromosome 8 loci . measuring the ratios of products from the test and reference loci ( see materials and methods ) allowed inference of defb4 copy number , which was linearly related to product ratio . in the absence of gold standard , error - free methods to count defb4 copy numbers for reference samples , we validated the method by comparisons with results from three other established techniques . prt was used to type 591 genomic dna samples which had already been typed for defb4 copy number by the maph / redvr method . the maph / redvr approach uses a primary copy number estimate by maph ( 23 ) , refined by examining ratios of sequence variants from the repeat unit ( 10,11 ) . for 486 samples ( 82% ) , a single prt assay gave the same integer copy number as maph / redvr ; for a further 64 samples ( 11% ) , the maph / redvr copy number was confirmed by prt on repeat testing . thus for 93% of samples , the maph / redvr value was confirmed by prt either on first - pass testing or after a single repeat test . for 25 samples ( 4.2% ) prt consistently gave a value different from maph / redvr , and for these samples maph / redvr was assumed to have been in error . a subset of 135 of these samples was also typed by mlpa ( 19 ) , so that this smaller sample set was typed by three independent methods : maph / redvr , prt and mlpa . for samples which had undergone more than one prt assay , these three methods appear to be comparable in their accuracy ; all three methods agreed on the integer copy number for 113 out of 135 samples ( 84% ) , and of the 22 remaining samples , the result from one method disagreed with the other two first - pass prt gave the discordant result for seven samples , maph / redvr for 6 samples , and mlpa for nine samples . from the 135 mlpa results , 119 ( 88% ) agreed with the integral copy number measured by a single prt assay . comparisons between unrounded mlpa copy number estimates and unrounded copy number values from first - pass prt are shown in figure 3 . as expected , clusters of values corresponding to integral copy numbers are seen , and the spread of measured values is higher at higher copy number . comparison between unrounded copy number estimates from prt , with unrounded data from mlpa for the same 135 samples . we estimated rates of error for single - pass prt typing from a larger data set , combining results from maph / redvr and prt data , plus the mlpa results for the subset of 135 samples , to define a consensus copy number for all 575 samples ( after removing the 16 samples for which no consistent data were obtained ) . there were two striking patterns in the distribution of error for single - pass prt typing . first , the highest copy numbers had the greatest error , as expected for a method that relies on ratios of measurements . estimated error rates were < 10% for copy numbers of 2 , 3 and 4 , but rose to 20% or higher for samples with copy numbers of six or more ( figure 4a ) . second , unrounded prt values close to an integer value were more likely to give the correct integer value ; values within 0.2 of an integer had estimated error rates < 10% , but if the unrounded value was as much as 0.4 from an integer value , the error rate was > 30% ( figure 4b ) . overall , the analyses suggested that a single prt test yielded the correct integral value in 89% of samples ( 511 out of 575 ) , a figure that can be further improved by confirmatory and repeat testing as recommended below . correlation between error attributable to first - pass prt and ( a ) copy number or ( b ) absolute difference between the unrounded and integer values measured ( residual ) . in each case shaded columns show the relative frequencies of each copy number or residual category in the data set . as a further comparison of prt with established methodology , these data were then compared with array - cgh data from a clone ( 10c3 ) within the defb4 repeat unit . figure 5 shows the comparison between unrounded prt results and normalized signal from array - cgh of clone 10c3 , demonstrating clusters of data - points corresponding to integral copy numbers of 2 , 3 , 4 and 5 , with a greater spread at copy numbers of 6 and 7 . integral copy numbers from array - cgh data were assigned to these samples based on ranges defined by the clusters , and agreed with the prt - derived integer copy number for 202 samples ( 77% ) . this relatively low figure combines error attributable to prt with an unknown but presumably higher rate of error from array - cgh . analysis of flanking snp haplotypes in the hapmap samples showed very weak evidence for association with flanking haplotypes , and no snps or haplotypes were found that could reliably be used as a surrogate for copy number typing . comparison between first - pass prt typing ( unrounded values ) and normalized signal from a clone from the defb4 region in array - cgh for 261 samples from the hapmap study . finally , a total of 99 samples underwent duplicate testing by prt without any prior selection for concordance or disagreement with expected values , and 83 ( 83.8% ) gave the same integer values on both tests . because the frequency of samples with discordant duplicate tests is expected to be approximately twice the rate of error for a single test , this suggests that the error rate for integer values from a single prt test is 8.1% ( 95% confidence interval 5.611.8% ) . all these analyses together suggest that a single prt test would on its own return the correct integer value in 8595% of tests . in typing large numbers of samples for case - control association studies using any error - prone measurement or genotyping method , a compromise has to be made between throughput and the cost and replication work required to obtain accurate data . the accuracy of prt testing for copy number could clearly be improved by requiring concordant duplicate testing of all samples , but for many large - scale studies such a doubling of the genotyping cost and effort may not be justified . the patterns discovered in our analyses suggest that a low error rate can be achieved with selective re - testing of samples . for example , if re - testing was triggered by integral copy numbers of five or more with unrounded prt values > 0.3 from the nearest integer , then the data predict that only 15% of samples would require re - testing , and of the 85% of results accepted by these criteria , 92% would return the correct integral value . depending on the balance required between throughput , cost , labour and accuracy , alternative criteria for acceptance or re - testing can be formulated . in the presence of somatic mosaicism , the true copy number in a dna sample may not be an integer , but instead an intermediate value reflecting the mean copy number of the cell population . in such a case , if the correct measure of copy number is frequently non - integral , it is predicted that multiple measurements of the same samples ( typed by several methods , or in duplicate by a single method ) should have a tendency to cluster around non - integer values . in our data set , common mosaicism should therefore be detectable as a significant correlation between residuals ( i.e. the unrounded measurement minus the rounded integer value ) between duplicate prt tests or between different methods . no such correlation was detected in our data ( not shown ) , suggesting that determination of a whole number of repeats is the biologically relevant measurement result , and in turn that differences between the measured values and integer values are the results of measurement error . the analysis assumes that the reference locus ( in this case on chromosome 5 ) does not itself vary in copy number , and that substitutional mismatches at either the test or reference locus primer binding sites do not lead to ` drop - out ' of one or more copies . in addition to their absence from all available sequence databases and sequence trace archives , substitutional variants do not appear to pose a problem at the chromosome 5 reference locus for defb4 measurement ; drop - out of one copy at the reference locus should lead to apparent doubling of the copy number of the test locus , and no prt results showed a discrepancy of exactly double the consensus value . drop - out at the test locus may lead to subtler anomalies , including failure of only a single copy to amplify , reducing the apparent copy number by one . in particular , gene conversion among diverged members of a copy - variable array ( 3 ) could act to propagate even quite rare substitutional variants , leading to errors in the estimation of copy number . it is unlikely that this is a significant source of error at defb4 ; in addition to the close agreement of results from different methods , where the methods disagreed , there was no tendency for prt to record systematically lower values than maph / redvr or mlpa . further reassurance on both these sources of drop - out may be obtained by use of a second prt system with a different reference locus , and using a different part of the repeat element ; at defb4 a second prt system can be generated using a reference locus on chromosome 4 . although in practice the assay does not frequently give rise to partial digestion products , incomplete action of haeiii might in principle distort the product ratios . however , one would predict that ( unless incomplete digestion affected the test and reference loci differentially ) the correct product ratios would nevertheless be preserved in the population of completely digested molecules . furthermore , there is no absolute need to use restriction digestion as the method to discriminate test and reference products ; the assay could easily be modified to determine the ratios using other methods such as pyrosequencing . although many genes and regions have been defined as copy variable , the true extent of the variable region has been accurately determined for only a few . consequently , the precise extent of the dna to be measured can not be defined for most loci . furthermore , even when there are appropriately diverged dispersed repeats , without extensive individual examination it is not easy to assess the critical requirement for a pair of primers specific to precisely two loci . compounding this problem is the uncertainty surrounding the extent to which primer mismatches can be relied upon to discriminate against additional , alternative amplification products . finally , we have not yet determined whether using two or more co - amplified reference loci may be as accurate as using a single reference locus ; if so , then the scope for developing accurate prt systems may be even wider than we suggest here . where there are closely linked paralogous sequences , as may frequently happen in the evolution of gene family clusters , paralogues assumed to be present at constant copy number may be used as specific reference loci . thus , for example , the cyp2d8p pseudogene has been used as a reference locus in the accurate measurement of gene copy number for cyp2d6 ( 25 ) , and the rhce gene , usually present at two copies , could be used as an effective reference locus for rhd . however , it is very difficult to be sure that gene conversion does not exchange test and reference sequences in such local tandem arrays , and at other loci such as defa1a3 it is known that the paralogous variants can interchange locations ( 11 ) . for this reason an unlinked reference locus is desirable . to examine how frequently a copy - variable locus would harbour a sufficiently diverged repeat element to allow a prt assay , we examined 20 well - characterized examples of gene - containing copy - variable regions in addition to defb4 . we found 18 loci at which primers could be designed to amplify a test product plus exactly one reference product , with mismatches to other genomic loci ( table 1 ) . in many cases , the two products differ in size , so that no restriction enzyme digestion or other sequence - specific processing is required to distinguish them . some of the systems proposed involve diverged members of sequence families present in the genome at very high copy number ( alu , flam - c , l1 ) for which it may be difficult to generate the locus - specificity required . nevertheless , although they remain to be tested in practice , at many of these loci it is clear that the primers are very likely to form the basis of a specific and accurate prt assay for copy number . we may therefore conservatively suggest that useful prt systems could be designed for at least 50% of copy - variable genes . potential prt systems for defb4 and 20 further copy variable genes the final coordinates column specifies the reference locus and the placement of primers that should discriminate against other copies in the genome . in column 4 , ( dup) indicates a duplicon not associated with an identifiable dispersed repeat element . where there is a potentially useful reference locus closely linked to the test locus , this is shown in the third column ; in the specific cases of c4b and smn2 , the paralogues are not distinguished by substitutional divergence .

recent work has demonstrated an unexpected prevalence of copy number variation in the human genome , and has highlighted the part this variation may play in predisposition to common phenotypes . some important genes vary in number over a high range ( e.g. defb4 , which commonly varies between two and seven copies ) , and have posed formidable technical challenges for accurate copy number typing , so that there are no simple , cheap , high - throughput approaches suitable for large - scale screening . we have developed a simple comparative pcr method based on dispersed repeat sequences , using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci , which are subsequently distinguished and quantified via internal sequence differences . we have validated the method for the measurement of copy number at defb4 by comparison of results from > 800 dna samples with copy number measurements by maph / redvr , mlpa and array - cgh . the new paralogue ratio test ( prt ) method can require as little as 10 ng genomic dna , appears to be comparable in accuracy to the other methods , and for the first time provides a rapid , simple and inexpensive method for copy number analysis , suitable for application to typing thousands of samples in large case - control association studies .

INTRODUCTION MATERIALS AND METHODS Samples and formats MLPA and array-CGH data PRT assay Data analysis RESULTS AND DISCUSSION

several recent studies have demonstrated that some genes or groups of genes can show variation in copy number , and that this variation can have important functional consequences ( 16 ) . for example , the genes ccl3l1 , ccl4l1 and tbc1d3 are present on a segmental duplication that can vary between 0 and 10 copies per person ( 7 ) ; this variation appears to be a determinant of individual susceptibility to , and progression of , infection with hiv-1 ( 8) . similarly , a group of beta - defensin genes including defb4 commonly varies between two and seven copies per person , with occasional extremely expanded alleles containing 811 repeats visible as these beta - defensin genes , as well as the independently variable alpha - defensins defa1a3 ( 1012 ) , are candidate genes for variation in susceptibility to infectious disease , as well as autoimmune and inflammatory disorders ( 10 ) , and low copy number of the defb4 segmental duplication has been associated with crohn 's disease of the colon ( 13 ) . where frequent copy number variation encompasses the 03 copy number range , many established technologies can be used to provide rapid , cheap and accurate measurement of dna copy number . for example , combining maph with determination of restriction enzyme digest variant ratios ( maph / redvr ) has been capable of high reproducibility in determining copy number at defb4 ( 10 ) and defa1a3 ( 11 ) , but uses large amounts of genomic dna ( > 1 g ) and is labour - intensive . real - time pcr is gaining in popularity as a method of determining copy number ( 8,13 ) , but requires careful set - up and does not easily provide the highest throughput required for large association studies . an ideal high - throughput method for measuring copy number in large - scale association studies would be accurate , inexpensive , robust and use only small amounts of genomic dna , and the pressing need for methods with these properties is increasingly recognized ( 14 ) . the experimental variability of multiplex pcr is presumably due to the different amplification properties of the test and reference loci , and the differential sensitivity of the yield of each amplification reaction to the precise conditions . to obtain reliable results in multiplex pcr , great care needs to be taken with a number of experimental factors , including dna quality and ( as far as possible ) matching the amplification properties of test and reference amplicons . we have applied this method to the copy - variable defb4 repeat unit , and compare the results from this approach with results on the same samples from three independent alternative methods . this new prt method is comparable in accuracy to these alternative methods , and its simple format , and requirement for only small ( 1020 ng ) amounts of genomic dna , should allow accurate , inexpensive and rapid copy number typing of large cohorts of samples in association studies . genomic dna from dutch and hapmap samples was used at concentrations of 510 ng/l , and for most experiments samples were arrayed in 96-well microtitre plates and processed in batches of 96 . mlpa was carried out as described ( 19 ) using 250 ng genomic dna and the salsa mlpa kit p139 defensin from mrc - holland , and data were analyzed as described ( 20 ) . pcr was carried out using 5 ng input genomic dna , 0.5 m primer hspd5.8f ( ccagatgagaccagtgtcc ) and 0.5 m fam- or hex - labelled primer hspd5.8r ( ttttaagttcagcaattacagc ) ( figure 1 ) , in a buffer [ modified from ( 21 ) ] containing final concentrations of 50 mm tris hcl ( ph 8.8 ) , 12 mm ammonium sulphate , 5 mm magnesium chloride , 125 g / ml bsa ( non - acetylated , ambion inc ) , 7.4 mm 2-mercaptoethanol and 1.1 mm each dntp ( sodium salts ) , with 0.5u taq dna polymerase in a total volume of 10 l . products were amplified using 30 cycles of 95c for 30 s , 53c for 30 s and 70c for 30 s , followed by a single chase phase of 53c for 1 min/70c for 20 min to enhance complete extension in the final round and hence reduce levels of single - stranded dna products . defb4 and defb103107 , together with the locations of probes or amplicons used in maph , redvr , mlpa and prt , and the extent of the clone ( 10c3 ) used in array - cgh ( ' wt whole genome acgh ' ) . in the detailed display at the bottom , the primers amplify products from the hspdp3 pseudogene upstream of defb4 on chromosome 8 , and from a reference copy on chromosome 5 , but have multiple mismatches ( bold ) with other copies of the element . in this way a single primer pair can be used to amplify two very similar products , one from near defb4 , the other from chromosome 5 . two amplifications were carried out for each sample , one with a fluorescent fam label , the other with a fluorescent hex label ; 1 l of each pcr product was added , without further purification , to a 10 l digestion containing 1 react 2 buffer [ 50 mm tris hcl ( ph8.0 ) , 10 mm mgcl2 , 50 mm nacl ] ( invitrogen ) and 5u haeiii ( new england biolabs ) . after incubation at 37c for 416 h , 2 l was added to 10 l hidi formamide with rox-500 marker ( applied biosystems ) , and analyzed by electrophoresis on an abi3100 36 cm capillary using pop4 polymer and an injection time of 30 s. peak areas corresponding to the 302 bp haeiii fragment from near defb4 and the 315 bp fragment from chromosome 5 were recorded for both fam- and hex - labelled products using genescan and genotyper software ( applied biosystems ) . comparison of ratios ( chromosome 8 : chromosome 5 ) from fam- and hex - labelled products in a single experiment . although nave inference of a copy number equal to double the mean ratio would lead to reasonably accurate results , there were small but definite shifts between experiments in the relative amplification of the test and reference products . in most experiments maph / redvr copy numbers were used to calibrate prt assays ; subsequently , dna samples giving reproducible results from several prt assays have also been successfully used as calibration standards . genomic dna from dutch and hapmap samples was used at concentrations of 510 ng/l , and for most experiments samples were arrayed in 96-well microtitre plates and processed in batches of 96 . mlpa was carried out as described ( 19 ) using 250 ng genomic dna and the salsa mlpa kit p139 defensin from mrc - holland , and data were analyzed as described ( 20 ) . pcr was carried out using 5 ng input genomic dna , 0.5 m primer hspd5.8f ( ccagatgagaccagtgtcc ) and 0.5 m fam- or hex - labelled primer hspd5.8r ( ttttaagttcagcaattacagc ) ( figure 1 ) , in a buffer [ modified from ( 21 ) ] containing final concentrations of 50 mm tris hcl ( ph 8.8 ) , 12 mm ammonium sulphate , 5 mm magnesium chloride , 125 g / ml bsa ( non - acetylated , ambion inc ) , 7.4 mm 2-mercaptoethanol and 1.1 mm each dntp ( sodium salts ) , with 0.5u taq dna polymerase in a total volume of 10 l . products were amplified using 30 cycles of 95c for 30 s , 53c for 30 s and 70c for 30 s , followed by a single chase phase of 53c for 1 min/70c for 20 min to enhance complete extension in the final round and hence reduce levels of single - stranded dna products . not saturating ) amounts of product ; the annealing temperature was also chosen after empirical comparisons , and the choice of 53c is near the maximum annealing temperature that can be used , probably associated with a reduction in the efficiency of amplification . defb4 and defb103107 , together with the locations of probes or amplicons used in maph , redvr , mlpa and prt , and the extent of the clone ( 10c3 ) used in array - cgh ( ' wt whole genome acgh ' ) . in the detailed display at the bottom , the primers amplify products from the hspdp3 pseudogene upstream of defb4 on chromosome 8 , and from a reference copy on chromosome 5 , but have multiple mismatches ( bold ) with other copies of the element . in this way a single primer pair can be used to amplify two very similar products , one from near defb4 , the other from chromosome 5 . after incubation at 37c for 416 h , 2 l was added to 10 l hidi formamide with rox-500 marker ( applied biosystems ) , and analyzed by electrophoresis on an abi3100 36 cm capillary using pop4 polymer and an injection time of 30 s. peak areas corresponding to the 302 bp haeiii fragment from near defb4 and the 315 bp fragment from chromosome 5 were recorded for both fam- and hex - labelled products using genescan and genotyper software ( applied biosystems ) . comparison of ratios ( chromosome 8 : chromosome 5 ) from fam- and hex - labelled products in a single experiment . although nave inference of a copy number equal to double the mean ratio would lead to reasonably accurate results , there were small but definite shifts between experiments in the relative amplification of the test and reference products . in most experiments maph / redvr copy numbers were used to calibrate prt assays ; subsequently , dna samples giving reproducible results from several prt assays have also been successfully used as calibration standards . this principle has recently been successfully exploited in an innovative approach to the diagnosis of trisomy ; because some sequences present on chromosome 21 ( for example ) have nearly identical paralogues at another site in the genome , a single pair of primers can be used to amplify both test and reference loci , distinguishing them via minor differences of internal sequence ( 22 ) . although copy - variable loci are very unlikely to contain extensive regions with nearly identical counterparts at other locations , we were able to exploit the same advantages of amplifying both test and reference loci with a single primer pair by designing precisely placed primers in a diverged ( low copy number ) repetitive sequence , thereby allowing a paralogue ratio test ( prt ) . a heat - shock protein pseudogene of 2 kb ( hspdp3 ) is found 2 kb upstream of the defb4 gene ( figure 1 ) , and at 10 locations elsewhere in the genome . measuring the ratios of products from the test and reference loci ( see materials and methods ) allowed inference of defb4 copy number , which was linearly related to product ratio . in the absence of gold standard , error - free methods to count defb4 copy numbers for reference samples , we validated the method by comparisons with results from three other established techniques . prt was used to type 591 genomic dna samples which had already been typed for defb4 copy number by the maph / redvr method . the maph / redvr approach uses a primary copy number estimate by maph ( 23 ) , refined by examining ratios of sequence variants from the repeat unit ( 10,11 ) . for 486 samples ( 82% ) , a single prt assay gave the same integer copy number as maph / redvr ; for a further 64 samples ( 11% ) , the maph / redvr copy number was confirmed by prt on repeat testing . thus for 93% of samples , the maph / redvr value was confirmed by prt either on first - pass testing or after a single repeat test . for 25 samples ( 4.2% ) prt consistently gave a value different from maph / redvr , and for these samples maph / redvr was assumed to have been in error . a subset of 135 of these samples was also typed by mlpa ( 19 ) , so that this smaller sample set was typed by three independent methods : maph / redvr , prt and mlpa . for samples which had undergone more than one prt assay , these three methods appear to be comparable in their accuracy ; all three methods agreed on the integer copy number for 113 out of 135 samples ( 84% ) , and of the 22 remaining samples , the result from one method disagreed with the other two first - pass prt gave the discordant result for seven samples , maph / redvr for 6 samples , and mlpa for nine samples . we estimated rates of error for single - pass prt typing from a larger data set , combining results from maph / redvr and prt data , plus the mlpa results for the subset of 135 samples , to define a consensus copy number for all 575 samples ( after removing the 16 samples for which no consistent data were obtained ) . estimated error rates were < 10% for copy numbers of 2 , 3 and 4 , but rose to 20% or higher for samples with copy numbers of six or more ( figure 4a ) . overall , the analyses suggested that a single prt test yielded the correct integral value in 89% of samples ( 511 out of 575 ) , a figure that can be further improved by confirmatory and repeat testing as recommended below . in each case shaded columns show the relative frequencies of each copy number or residual category in the data set . as a further comparison of prt with established methodology , these data were then compared with array - cgh data from a clone ( 10c3 ) within the defb4 repeat unit . figure 5 shows the comparison between unrounded prt results and normalized signal from array - cgh of clone 10c3 , demonstrating clusters of data - points corresponding to integral copy numbers of 2 , 3 , 4 and 5 , with a greater spread at copy numbers of 6 and 7 . integral copy numbers from array - cgh data were assigned to these samples based on ranges defined by the clusters , and agreed with the prt - derived integer copy number for 202 samples ( 77% ) . analysis of flanking snp haplotypes in the hapmap samples showed very weak evidence for association with flanking haplotypes , and no snps or haplotypes were found that could reliably be used as a surrogate for copy number typing . comparison between first - pass prt typing ( unrounded values ) and normalized signal from a clone from the defb4 region in array - cgh for 261 samples from the hapmap study . because the frequency of samples with discordant duplicate tests is expected to be approximately twice the rate of error for a single test , this suggests that the error rate for integer values from a single prt test is 8.1% ( 95% confidence interval 5.611.8% ) . in typing large numbers of samples for case - control association studies using any error - prone measurement or genotyping method , a compromise has to be made between throughput and the cost and replication work required to obtain accurate data . the accuracy of prt testing for copy number could clearly be improved by requiring concordant duplicate testing of all samples , but for many large - scale studies such a doubling of the genotyping cost and effort may not be justified . for example , if re - testing was triggered by integral copy numbers of five or more with unrounded prt values > 0.3 from the nearest integer , then the data predict that only 15% of samples would require re - testing , and of the 85% of results accepted by these criteria , 92% would return the correct integral value . in such a case , if the correct measure of copy number is frequently non - integral , it is predicted that multiple measurements of the same samples ( typed by several methods , or in duplicate by a single method ) should have a tendency to cluster around non - integer values . the analysis assumes that the reference locus ( in this case on chromosome 5 ) does not itself vary in copy number , and that substitutional mismatches at either the test or reference locus primer binding sites do not lead to ` drop - out ' of one or more copies . in addition to their absence from all available sequence databases and sequence trace archives , substitutional variants do not appear to pose a problem at the chromosome 5 reference locus for defb4 measurement ; drop - out of one copy at the reference locus should lead to apparent doubling of the copy number of the test locus , and no prt results showed a discrepancy of exactly double the consensus value . drop - out at the test locus may lead to subtler anomalies , including failure of only a single copy to amplify , reducing the apparent copy number by one . in particular , gene conversion among diverged members of a copy - variable array ( 3 ) could act to propagate even quite rare substitutional variants , leading to errors in the estimation of copy number . it is unlikely that this is a significant source of error at defb4 ; in addition to the close agreement of results from different methods , where the methods disagreed , there was no tendency for prt to record systematically lower values than maph / redvr or mlpa . further reassurance on both these sources of drop - out may be obtained by use of a second prt system with a different reference locus , and using a different part of the repeat element ; at defb4 a second prt system can be generated using a reference locus on chromosome 4 . however , one would predict that ( unless incomplete digestion affected the test and reference loci differentially ) the correct product ratios would nevertheless be preserved in the population of completely digested molecules . furthermore , there is no absolute need to use restriction digestion as the method to discriminate test and reference products ; the assay could easily be modified to determine the ratios using other methods such as pyrosequencing . finally , we have not yet determined whether using two or more co - amplified reference loci may be as accurate as using a single reference locus ; if so , then the scope for developing accurate prt systems may be even wider than we suggest here . where there are closely linked paralogous sequences , as may frequently happen in the evolution of gene family clusters , paralogues assumed to be present at constant copy number may be used as specific reference loci . thus , for example , the cyp2d8p pseudogene has been used as a reference locus in the accurate measurement of gene copy number for cyp2d6 ( 25 ) , and the rhce gene , usually present at two copies , could be used as an effective reference locus for rhd . however , it is very difficult to be sure that gene conversion does not exchange test and reference sequences in such local tandem arrays , and at other loci such as defa1a3 it is known that the paralogous variants can interchange locations ( 11 ) . nevertheless , although they remain to be tested in practice , at many of these loci it is clear that the primers are very likely to form the basis of a specific and accurate prt assay for copy number . where there is a potentially useful reference locus closely linked to the test locus , this is shown in the third column ; in the specific cases of c4b and smn2 , the paralogues are not distinguished by substitutional divergence .

machine learning ( ml ) plays an essential role in the medical imaging field , including medical image analysis and computer - aided diagnosis ( cad ) [ 1 , 2 ] , because objects such as lesions and organs in medical images may be too complex to be represented accurately by a simple equation ; modeling of such complex objects often requires a number of parameters which have to be determined by data . for example , a lung nodule is generally modeled as a solid sphere , but there are nodules of various shapes and nodules with internal inhomogeneities , such as spiculated nodules and ground - glass nodules . a polyp in the colon is modeled as a bulbous object , but there are also polyps which exhibit a flat shape [ 4 , 5 ] . thus , diagnostic tasks in medical images essentially require learning from examples ( or data ) to determine a number of parameters in a complex model . one of the most popular uses of ml in medical image analysis is the classification of objects such as lesions into certain classes ( e.g. , abnormal or normal , lesions or non - lesions , and malignant or benign ) based on input features ( e.g. , contrast , area , and circularity ) obtained from segmented object candidates ( this class of ml is referred to feature - based ml . ) . the task of ml here is to determine optimal boundaries for separating classes in the multidimensional feature space which is formed by the input features . ml algorithms for classification include linear discriminant analysis , quadratic discriminant analysis , multilayer perceptron [ 8 , 9 ] , and support vector machines [ 10 , 11 ] . such ml algorithms were applied to lung nodule detection in chest radiography [ 1215 ] and thoracic ct [ 1619 ] , classification of lung nodules into benign or malignant in chest radiography and thoracic ct [ 21 , 22 ] , detection of microcalcifications in mammography [ 2326 ] , detection of masses in mammography , classification of masses into benign or malignant in mammography [ 2830 ] , polyp detection in ct colonography [ 3133 ] , determining subjective similarity measure of mammographic images [ 3436 ] , and detection of aneurysms in brain mri . recently , as available computational power increased dramatically , pixel / voxel - based ml ( pml ) emerged in medical image processing / analysis which uses pixel / voxel values in images directly instead of features calculated from segmented regions as input information ; thus , feature calculation or segmentation is not required . because the pml can avoid errors caused by inaccurate feature calculation and segmentation which often occur for subtle or complex objects , the performance of the pml can potentially be higher for such objects than that of common classifiers ( i.e. , feature - based mls ) . in this paper , pmls are surveyed and reviewed to make clear ( a ) classes of pmls , ( b ) the similarities and differences within different pmls and those between pmls and feature - based mls , ( c ) the advantages and limitations of pmls , and ( d ) their applications in medical imaging . pmls have been developed for tasks in medical image processing / analysis and computer vision . there are three classes of pmls : neural filters [ 38 , 39 ] ( including neural edge enhancers [ 40 , 41 ] ) , convolution neural networks ( nns ) [ 4248 ] ( including shift - invariant nns [ 4951 ] ) , and massive - training artificial neural networks ( mtanns ) [ 5256 ] ( including multiple mtanns [ 17 , 38 , 39 , 52 , 57 , 58 ] , a mixture of expert mtanns [ 59 , 60 ] , a multiresolution mtann , a laplacian eigenfunction mtann ( lap - mtann ) , and a massive - training support vector regression ( mtsvr ) ) . the class of neural filters has been used for image - processing tasks such as edge - preserving noise reduction in radiographs and other digital pictures [ 38 , 39 ] , edge enhancement from noisy images , and enhancement of subjective edges traced by a physician in left ventriculograms . the class of convolution nns has been applied to classification tasks such as false - positive ( fp ) reduction in cad schemes for detection of lung nodules in chest radiographs ( also known as chest x - rays ; cxrs ) [ 4244 ] , fp reduction in cad schemes for detection of microcalcifications and masses in mammography , face recognition , and character recognition . the class of mtanns has been used for classification , such as fp reduction in cad schemes for detection of lung nodules in cxr and ct [ 17 , 52 , 63 ] , distinction between benign and malignant lung nodules in ct , and fp reduction in a cad scheme for polyp detection in ct colonography [ 53 , 5962 ] . the mtanns have also been applied to pattern enhancement and suppression such as separation of bone from soft tissue in cxr [ 54 , 55 ] and enhancement of lung nodules in ct . an iterative , pixel - based , supervised , statistical classification method called iterated contextual pixel classification has been proposed for segmenting posterior ribs in cxr . a pixel - based , supervised regression filtering technique called filter learning has been proposed for separation of ribs from soft tissue in cxr . in the field of signal / image processing , supervised nonlinear filters based on a multilayer ann , called neural filters , have been studied [ 38 , 39 ] . the neural filter employs a linear - output ann model as a convolution kernel of a filter . the inputs to the neural filter are an object pixel value and spatially / spatiotemporally adjacent pixel values in a subregion ( or local window ) . the neural filter is trained with input images and corresponding teaching ( desired or ideal ) images . the training is performed by a linear - output backpropagation algorithm which is a back - propagation algorithm modified for the linear - output ann architecture . the input , output , and teacher ( desired output ) for neural filters are summarized in table 2 . neural filters have been applied to reduction of the quantum noise in x - ray fluoroscopic and radiographic images [ 38 , 39 ] . it was reported that the performance of the neural filter was superior to that of well - known nonlinear filters such as an adaptive weighted averaging filter . a study showed that adding features from the subregion to the input information improved the performance of the neural filter . neural filters have been extended to accommodate the task of enhancement of edges , and a supervised edge enhancer ( detector ) , called a neural edge enhancer , was developed . the neural edge enhancer can acquire the function of a desired edge enhancer through training . it was reported that the performance of the neural edge enhancer in the detection of edges from noisy images was far superior to that of well - known edge detectors such as the canny edge detector , the marr - hildreth edge detector , and the huckel edge detector . in its application to the contour extraction of the left ventricular cavity in digital angiography , it has been reported that the neural edge enhancer can accurately replicate the subjective edges traced by a cardiologist . an mtann was developed by extension of neural filters to accommodate various pattern - recognition tasks . a two - dimensional ( 2d ) mtann was first developed for distinguishing a specific opacity ( pattern ) from other opacities ( patterns ) in 2d images . the 2d mtann was applied to reduction of fps in computerized detection of lung nodules on 2d ct slices in a slice - by - slice way [ 17 , 52 , 63 ] and in cxr , the separation of ribs from soft tissue in cxr [ 54 , 55 , 70 ] , and the distinction between benign and malignant lung nodules on 2d ct slices . for processing of three - dimensional ( 3d ) volume data , a 3d mtann was developed by extending the structure of the 2d mtann , and it was applied to 3d ct colonography data [ 53 , 5962 ] . the generalized architecture of an mtann which unifies 2d and 3d mtanns is shown in figure 1 . the input , output , and teacher for mtanns an mtann consists of an ml model such as a linear - output ann regression model and a support vector regression model , which is capable of operating on pixel / voxel data directly . the linear - output ann regression model employs a linear function instead of a sigmoid function as the activation function of the unit in the output layer because the characteristics of an ann were improved significantly with a linear function when applied to the continuous mapping of values in image processing . note that the activation functions of the units in the hidden layer are a sigmoid function for nonlinear processing , and those of the unit in the input layer are an identity function , as usual . the pixel / voxel values of the input images / volumes may be normalized from 0 to 1 . the input to the mtann consists of pixel / voxel values in a subregion / subvolume , r , extracted from an input image / volume . the output of the mtann is a continuous scalar value , which is associated with the center voxel in the subregion , and is represented by ( 1)o(x , y , z or t ) = ml{i(xi , yj , zk or tk ) ( i , j , k)r } , where x , y , and z or t are the coordinate indices , ml( ) is the output of the ml model , and i(x , y , z or t ) is a pixel / voxel value of the input image / volume . a three - layer structure may be selected as the structure of the ann , because it has been proved that any continuous mapping can be approximated by a three - layer ann [ 71 , 72 ] . the structure of input units and the number of hidden units in the ann may be designed by use of sensitivity - based unit - pruning methods [ 73 , 74 ] . other ml models such as support vector regression [ 10 , 11 ] can be used as a core part of the mtann . ml regression models rather than ml classification models would be suited for the mtann framework , because the output of the mtann is continuous scalar values ( as opposed to nominal categories or classes ) . the entire output image / volume is obtained by scanning with the input subvolume of the mtann on the entire input image / volume . the input subregion / subvolume and the scanning with the mtann can be analogous to the kernel of a convolution filter and the convolutional operation of the filter , respectively . the mtann is trained with input images / volumes and the corresponding teaching images / volumes for enhancement of a specific pattern and suppression of other patterns in images / volumes . images / volumes are ideal or desired images for the corresponding input images / volumes . for enhancement of lesions and suppression of nonlesions , the teaching volume contains a map for the likelihood of being lesions , represented by ( 2)t(x , y , z or t)={a certain distribution , for a lesion,0,otherwise . to enrich the training samples , a training region , rt , extracted from the input images the mtann is massively trained by use of each of a large number of input subregions together with each of the corresponding teaching single pixels , hence the term massive - training ann . the error to be minimized by training of the mtann is represented by ( 3)e=1pc(x , y , z or t)rt{tc(x , y , z or t)oc(x , y , z or t)}2 , where c is a training case number , oc is the output of the mtann for the cth case , tc is the teaching value for the mtann for the cth case , and p is the number of total training voxels in the training region for the mtann , rt . the expert 3d mtann is trained by a linear - output backpropagation ( bp ) algorithm which was derived for the linear - output ann model by use of the generalized delta rule . after training , the mtann is expected to output the highest value when a lesion is located at the center of the subregion of the mtann , a lower value as the distance from the subregion center increases , and zero when the input subregion contains a nonlesion . a scoring method is used for combining output pixels from the trained mtanns . a score for a given region of interest ( roi ) from the mtann is defined as ( 4)s=(x , y , z or t)refw(x , y , z or t)o(x , y , z or t ) , where fw is a weighting function for combining pixel - based output responses from the trained mtann into a single score , which may often be the same distribution function used in the teaching images , and with its center corresponding to the center of the region for evaluation , re ; and o is the output image of the trained mtann , where its center corresponds to the center of re . this score represents the weighted sum of the estimates for the likelihood that the roi ( e.g. , a lesion candidate ) contains a lesion near the center ; that is , a higher score would indicate a lesion and a lower score would indicate a non - lesion . thresholding is then performed on the scores for distinction between lesions and non - lesions . the input , output , and teacher for convolution nns are summarized in table 2 . the convolution nn can be considered as a simplified version of the neocognitron model [ 7678 ] which was proposed to simulate the human visual system in 1980 . the input and output of the convolution nn are images and nominal class labels , respectively . the convolution nn consists of one input layer , several hidden layers , and one output layer . the layers are connected with local shift - invariant interconnections ( or convolution with a local kernel ) . unlike the neocognitron , the convolution nn has no lateral interconnections or feedback loops , and the error bp algorithm is used for training of the convolution nn . each unit in a subsequent layer is connected with the units of a small region in each group in the preceding layer . the groups between adjacent layers are interconnected by weights that are organized in kernels . for obtaining the shift - invariant responses , connection weights between any two groups in two layers are constrained to be shift - invariant ; in other words , forward signal propagation is similar to a shift - invariant convolution operation . the signals from the units in a certain layer are convolved with the weight kernel , and the resulting value of the convolution is collected into the corresponding unit in the subsequent layer . this value is further processed by the unit through an activation function and produces an output signal . the activation function between two layers is a sigmoid function . for deriving the training algorithm for the convolution nn , the generalized delta rule is applied to the architecture of the convolution nn . for distinguishing an roi containing a lesion from an roi containing a non - lesion , a class label ( e.g. , 1 for a lesion , 0 for a non - lesion ) the dual - kernel approach , which employs central kernels and peripheral kernels in each layer , was proposed for distinction between lung nodules and nonnodules in chest radiographs [ 42 , 43 ] and distinction between microcalcifications and other anatomic structures in mammograms . this dual - kernel - based convolution nn has several output units ( instead of one or two output units in the standard convolution nn ) for two - class classification . the fuzzy association was employed for transformation of output values from the output units to two classes ( i.e. , nodules or nonnodules ; microcalcifications or other anatomic structures ) . some convolution nns have one output unit [ 48 , 79 ] , some have two output units , and some have more than two output units [ 42 , 43 , 45 , 47 ] for two - class classification . shift - invariant nns [ 50 , 51 ] are mostly the same as convolution nns except for the output layer , which outputs images instead of classes . the shift - invariant nns were used for localization ( detection ) of lesions in images , for example , detection of microcalcifications in mammograms [ 50 , 51 ] and detection of the boundaries of the human corneal endothelium in photomicrographs . a multilayer perceptron has been proposed for character recognition from an optical card reader [ 82 , 83 ] . the input , output , and teacher for the multilayer perceptron for character recognition are summarized in table 2 . the input and output of the multilayer perceptron are pixel values in a given binary image that contains a single character ( e.g. , a , b , or c ) and a class to which the given image belongs , respectively . the number of input units equals the number of pixels in the given binary image ( e.g. , 16 16 pixels ) . the number of output units equals the number of classes ( i.e. , 26 for small - letter alphabetic characters ) . each output unit is assigned to one of the classes . the class to which the given image belongs is determined as the class of the output unit with the maximum value . in the teaching data , a class label of 1 is assigned the corresponding output unit when a training sample belongs to that class ; 0 is assigned to the other output units . characters in given images are geometrically normalized before they are entered to the multilayer perceptron , because the architecture is not designed for being scale - invariant . because character recognition with this type of the multilayer perceptron architecture is not shift- , rotation- , or scale - invariant , a large number of training samples is generally required . to enrich training samples , shifting , rotating , and scaling of training characters this type of multilayer perceptron has been applied to the classification of microcalcifications in mammography . in this application , pixel values in rois in mammograms or those in the fourier - transformed rois were entered as input to the multilayer perceptron . in that study , the performance of the multilayer perceptrons based on rois in the spatial domain and the fourier domain was found to be comparable . one of most popular uses of ml algorithms would probably be classification . in this use , an ml algorithm the input , output , and teacher for a classifier with features are summarized in table 2 . next , features are extracted from the segmented objects . then , extracted features are entered as input to an ml model such as linear discriminant analysis , quadratic discriminant analysis , a multilayer perceptron [ 8 , 9 ] , and a support - vector machine [ 10 , 11 ] . the ml model is trained with sets of input features and correct class labels . a class label of 1 is assigned to the corresponding output unit when a training sample belongs to that class , and 0 is assigned to the other output units . after training , the class of the unit with the maximum value is determined to be the corresponding class to which an unknown sample belongs . for details of feature - based classifiers , refer to one of many textbooks in pattern recognition such as [ 68 , 10 , 84 ] . mtanns were developed by extension of neural filters to accommodate various pattern - recognition tasks . in other words , the applications and functions of neural filters are limited to noise reduction [ 38 , 39 ] and edge enhancement [ 40 , 41 ] , whereas those of mtanns were extended to include classification [ 5254 , 5762 ] , pattern enhancement and suppression , and object detection . the input information to mtanns , which is the pixel values in a subregion , is the same as that to neural filters . however , the output of ( thus , teacher for ) neural filters is the desired pixel values in a given image , whereas that of mtanns is a map for the likelihood of being a specific pattern in a given image , as summarized in table 2 . both convolution nns and the perceptron used for character recognition are in the class of pml . input information to the convolution nns and the perceptron is the pixel values in a given image , whereas the output of ( thus , teacher for ) both algorithms is a nominal class label for the given image . however , the input images for the perceptron for character recognition are limited to be binary , although the perceptron itself is capable of processing gray - scale images . the major difference between convolution nns and the perceptron used for character recognition is their internal architectures . units in layers of the perceptron are fully connected , whereas the connections in the convolution nn are spatially ( locally ) limited . because of this architecture , forward signal propagation in the convolution nn is realized by a convolution operation . this convolution operation offers a shift - invariant property which is desirable for image classification . the applications and functions of the perceptron are limited to character recognition such as zip code recognition and optical character recognition , whereas those of convolution nns are general classification of images into known classes such as classification of lesion candidates into lesions or nonlesions [ 4246 ] , classification of faces , and classification of characters . shift - invariant nns are mostly the same as convolution nns except for the output layer , which outputs images instead of classes . the shift - invariant nns can be used for localization ( detection ) of objects in images in addition to classification [ 50 , 51 ] . convolution nns , shift - invariant nns , and mtanns perform convolution operations . in convolution nns and shift - invariant nns , convolution operations are performed within the network , as shown in figure 3 , whereas the convolutional operation is performed outside the network in the mtann , as shown in figure 1 . the major difference between pmls and ordinary classifiers ( i.e. , feature - based classifiers ) is the input information . ordinary classifiers use features extracted from a segmented object in a given image , whereas pmls use pixel values in a given image as the input information . although the input information to pmls can be features ( see addition of features to the input information to neural filters in , i.e. ) , these features are obtained pixel by pixel ( rather than by object ) . in other words , features for pmls are features at each pixel in a given image , whereas features for ordinary classifiers are features from a segmented object . in that sense , feature - based classifiers may be referred to as object - based classifiers . because pmls use pixel / voxel values in images directly instead of features calculated from segmented objects as the input information , feature calculation or segmentation is not required . although the development of segmentation techniques has been studied for a long time , segmentation of objects is still challenging , especially for complicated objects , subtle objects , and objects in a complex background . because , with pmls , errors caused by inaccurate feature calculation and segmentation can be avoided , the performance of pmls can be higher than that of ordinary classifiers for some cases , such as complicated objects . the output information from ordinary classifiers , convolution nns , and the perceptron used for character recognition is nominal class labels , whereas that from neural filters , mtanns , and shift - invariant nns is images . with the scoring method in mtanns , output images of the mtanns are converted to likelihood scores for distinguishing among classes , which allow mtanns to do classification . in addition to classification , mtanns can perform pattern enhancement and suppression as well as object detection , whereas the other pmls can not . quantum noise is dominant in low - radiation - dose x - ray images used in diagnosis . for training a neural filter to reduce quantum noise in diagnostic x - ray images while preserving image details such as edges , noisy input images and corresponding teaching when a high radiation dose is used , x - ray images with little noise can be acquired and used as the teaching images . a noisy input image can be synthesized by addition of simulated quantum noise ( which is modeled as signal - dependent noise ) to a noiseless original high - radiation - dose image fo(x , y ) , represented by ( 5)fn(x , y)=fo(x , y)+n[{fo(x , y ) } ] , where n[{fo(x , y ) } ] is noise with standard deviation {fo(x , y)}=knfo(x , y ) and kn is a parameter determining the amount of noise . a synthesized noisy x - ray image obtained with this method and a noiseless original high - radiation - dose x - ray image they were used as the input image and as the teaching image for training of a neural filter . for sufficient reduction of noise , for efficient training of the entire image , 5,000 training pixels were sampled randomly from the input and teaching images . the output image of the trained neural filter for a nontraining case is shown in figure 6(b ) . the noise in the input image is reduced while image details such as the edges of arteries are maintained . when an averaging filter was used for noise reduction , although conventional edge enhancers can very well enhance edges in images with little noise , they do not work well on noisy images . to address this issue , the neural edge enhancer is based on a neural filter and can be trained with input images and corresponding teaching edge images . figure 7(a ) shows a way of creating noisy input images and corresponding teaching a sobel edge enhancer was applied to the original noiseless images to create teaching edge images . the key here is that the sobel edge enhancer works very well for noiseless images . the neural edge enhancer was trained with the noisy input images together with the corresponding teaching edge images . for comparison , the trained neural edge enhancer and the sobel edge enhancer were applied to nontraining noisy images . edges are enhanced clearly in the output image of the neural edge enhancer while noise is suppressed , whereas the sobel edge enhancer enhances not only edges but also noise . cxr is the most frequently used diagnostic imaging examination for chest diseases such as lung cancer , tuberculosis , and pneumonia . lung cancer causes 945,000 deaths and is the leading cause of cancer deaths in the world and in countries such as the united states , the united kingdom , and japan . lung nodules ( i.e. , potential lung cancers ) in cxr , however , can be overlooked by radiologists in from 12 to 90% of cases that have nodules visible in retrospect [ 88 , 89 ] . studies showed that 82 to 95% of the missed lung cancers were partly obscured by overlying bones such as ribs and/or a clavicle [ 88 , 89 ] . to address this issue , dual - energy imaging uses the energy dependence of the x - ray attenuation by different materials ; it can produce two tissue - selective images , that is , a bone image and a soft - tissue image [ 9294 ] . major drawbacks of dual - energy imaging , however , are that ( a ) the radiation dose can be double , ( b ) specialized equipment for obtaining dual - energy x - ray exposures is required , and ( c ) the subtraction of two - energy images causes an increased noise level in the images . for resolving the above drawbacks with dual - energy images , mtanns have been developed as an image - processing technique for separation of ribs from soft tissue [ 54 , 70 ] . the basic idea is to train the mtann with soft - tissue and bone images acquired with a dual - energy radiography system [ 92 , 95 , 96 ] . for separation of ribs from soft tissue , the mtann was trained with input cxrs and the corresponding teaching dual - energy bone images , as illustrated in figure 8(a ) . figure 8(b ) shows a nontraining original cxr and a soft - tissue image obtained by use of the trained mtann . the contrast of ribs is suppressed substantially in the mtann soft - tissue image , whereas the contrast of soft tissue such as lung vessels is maintained . computer - aided diagnosis ( cad ) has been an active area of study in medical image analysis [ 1 , 2 , 97 , 98 ] . some cad schemes employ a filter for enhancement of lesions as a preprocessing step for improving sensitivity and specificity , but some do not employ such a filter . the filter enhances objects similar to a model employed in the filter ; for example , a blob - enhancement filter based on the hessian matrix enhances sphere - like objects . actual lesions , however , often differ from a simple model ; for example , a lung nodule is generally modeled as a solid sphere , but there are nodules of various shapes and inhomogeneous nodules such as nodules with spiculation and ground - glass nodules . thus , conventional filters often fail to enhance such actual lesions . to address this issue , a lesion - enhancement filter based on mtanns has been developed for enhancement of actual lesions in a cad scheme for detection of lung nodules in ct . for enhancement of lesions and suppression of non - lesions in ct images , the teaching image contains a map for the likelihood of being lesions . for enhancement of a nodule in an input ct image , a 2d gaussian distribution was placed at the location of the nodule in the teaching image , as a model of the likelihood of being a lesion . for testing of the performance , as shown in figure 9 , the nodule is enhanced in the output image of the trained mtann filter , while normal structures such as lung vessels are suppressed . note that small remaining regions due to vessels can easily be separated from nodules by use of their area information which can be obtained by use of connected - component labeling [ 100102 ] . a major challenge in cad development is to reduce the number of fps [ 27 , 103107 ] , because there are various normal structures similar to lesions in medical images . to address this issue , an fp - reduction technique based on an mtann has been developed for a cad scheme for lung nodule detection in ct . for enhancement of nodules ( i.e. , true positives ) and suppression of nonnodules ( i.e. , fps ) on ct images , the teaching image contains a distribution of values that represent the likelihood of being a nodule . for example , the teaching volume contains a 3d gaussian distribution with standard deviation t for a lesion and zero ( i.e. , completely dark ) for non - lesions , as illustrated in figure 10 . this distribution represents the likelihood of being a lesion : ( 6)t(x , y , z or t ) = { 12t exp{(x2+y2+z2 or t2)2t2},for a lesion,0,otherwise . the mtann involves training with a large number of subvolume - voxel pairs , which is called a massive - subvolumes training scheme . a scoring method is used for combining of output voxels from the trained mtanns , as illustrated in figure 11 . a score for a given roi from the mtann is defined as ( 7)s=(x , y , z or t)refw(x , y , z or t)o(x , y , z or t ) , where ( 8)fw(x , y , z or t ) = fg(x , y , z or t;)=12e(x2+y2+z2 or t2)/22 is a 3d gaussian weighting function with standard deviation and with its center corresponding to the center of the volume for evaluation , re , and o is the output image of the trained mtann , where its center corresponds to the center of re . the use of the 3d gaussian weighting function allows us to combine the responses ( outputs ) of a trained mtann as a 3d distribution . a 3d gaussian function is used for scoring , because the output of a trained mtann is expected to be similar to the 3d gaussian distribution used in the teaching images . this score represents the weighted sum of the estimates for the likelihood that the roi ( lesion candidate ) contains a lesion near the center ; that is , a higher score would indicate a lesion and a lower score would indicate a nonlesion . thresholding is then performed on the scores for distinction between lesions and non - lesions . an mtann was trained with typical nodules and typical types of fps ( nonnodules ) and corresponding teaching images . the trained mtann was applied to 57 true positives ( nodules ) and 1,726 fps ( nonnodules ) produced by a cad scheme . figure 12 shows various types of nodules and nonnodules and the corresponding output images of the trained mtann . nodules such as a solid nodule , a part - solid ( mixed - ground - glass ) nodule , and a non - solid ( ground - glass ) nodule are enhanced , whereas nonnodules such as different - sized lung vessels and soft - tissue opacity are suppressed around the centers of rois . for combining output pixels into a single score for each nodule candidate , a scoring method was applied to the output images for distinction between a nodules and a nonnodule . free - response receiver operating characteristic ( froc ) analysis was carried out for evaluation of the performance of the trained mtann . the froc curve for the mtann indicates 80.3% overall sensitivity ( 100% classification performance ) and a reduction in the fp rate from 0.98 to 0.18 per section , as shown in figure 13 . convolution nns have been used for fp reduction in cad schemes for lung nodule detection in cxrs [ 4244 ] . a convolution nn was trained with 28 chest radiographs for distinguishing lung nodules from nonnodules ( i.e. , fps produced by an initial cad scheme ) . the trained convolution nn reduced 79% of fp detections ( which is equivalent to 2 - 3 fps per patient ) , while 80% of true - positive detections were preserved . convolution nns have been applied to fp reduction in cad schemes for detection of microcalcifications and masses in mammography . the trained convolution nn reduced 90% of fp detections , which resulted in 0.5 fp detections per image , while a true - positive detection rate of 87% was preserved . shift - invariant nns have been used for fp reduction in cad for detection of microcalcifications [ 50 , 51 ] . microcalcifications were detected by thresholding of the output images of the trained shift - invariant nn . when the number of detected microcalcifications was greater than a predetermined number , the roi was considered as a microcalcification roi . with the trained shift - invariant nn , 55% of fps was removed without any loss of true positives . as described earlier , the major difference between pmls and ordinary classifiers is the direct use of pixel values with pml . in other words , unlike ordinary classifiers , feature calculation from segmented objects is not necessary . because the pml can avoid errors caused by inaccurate feature calculation and segmentation , the performance of the pml can potentially be higher than that of ordinary feature - based classifiers for some cases . pmls learn pixel data directly , and thus all information on pixels should not be lost before the pixel data are entered into the pml , whereas ordinary feature - based classifiers learn the features extracted from segmented lesions and thus important information can be lost with this indirect extraction ; also , inaccurate segmentation often occurs for complicated patterns . in addition , because feature calculation is not required for pml , development and implementation of segmentation and feature calculation , and selection of features are unnecessary . ordinary classifiers such as linear discriminant analysis , anns , and support vector machines can not be used for image processing , detection ( localization ) of objects , or enhancement of objects or patterns , whereas mtanns can do those tasks . for example , mtanns can separate bones from soft tissue in cxrs , and mtann can enhance and detect lung nodules on ct images . the characteristics of pmls which use pixel data directly should differ from those of ordinary feature - based classifiers . therefore , combining an ordinary feature - based classifier with a pml would yield a higher performance than that of a classifier alone or a pml alone . indeed , in previous studies , both classifier and pml were used successfully for classification of lesion candidates into lesions and non - lesions [ 17 , 45 , 46 , 4953 , 5863 ] . a limitation of pmls is the relatively long time for training because of the high dimensionality of input data . because pmls use pixel data in images directly , the number of input dimensions is generally large . for example , a 3d mtann for 3d ct data requires 171 dimensions for its input [ 53 , 60 ] . the ordinary feature - based classifiers are more efficient than pmls . in an application of pmls and feature - based classifiers to cad schemes , a feature - based classifier should be applied first , because the number of lesion candidates that need to be classified is larger at an earlier stage . after the number of lesion candidates is reduced by use of the feature - based classifier , a pml should be applied for further reduction of fps . indeed , previous studies employed this strategy [ 17 , 52 , 53 , 5861 ] . to address the issue of training time for pml , dimensionality reduction methods for pml have been proposed . with the use of the laplacian - eigenfunction - based dimensionality reduction of the input vectors to a 3d mtann in this paper , pmls were surveyed and compared with each other as well as with other non - pml algorithms ( i.e. , ordinary feature - based classifiers ) to make the similarities , differences , advantages , and limitations clear . the major difference between pmls and non - pml algorithms ( e.g. , classifiers ) is a need for segmentation and feature calculation with non - pml algorithms . the major advantage of pmls over non - pml algorithms is that no information is lost due to inaccurate segmentation and feature calculation , which would result in a higher performance for some cases such as complicated patterns . with the combination of pmls with non - pml algorithms , in addition to a classification task , mtanns can be used for enhancement ( and suppression ) and detection ( i.e. , localization ) of objects ( or patterns ) in images .

machine learning ( ml ) plays an important role in the medical imaging field , including medical image analysis and computer - aided diagnosis , because objects such as lesions and organs may not be represented accurately by a simple equation ; thus , medical pattern recognition essentially require learning from examples . one of the most popular uses of ml is classification of objects such as lesions into certain classes ( e.g. , abnormal or normal , or lesions or nonlesions ) based on input features ( e.g. , contrast and circularity ) obtained from segmented object candidates . recently , pixel / voxel - based ml ( pml ) emerged in medical image processing / analysis , which use pixel / voxel values in images directly instead of features calculated from segmented objects as input information ; thus , feature calculation or segmentation is not required . because the pml can avoid errors caused by inaccurate feature calculation and segmentation which often occur for subtle or complex objects , the performance of the pml can potentially be higher for such objects than that of common classifiers ( i.e. , feature - based mls ) . in this paper , pmls are surveyed to make clear ( a ) classes of pmls , ( b ) similarities and differences within ( among ) different pmls and those between pmls and feature - based mls , ( c ) advantages and limitations of pmls , and ( d ) their applications in medical imaging .

1. Introduction 2. Pixel/Voxel-Based Machine Learning (PML) 3. Similarities and Differences 4. Applications of PML Algorithms in Medical Images 5. Advantages and Limitations of PML Algorithms 6. Conclusion

machine learning ( ml ) plays an essential role in the medical imaging field , including medical image analysis and computer - aided diagnosis ( cad ) [ 1 , 2 ] , because objects such as lesions and organs in medical images may be too complex to be represented accurately by a simple equation ; modeling of such complex objects often requires a number of parameters which have to be determined by data . thus , diagnostic tasks in medical images essentially require learning from examples ( or data ) to determine a number of parameters in a complex model . one of the most popular uses of ml in medical image analysis is the classification of objects such as lesions into certain classes ( e.g. , abnormal or normal , lesions or non - lesions , and malignant or benign ) based on input features ( e.g. , contrast , area , and circularity ) obtained from segmented object candidates ( this class of ml is referred to feature - based ml . ) the task of ml here is to determine optimal boundaries for separating classes in the multidimensional feature space which is formed by the input features . ml algorithms for classification include linear discriminant analysis , quadratic discriminant analysis , multilayer perceptron [ 8 , 9 ] , and support vector machines [ 10 , 11 ] . recently , as available computational power increased dramatically , pixel / voxel - based ml ( pml ) emerged in medical image processing / analysis which uses pixel / voxel values in images directly instead of features calculated from segmented regions as input information ; thus , feature calculation or segmentation is not required . because the pml can avoid errors caused by inaccurate feature calculation and segmentation which often occur for subtle or complex objects , the performance of the pml can potentially be higher for such objects than that of common classifiers ( i.e. , feature - based mls ) . in this paper , pmls are surveyed and reviewed to make clear ( a ) classes of pmls , ( b ) the similarities and differences within different pmls and those between pmls and feature - based mls , ( c ) the advantages and limitations of pmls , and ( d ) their applications in medical imaging . pmls have been developed for tasks in medical image processing / analysis and computer vision . there are three classes of pmls : neural filters [ 38 , 39 ] ( including neural edge enhancers [ 40 , 41 ] ) , convolution neural networks ( nns ) [ 4248 ] ( including shift - invariant nns [ 4951 ] ) , and massive - training artificial neural networks ( mtanns ) [ 5256 ] ( including multiple mtanns [ 17 , 38 , 39 , 52 , 57 , 58 ] , a mixture of expert mtanns [ 59 , 60 ] , a multiresolution mtann , a laplacian eigenfunction mtann ( lap - mtann ) , and a massive - training support vector regression ( mtsvr ) ) . the class of neural filters has been used for image - processing tasks such as edge - preserving noise reduction in radiographs and other digital pictures [ 38 , 39 ] , edge enhancement from noisy images , and enhancement of subjective edges traced by a physician in left ventriculograms . the class of convolution nns has been applied to classification tasks such as false - positive ( fp ) reduction in cad schemes for detection of lung nodules in chest radiographs ( also known as chest x - rays ; cxrs ) [ 4244 ] , fp reduction in cad schemes for detection of microcalcifications and masses in mammography , face recognition , and character recognition . an iterative , pixel - based , supervised , statistical classification method called iterated contextual pixel classification has been proposed for segmenting posterior ribs in cxr . in the field of signal / image processing , supervised nonlinear filters based on a multilayer ann , called neural filters , have been studied [ 38 , 39 ] . it was reported that the performance of the neural filter was superior to that of well - known nonlinear filters such as an adaptive weighted averaging filter . a study showed that adding features from the subregion to the input information improved the performance of the neural filter . it was reported that the performance of the neural edge enhancer in the detection of edges from noisy images was far superior to that of well - known edge detectors such as the canny edge detector , the marr - hildreth edge detector , and the huckel edge detector . in its application to the contour extraction of the left ventricular cavity in digital angiography , it has been reported that the neural edge enhancer can accurately replicate the subjective edges traced by a cardiologist . the 2d mtann was applied to reduction of fps in computerized detection of lung nodules on 2d ct slices in a slice - by - slice way [ 17 , 52 , 63 ] and in cxr , the separation of ribs from soft tissue in cxr [ 54 , 55 , 70 ] , and the distinction between benign and malignant lung nodules on 2d ct slices . the input , output , and teacher for mtanns an mtann consists of an ml model such as a linear - output ann regression model and a support vector regression model , which is capable of operating on pixel / voxel data directly . the linear - output ann regression model employs a linear function instead of a sigmoid function as the activation function of the unit in the output layer because the characteristics of an ann were improved significantly with a linear function when applied to the continuous mapping of values in image processing . note that the activation functions of the units in the hidden layer are a sigmoid function for nonlinear processing , and those of the unit in the input layer are an identity function , as usual . the pixel / voxel values of the input images / volumes may be normalized from 0 to 1 . the input to the mtann consists of pixel / voxel values in a subregion / subvolume , r , extracted from an input image / volume . the output of the mtann is a continuous scalar value , which is associated with the center voxel in the subregion , and is represented by ( 1)o(x , y , z or t ) = ml{i(xi , yj , zk or tk ) ( i , j , k)r } , where x , y , and z or t are the coordinate indices , ml( ) is the output of the ml model , and i(x , y , z or t ) is a pixel / voxel value of the input image / volume . a three - layer structure may be selected as the structure of the ann , because it has been proved that any continuous mapping can be approximated by a three - layer ann [ 71 , 72 ] . the structure of input units and the number of hidden units in the ann may be designed by use of sensitivity - based unit - pruning methods [ 73 , 74 ] . ml regression models rather than ml classification models would be suited for the mtann framework , because the output of the mtann is continuous scalar values ( as opposed to nominal categories or classes ) . the error to be minimized by training of the mtann is represented by ( 3)e=1pc(x , y , z or t)rt{tc(x , y , z or t)oc(x , y , z or t)}2 , where c is a training case number , oc is the output of the mtann for the cth case , tc is the teaching value for the mtann for the cth case , and p is the number of total training voxels in the training region for the mtann , rt . after training , the mtann is expected to output the highest value when a lesion is located at the center of the subregion of the mtann , a lower value as the distance from the subregion center increases , and zero when the input subregion contains a nonlesion . a score for a given region of interest ( roi ) from the mtann is defined as ( 4)s=(x , y , z or t)refw(x , y , z or t)o(x , y , z or t ) , where fw is a weighting function for combining pixel - based output responses from the trained mtann into a single score , which may often be the same distribution function used in the teaching images , and with its center corresponding to the center of the region for evaluation , re ; and o is the output image of the trained mtann , where its center corresponds to the center of re . this score represents the weighted sum of the estimates for the likelihood that the roi ( e.g. unlike the neocognitron , the convolution nn has no lateral interconnections or feedback loops , and the error bp algorithm is used for training of the convolution nn . the signals from the units in a certain layer are convolved with the weight kernel , and the resulting value of the convolution is collected into the corresponding unit in the subsequent layer . for distinguishing an roi containing a lesion from an roi containing a non - lesion , a class label ( e.g. this dual - kernel - based convolution nn has several output units ( instead of one or two output units in the standard convolution nn ) for two - class classification . the fuzzy association was employed for transformation of output values from the output units to two classes ( i.e. the shift - invariant nns were used for localization ( detection ) of lesions in images , for example , detection of microcalcifications in mammograms [ 50 , 51 ] and detection of the boundaries of the human corneal endothelium in photomicrographs . the input and output of the multilayer perceptron are pixel values in a given binary image that contains a single character ( e.g. the number of input units equals the number of pixels in the given binary image ( e.g. the number of output units equals the number of classes ( i.e. characters in given images are geometrically normalized before they are entered to the multilayer perceptron , because the architecture is not designed for being scale - invariant . because character recognition with this type of the multilayer perceptron architecture is not shift- , rotation- , or scale - invariant , a large number of training samples is generally required . to enrich training samples , shifting , rotating , and scaling of training characters this type of multilayer perceptron has been applied to the classification of microcalcifications in mammography . in this application , pixel values in rois in mammograms or those in the fourier - transformed rois were entered as input to the multilayer perceptron . in that study , the performance of the multilayer perceptrons based on rois in the spatial domain and the fourier domain was found to be comparable . one of most popular uses of ml algorithms would probably be classification . in this use , an ml algorithm the input , output , and teacher for a classifier with features are summarized in table 2 . then , extracted features are entered as input to an ml model such as linear discriminant analysis , quadratic discriminant analysis , a multilayer perceptron [ 8 , 9 ] , and a support - vector machine [ 10 , 11 ] . for details of feature - based classifiers , refer to one of many textbooks in pattern recognition such as [ 68 , 10 , 84 ] . in other words , the applications and functions of neural filters are limited to noise reduction [ 38 , 39 ] and edge enhancement [ 40 , 41 ] , whereas those of mtanns were extended to include classification [ 5254 , 5762 ] , pattern enhancement and suppression , and object detection . the input information to mtanns , which is the pixel values in a subregion , is the same as that to neural filters . however , the output of ( thus , teacher for ) neural filters is the desired pixel values in a given image , whereas that of mtanns is a map for the likelihood of being a specific pattern in a given image , as summarized in table 2 . input information to the convolution nns and the perceptron is the pixel values in a given image , whereas the output of ( thus , teacher for ) both algorithms is a nominal class label for the given image . units in layers of the perceptron are fully connected , whereas the connections in the convolution nn are spatially ( locally ) limited . because of this architecture , forward signal propagation in the convolution nn is realized by a convolution operation . the applications and functions of the perceptron are limited to character recognition such as zip code recognition and optical character recognition , whereas those of convolution nns are general classification of images into known classes such as classification of lesion candidates into lesions or nonlesions [ 4246 ] , classification of faces , and classification of characters . the major difference between pmls and ordinary classifiers ( i.e. , feature - based classifiers ) is the input information . ordinary classifiers use features extracted from a segmented object in a given image , whereas pmls use pixel values in a given image as the input information . although the input information to pmls can be features ( see addition of features to the input information to neural filters in , i.e. ) in other words , features for pmls are features at each pixel in a given image , whereas features for ordinary classifiers are features from a segmented object . in that sense , feature - based classifiers may be referred to as object - based classifiers . because pmls use pixel / voxel values in images directly instead of features calculated from segmented objects as the input information , feature calculation or segmentation is not required . although the development of segmentation techniques has been studied for a long time , segmentation of objects is still challenging , especially for complicated objects , subtle objects , and objects in a complex background . because , with pmls , errors caused by inaccurate feature calculation and segmentation can be avoided , the performance of pmls can be higher than that of ordinary classifiers for some cases , such as complicated objects . cxr is the most frequently used diagnostic imaging examination for chest diseases such as lung cancer , tuberculosis , and pneumonia . lung cancer causes 945,000 deaths and is the leading cause of cancer deaths in the world and in countries such as the united states , the united kingdom , and japan . studies showed that 82 to 95% of the missed lung cancers were partly obscured by overlying bones such as ribs and/or a clavicle [ 88 , 89 ] . major drawbacks of dual - energy imaging , however , are that ( a ) the radiation dose can be double , ( b ) specialized equipment for obtaining dual - energy x - ray exposures is required , and ( c ) the subtraction of two - energy images causes an increased noise level in the images . computer - aided diagnosis ( cad ) has been an active area of study in medical image analysis [ 1 , 2 , 97 , 98 ] . actual lesions , however , often differ from a simple model ; for example , a lung nodule is generally modeled as a solid sphere , but there are nodules of various shapes and inhomogeneous nodules such as nodules with spiculation and ground - glass nodules . for enhancement of a nodule in an input ct image , a 2d gaussian distribution was placed at the location of the nodule in the teaching image , as a model of the likelihood of being a lesion . for testing of the performance , as shown in figure 9 , the nodule is enhanced in the output image of the trained mtann filter , while normal structures such as lung vessels are suppressed . , true positives ) and suppression of nonnodules ( i.e. for example , the teaching volume contains a 3d gaussian distribution with standard deviation t for a lesion and zero ( i.e. a 3d gaussian function is used for scoring , because the output of a trained mtann is expected to be similar to the 3d gaussian distribution used in the teaching images . free - response receiver operating characteristic ( froc ) analysis was carried out for evaluation of the performance of the trained mtann . a convolution nn was trained with 28 chest radiographs for distinguishing lung nodules from nonnodules ( i.e. as described earlier , the major difference between pmls and ordinary classifiers is the direct use of pixel values with pml . in other words , unlike ordinary classifiers , feature calculation from segmented objects is not necessary . because the pml can avoid errors caused by inaccurate feature calculation and segmentation , the performance of the pml can potentially be higher than that of ordinary feature - based classifiers for some cases . pmls learn pixel data directly , and thus all information on pixels should not be lost before the pixel data are entered into the pml , whereas ordinary feature - based classifiers learn the features extracted from segmented lesions and thus important information can be lost with this indirect extraction ; also , inaccurate segmentation often occurs for complicated patterns . in addition , because feature calculation is not required for pml , development and implementation of segmentation and feature calculation , and selection of features are unnecessary . ordinary classifiers such as linear discriminant analysis , anns , and support vector machines can not be used for image processing , detection ( localization ) of objects , or enhancement of objects or patterns , whereas mtanns can do those tasks . the characteristics of pmls which use pixel data directly should differ from those of ordinary feature - based classifiers . therefore , combining an ordinary feature - based classifier with a pml would yield a higher performance than that of a classifier alone or a pml alone . indeed , in previous studies , both classifier and pml were used successfully for classification of lesion candidates into lesions and non - lesions [ 17 , 45 , 46 , 4953 , 5863 ] . a limitation of pmls is the relatively long time for training because of the high dimensionality of input data . because pmls use pixel data in images directly , the number of input dimensions is generally large . the ordinary feature - based classifiers are more efficient than pmls . in an application of pmls and feature - based classifiers to cad schemes , a feature - based classifier should be applied first , because the number of lesion candidates that need to be classified is larger at an earlier stage . after the number of lesion candidates is reduced by use of the feature - based classifier , a pml should be applied for further reduction of fps . with the use of the laplacian - eigenfunction - based dimensionality reduction of the input vectors to a 3d mtann in this paper , pmls were surveyed and compared with each other as well as with other non - pml algorithms ( i.e. , ordinary feature - based classifiers ) to make the similarities , differences , advantages , and limitations clear . the major difference between pmls and non - pml algorithms ( e.g. the major advantage of pmls over non - pml algorithms is that no information is lost due to inaccurate segmentation and feature calculation , which would result in a higher performance for some cases such as complicated patterns . with the combination of pmls with non - pml algorithms , in addition to a classification task , mtanns can be used for enhancement ( and suppression ) and detection ( i.e. , localization ) of objects ( or patterns ) in images .

tuberculosis ( tb ) continues to haunt as a major health concern , and its spread has been aggravated by the onset of multidrug - resistant strains of mycobacterium tuberculosis ( mtb ) . tb , one of the oldest reemerging diseases , remains to be eliminated . in 2012 , tb worldwide accounted for 8.6 million cases and 1.3 million deaths from tb among hiv - negative people . however , in spite of this endemic occurrence , the underlying mechanisms of pathogenesis of tb are not fully delineated . to deal with this issue , it is essential to identify newer ways to eliminate intracellular mycobacteria in multidrug - resistant tb infected individuals and to come up with highly potent therapeutic processes for treating such tb infected individuals . apoptosis is a cellular decision that involves the synthesis of a succession of cellular proteins to elicit an apoptotic signal . although apoptosis in microbial infections leads to tissue damage , the initiation of apoptosis may be advantageous to the host , as it also ends up eliminating the microorganisms . mtb emerges as one of the highly successful human pathogens , due to its ability to survive by manipulating host cells by manipulation of multiple pathways [ 46 ] . recent studies have revealed that macrophages employ apoptosis as an innate defense response against mtb . however , virulent mtb does not elicit macrophage apoptosis and instead initiates necrosis at excessive intracellular bacterial cargoes . lim et al . have shown that the endoplasmic reticulum ( er ) stress response may be linked to mtb - induced apoptosis , and this process has an important role in controlling the survival of intracellular mtb . the er has an essential role in folding secretory and cellular proteins during their transit , and er chaperone proteins avert the toxic buildup of incorrectly folded secretory proteins . in addition to this critical role in protein folding , quality control , and targeting , the er is also involved in the synthesis of a wide range of cellular lipids [ 8 , 9 ] along with regulation of ca homeostasis . any malfunction in er can lead to cell death by activating a series of er chaperones that participate in the regulation of protein folding and the induction of cell death . even though apoptosis favoring the host protection is inferred and normally considered to be responsible for the modulation of intracellular mtb survival , the biological significance of this event remains to be clarified . choi et al . showed esat-6 antigen stimulates er stress - mediated apoptosis in mtb infected macrophages that induce growth arrest by dna damage - induced gene-153 ( gadd153 ) production . seimon et al . using tb granuloma macrophages demonstrated that er stress is induced in areas where apoptotic cells concentrate . sohn et al . recently showed that heparin - binding haemagglutinin antigen ( hbha ) enters macrophages and stimulates apoptosis by effecting loss of mitochondrial transmembrane potential with concomitant reactive oxygen species ( ros ) generation . however , choi et al . demonstrated that hbha induces ros production by the disruption of intracellular ca homeostasis . the ros stimulate excess of proinflammatory cytokines thereby leading to er stress - induced apoptosis . performed differential mitochondrial proteomics on human macrophages that were infected with either the avirulent mtb strain h37ra or its virulent counterpart h37rv to identify several host proteins as virulence factors that induce either apoptosis in h37ra infected macrophages or survival in h37rv infected host cells . these proteins were capable of differentially influencing several biochemical pathways such as atp production ( atp50 subunit ) , citric acid cycle ( uqcrh and dld ) and associated electron transport chain , voltage - dependent anion channels ( vdac2 ) , ros ( prdx1 ) , no production ( oas2 , sqrdl ) , phospholipid synthesis ( acsl1 , acsl4 , and acat1 ) , and fatty acid metabolism ( hadha ) , and lipid bodies ( lbs ) synthesis eventually leading to foamy macrophages formation in h37rv infected macrophages . the present study is designed to monitor the changes brought about by mtb at the er organelle level by observing the ultrastructural features of the host cell employing transmission electron microscopy to probe for alterations in morphology of the er , as a function of the duration of infection . the basis behind this is that , by this comparison between the consequences of a virulent versus an avirulent infection , it should be possible to identify those functional changes that would contribute towards eventual survival and persistence of the intracellular pathogen . mtb productively infects macrophages by upsetting the maturation of its phagosome , generating an intracellular compartment with endosomal rather than lysosomal characteristics . ca plays a significant role in different apoptotic pathways and is responsible for the phagosome - lysosome fusion . er is the primary site for maintaining cellular calcium homeostasis , decisions on cell survival versus death , and all processes involved in maintaining homeostasis of the various molecular constituents of the cell . therefore , er can be one of the targets of mycobacterial manipulation in the macrophage . accordingly , our analysis is directed at the perturbations induced in the er proteome and lipidome of infected cells . here again it was decided to compare between the effects of a virulent versus an avirulent infection so as to distinguish those effects that specifically relate to the virulence properties of the pathogen . for proteomic studies we employed the technique of stable isotope labeling of amino acids in cell culture ( silac ) wherein macrophages were first equilibrated with isotopically labeled lysine prior to infection . this has enabled a quantitative comparison of the er proteome by 2d - lc - ms / ms . for lipidomics study we employed shotgun lipidomics approach to quantitate the er lipidome from the organic phase of the crude extract based on their distinct headgroups released upon ms / ms tandem mass spectrometry . it is expected that such a comparison would provide mechanistic information on the nature of crosstalk between the host and the pathogen . thp-1 is a human monocyte leukemia cell line which was derived from a 1-year - old boy with acute monocytic leukemia . thp-1 cells were cultured in rpmi 1640 supplemented with 10% fbs at a density of 210 10 cells / ml at 37c in a humidified , 5% co2 atmosphere . forty - eight hours before mtb infection cells were seeded in a t-175 flask at 2025 10 cells per flask per 30 ml and differentiated using 30 ng of pma washed with rpmi without serum and medium was replenished . two strains of mtb , namely , h37ra and h37rv , were used for the study . tuberculosis aerosol challenge facility ( tacf ) is equipped with state - of - the - art infrastructure for safe handling of pathogenic tubercle bacilli . thp-1 cells were infected with designated mtb strains at the multiplicity of infection ( moi ) of ~10 . thp-1 cells were seeded at density of 25 10/30 ml of t-175 flask or 10 10/12 ml of 6-well plate . mtb culture for infection purposes was prepared by spinning the bacterial culture at 3000 rpm/10 min , followed by resuspension of pellets in rpmi without fbs . suspension was passed through series of needles , that is , 23 gauge , 26 gauge , and 30 gauge , in successive order to break the bacterial clumps to a single cell suspension for accurate moi . bacterial cell density was measured at 600 nm and the required number of bacteria was resuspended in an appropriate volume of medium ( rpmi ) of differentiated thp-1 at an moi of 10 bacilli / cell to differentiated thp-1 cell . the bacteria were allowed to invade the cells for 4 hrs at 37c in 5% co2 ; cells were washed twice with rpmi after internalization to remove the extracellular bacteria . amikacin ( 200 ng / ml ) was added to infected cells which were incubated for 2 hrs , 37c , in 5% co2 to kill any extracellular bacteria . the mtb infected cells were washed and harvested at specific time points after infection . thp-1 cells ( 200 10 ) were grown in rpmi 1640 medium according to manufacturer protocol ( sigma aldrich ) . after 24 hrs of infection , thp-1 cells were trypsinized and centrifuged at 600 g for 5 min . packed cell volume ( pcv ) was measured and 1x hypotonic extraction buffer equivalent to 3 times the pcv was added and incubated for 20 min at 4c to allow the cells to swell . the swollen cells were spin at 600 g for 5 min to remove the supernatant . to this , 1x isotonic extraction buffer equivalent to 2 times the new pcv was added and samples passed 10 times through 23 gauge needle to break the cells . the homogenate thus obtained was centrifuged at 1000 g for 10 min at 4c to separate the post nuclear supernatant that contains mitochondrial as well as microsomal fractions . it was further fractionated by centrifugation at 12000 g for 15 min at 4c to get the post mitochondrial supernatant which is the source of microsomes . at 24 hrs after infection , the uninfected or infected thp-1 cells were used to monitor the ultrastructural changes with the assistance of transmission electron microscope ( technai 12 biotwin tm , fei co. , the netherlands ) . briefly , 10 10 cells infected with either h37rv or h37ra and the third group that was left without infection which acts as a control were taken . cells ( ~100 ) were taken for imaging under 80 and 100 kev operating voltages of a transmission electron microscope ( technai 12 biotwin tm , fei co. , the netherlands ) . images were recorded digitally using a side - mounted 2k 2k ccd camera ( sis , germany ) . cells were maintained in the appropriate heavy labeled medium for 5 passages prior to differentiation with pma and subsequent infection ( supplementary figure 1 , in supplementary material available online at http://dx.doi.org/10.1155/2015/270438 ) . lys-6 labeled media were chosen for cells to be infected with h37ra ; lys-8 labeled media for those to be infected with h37rv and parallel set of uninfected cells were maintained in medium containing normal lysine . at 24 hrs after infection , equal numbers ( 10 10 ) of uninfected , this cell pool was then employed for isolation of endoplasmic reticulum by using the er isolation kit adhering to the laid down protocol recommended by the manufacturer . from the suspension of isolated er , protein lysate was prepared , lyophilized , and then resuspended in 100 mm ammonium bicarbonate buffer . the proteins were then subjected to denaturation and reductive alkylation prior to digestion with trypsin . the labeled peptide mixtures was separated by offline strong cation exchange ( scx ) chromatography using an hplc with a uv detector ( 1260 , binary pumps , agilent ) . briefly , labeled samples ( 50 g ) were reconstituted in scx running buffer ( 5 mm ammonium formate/30% acetonitrile ) and loaded onto a poly - lc polysulfethyl zorbax 300 scx column , 5 m ( 2.1 mm 150 mm , agilent ) . peptides were eluted with increasing concentrations of 5 mm ammonium formate , 30% acn ( solvent a ) , to 500 mm ammonium formate , 30% acn ( solvent b ) , using a gradient : 05 min 100% a and 0% b , 535 min 30% a and 70% b , 3555 min 0% a and 100% b , 5560 min 100% a and 0% b , and finally till 65 min on 100% a to reequilibrate the column with solvent a. fifteen fractions were collected at a flow rate of 400 l / min according to uv trace at 220 nm . fractions were lyophilized and were further analyzed offline on a tempo nano - lc and spotted on maldi plates . the peptide mixtures were analyzed by reverse - phase liquid chromatography ( tempo nano - lc from applied biosystems , foster city , ca ) offline coupled to ab sciex 5800 proteomics analyzer maldi - tof / tof mass spectrometer ( applied biosystems , foster city , ca ) . each fraction was reconstituted in 15 l of 1a buffer ( 98% water , 2% acn and 0.1% tfa ) and 12 l was picked up by auto sampler and directly loaded on to lc tempo column ( chromolith monolithic capillary , rp c18 , 150 0.1 mm ) and separated using 50 min gradient : 5% acn to 50% acn . the column elutes were mixed with 5 mg / ml chca ( -cyano-4 hydroxycinnamic acid ) matrix in 85% acetonitrile ( acn ) ; 0.1% tfa was spotted at a 1.5 l / min flow rate on 1364-well ( 44 31 ) lc - maldi stainless steel plate . protein identification was performed on an ab sciex maldi toftof 5800 analyzer ( ab sciex , foster city , ca ) with the mass tolerance kept at 50 ppm . for ms mode , peptide mass maps were acquired in positive reflection mode , and 8004000 m / z mass range was used with 1500 laser shots per spectrum . the pmf peak detection criteria used include minimum signal - to - noise ( s / n ) of 20 , local noise window width mass / charge ( m / z ) of 250 , and minimum full - width half maximum ( bins ) of 1 . a maximum of 30 precursors per spot with a minimum signal / noise ratio of 20 were selected for ms / ms analysis . energy of 1 kv was used for collision - induced dissociation ( cid ) , and 4000 acquisitions were accumulated for each ms / ms spectrum with dynamic exclusion mode of captured peptides . the peak detection criteria used were minimum s / n of 10 , local noise window width ( m / z ) of 200 , and minimum full - width half - maximum ( bins ) of 2.9 . the interpretation for ms / ms analysis includes the exclusion of contaminant m / z peaks originating from human keratin , trypsin autodigestion , and matrix . all automatic data analysis ( ms and ms / ms ) and database searching were conducted against the uniprot database ( version 02 - 15 - 2014 ) using the proteinpilot software ( version 4.0 , revision 148085 , applied biosystems ) with the paragon method utilizing the following search parameters : homo sapiens as species , trypsin as enzyme ( one missed cleavage allowed ) , with fixed modification of methyl methanethiosulfonate ( mmts ) labeled cysteine parameter enabled , silac - lys6 , and lys8 labeled as sample type and the search effort parameter thorough i d , which provides a broad search of various protein modifications , were chosen . the raw peptide identification results from the paragon algorithm ( applied biosystems ) searches were further processed by the progroup algorithm ( applied biosystems ) within the proteinpilot software before final display . confidence threshold cut off of 95% ( unused - protscore > 1.3 ) with at least one peptide for identification and two different peptides for quantification was used . samples from two biological replicates were analyzed together and identified a total of 133 unique human er proteins ( supplementary table 1 ) . we used the uniprot knowledgebase ( uniprotkb ) and gene ontology ( go ) database information of the identified proteins to fix its er localization , molecular function , and biological processes . briefly , 10 10 thp-1 cells were cultured and infected with a virulent or avirulent strains of mtb . at 24 hrs after infection , thp-1 cells infected with either h37rv or h37ra were collected in a centrifuge tube separately . to each of these , 3.75 ml chcl3 : meoh 1 : 2 ( v / v ) mixture was added and vortexed . subsequently , 1.25 ml chcl3 was added and vortexed vigorously . then again , 1.25 ml ms grade h2o was added and the sample vortexed for 5 min followed by centrifugation ( 1000 rpm , 5 min , room temperature ) . collected chloroform - methanol phase was further extracted once with ms grade water . organic phase was separated and subjected to mass spectrometry through shotgun lipidomic analysis . 4000 qtrap ( absciex , concord , ontario , canada ) were used for phospholipids analysis by direct infusion method . different precursor ions and neutral ions loss were assigned in our study ( supplementary table 2 ) . source and compound parameters were optimized by injecting the lipids standards purchased from avanti polar lipids . the source parameters were ionization spray voltage ( is ) 3300 , curtain gas ( cur ) , gas 1 ( gs1 ) , and gas 2 ( gs2)10 , 10 , and 5 , respectively ( arbitrary unit ) . the compound dependent parameters were declustering potential ( dp ) , 80 , entrance potential ( ep ) , 10 v in negative polarity . positive polarity source parameters were is + 3000 , cur , gs1 , and gs210 , 10 , and 5 , respectively ( arbitrary unit ) . samples were introduced with the help of model 11 plus syringe pump ( harvard apparatus , holliston , usa ) at the flow rate of 10 l / min . spectra were obtained for 100 cycles and step size is 0.1 da with mass ranges from m / z 550 to 1000 . 4000 qtrap instrument was calibrated by the mass spectrometer standard kit provided by absciex ( foster city , ca ) . phospholipids obtained from experimental sets were quantified against phospholipids standards procured from avanti polar lipids ( supplementary table 3 ) . cells on 96-well plates ( 15000/well/100 l ) were loaded with fluo-4 , am , ( 1 m , ca indicator ) for 30 min at 37c . excess dye was washed ; cells were resuspended with complete rpmi media and kept for 30 min to complete deesterification according to manufacturer 's instructions . images were obtained by confocal microscopy ( nikon eclipse ti - e laser scanning ) equipped with 60x/1.4 na planapochromat . dic objective lens was excited at 488 nm and emission was recorded through emission filter set at 515/30 nm with a scanning mode format ( 512 512 pixels ) . the mean fluorescence intensity / cell was determined with the help of density sum and area tools in the software . density sum refers to the sum of intensity values of all the pixels of a counted spot . after obtaining mean fluorescence intensity / cell , the values fold increase in calcium content refers to the ratio of the averaged intensity of infected thp-1 cells versus that of uninfected thp-1 cells . pma differentiated thp-1 cells were infected with either h37ra or h37rv and another aliquot kept as uninfected . at 24 hrs after infection , the cells were homogenized in 400 l of ice - cold lysis buffer ( 150 mm nacl , 50 mm hepes ph 7.4 , 1% np-40 , and 25 g / ml digitonin ) and incubated on ice for 30 min . the homogenate was further centrifuged at 8500 rpm for 10 min at 4c to get the supernatant . protein concentrations in the supernatants were measured . approximately 100 g of proteins from each sample was analyzed by sds - page using an 8% or a 12% resolving gel at 20 ma for 2 - 3 hrs . protein bands were electroblotted onto nitrocellulose membranes ( hybond - c extra , amersham biosciences ) . blocking was performed for 1 hr at room temperature using odyssey blocking buffer ( licor ) . for immunoblot analyses , antibodies of the following descriptions were used to probe the corresponding proteins : anti - cyclophilin b ( 1 : 1000 ) , anti - grp94 ( 1 : 2000 ) , anti - atp2a2 ( 1 : 1000 ) , anti - liver carboxylesterase 1 ( 1 : 1000 ) , anti - amrp ( 1 : 1000 ) , anti - calnexin ( 1 : 5000 ) , and anti - hyou1 ( 1 : 1000 ) . -actin was used as a loading control . detection involved using the goat anti - rabbit ir - dye 800 cw and goat anti - mouse ir - dye 800 cw ( 1 : 15000 , licor ) as secondary antibodies for 90 min . odyssey image scanner was used for scanning the images and these images were analyzed with the help of a software ( odyssey version 3.0 ) . thp-1 cells were seeded at a density of 9 10/6-well plate ( thermo scientific ) . after 24 hrs of infection , cells were scrapped and transferred to an eppendorf tube . caspase activity was measured as per the manufacturer 's protocol ( abcam , cambridge , ma , usa ) . briefly cells were homogenized with 50 l of ice - cold cell lysis buffer and further incubated the cells on ice for 10 min . after incubation , cells were centrifuged at 10,000 g for 1 min at 4c . each aliquot was then solubilized in 50 l of 2x reaction buffer containing 10 mm dtt and to this 5 l of the 4 mm ietd - p - na substrate ( resulting in 200 m final concentration ) was added and incubated for 2 hrs at 37c . after , incubation samples were read at 405 nm in a microtiter plate reader . thp-1 cells ( differentiated with pma ) were infected either with h37ra or with h37rv and were scanned to capture for any changes occurring in ultrastructure by tem . while infection - induced alterations were evident as early as 12 hrs after infection , these changes subsequently were more prominent by 24 hrs . the functional relevance of the 24 hrs time point of observation is also supported by recent findings , as evidenced from a study of the perturbations induced in mitochondrial function . we could observe prominent modifications induced in the morphology of er on infection with avirulent h37ra . with respect to the er , evidence for hyperproliferation of smooth form of er ( ser ) could be detected ( figure 1(a ) ) . in contrast to the observation made with h37ra , examination of over 100 cells profiles by tem showed significant morphological changes in er in response to infection with virulent h37rv ( figure 1(b ) ) which had shown the proliferation of rough form of endoplasmic reticulum ( rer ) . this suggests that virulent mtb induces an increase in protein synthesis and trafficking in the host cell soon after its entry . previously , it has been shown that er has the ability to modify according to the differentiation and functional state of the cells . consequently , this helps in adaptation of the er to its various synthetic and metabolic functions . for comparison , an ultrastructure of er ( microsome ) of uninfected thp-1 cells was shown ( figure 1(c ) ) . further to examine the adaptability of er , we chose the proteomics and lipidomics approach to check as to whether virulence is having any impact on the host cell er functionality . consistent with our tem interpretation , proteomics analysis performed on two replicate samples identified a total of 133 proteins that were known to be associated with er ( supplementary table 1 ) . when a cut - off difference of at least 10-fold was applied for up- or downregulated proteins as compared to that of in uninfected cells , the relative levels of 18 proteins ( table 1 ) were significantly altered in response to infection and differential effects induced by h37ra versus h37rv were clearly evident . similarly , lipids ( phospholipids ) which were differentially perturbed were enlisted in table 2 and among them phosphatidic acid ( pa ) , phosphatidylinositol bisphosphate ( pip2 ) , sphingomyelin ( sm ) , and phosphatidyl choline / phosphatidyl ethanolamine ( pc / pe ) were most prominent . after extensive literature survey these sets of proteins and lipids it is evident that serca has been shown to be involved in ca transport and maintaining the intracellular calcium levels . we found that the expression levels of atp2a2 protein ( table 1 ) were strongly attenuated in host cells infected with h37ra . however , atp2a2 is responsible for reducing the accumulation of cytosolic calcium by transferring it to er lumen from cytosol . to examine the relative expression of atp2a2 in thp-1 cells after an infection with either h37ra or h37rv , western blot analysis was employed using anti - atp2a2 primary antibody to estimate the abundance of er resident atp2a2 proteins in corresponding cell lysates . the representative western blot of the atp2a2 proteins expressed in thp-1 cells infected with either h37ra or h37rv was shown in figure 2(a)(i ) , which is consistent with our omics results . it is evident from figure 2(a)(ii ) ; the relative abundance of atp2a2 protein in thp-1 cells infected with h37ra was markedly reduced ( 0.6 0.14 ) when compared with the h37rv infected thp-1 . further , consistency seen with the perturbation at the level of protein , our lipids , specifically pc / pe ratio ( table 2 ) was found to be more than sixfold in thp-1 cells infected with h37ra compared to h37rv ( figure 2(b ) ) . these results indicated malfunctioning of serca and an insight of the accumulation of cytosolic calcium levels . so , the obvious next step was the quantitative estimation of cytosolic calcium level after an infection with either case of virulent or avirulent strain of mtb . confocal microscopy was used to monitor the intracellular levels of calcium by calcium binding fluo-4 , am green fluorescent dye . it was observed that the cytosolic calcium level was significantly induced in the h37ra infected thp-1 cells as shown in figure 2(c ) . the quantitative estimation of the level of cytosolic calcium was obtained by calculating the mean fluorescence intensity per cell . thus , from the relative intensity of the fluo-4 dye which irreversibly binds to intracellular calcium it was found to be twofold higher in thp-1 cells after infection with h37ra ( figure 2(d ) ) . our lipidomics results showed that the relative levels of pip2 were found to be more than fivefold ( 5.5 1.8 ) in case of thp-1 cells infected with h37rv ( figure 3(a ) ) . pip2 is an established agent for the inhibition of caspase 8 and caspase 9 enzymatic activities . next , the activities of caspase 8 and caspase 9 on infection with either h37ra or h37rv were monitored at 24 hrs of infection , using calorimetric assay , and we found that the expression level of caspase 8 was reduced to 50% ( 0.8 0.3 ) in host cells infected with h37rv . but surprisingly the level of expression of caspase 9 was unaltered ( figure 3(b ) ) . here , we can only speculate that h37rv strain of mtb might use some other pathway to counteract the host cell apoptotic challenge . further , our proteomics results exhibited the differential perturbations of est1 and amrp on the onset of infection with h37ra and h37rv in thp-1 cell ( table 1 ) . to confirm this , we employed western blot technique . figure 3(c ) shows the western blot image of the er resident est1 and amrp proteins . -actin was used as a loading control in each experiment . the representative western blot for visual comparison the relative intensity values of est1 and amrp proteins were quantitatively measured from three independent experiments . as it is evident from figure 3(d)(ii ) , while the relative expression of est1 protein was reduced significantly ( 0.8 0.3 ) , the relative expression of amrp protein was down to half - fold ( 0.6 0.2 ) in h37rv infected thp-1 cells . these results point to the concurrent efforts of h37rv , the virulent strain of mtb to avoid the apoptosis and prepare host cells for their latent infection . it was found that several proteins ( table 1 ) were either directly or indirectly modulated various cellular functions which impart a possible role for host cell and bacterial cross - talk . virulent strain of mtb , that is , h37rv , upregulated the proteins like endoplasmin ( enpl ) , protein disulphide - isomerase a4 ( pdia4 ) , calnexin ( canx ) , peptide - prolyl cis - trans isomerase b ( ppib ) , glucosidase ii subunit beta ( glu2b ) , erlin-2 ( erln2 ) , and cathepsin z ( ctsz ) . upregulation of enpl inhibits calpains and caspase activities by preventing an increase in calcium which leads to suppression of oxidative stress apoptosis . observed increase in pdia4 ( er resident chaperone ) levels can be associated with the termination of the classical mitochondrial apoptotic pathway . similarly , overexpression of canx may stop the er stress mediated apoptosis in preventing the accumulation of the nascent polypeptides [ 30 , 31 ] . reduced level of ppib is shown to be responsible for cellular vacuolation by inducing the persistent er stress , and it also activates p44/p42 mapk and nf-b pathways which are known to reduce the proinflammatory mediators by er stress . on the other hand , glu2b is a novel interacting protein of ip3r and enhances the activity of ip3r in vivo and enhances calcium release activity . changes in calcium concentration in the lumen of the er play a crucial role in modulating cell sensitivity to apoptosis . thus , to counteract the higher activity of glu2b and maintain intracellular ca levels , h37rv infected macrophages upregulated the erln2 protein also which facilitates the er - associated degradation of inactivated inositol 1,4,5-trisphosphate receptor ( ip3r ) . higher levels of catz ( cysteine proteinases ) activate mac-1 that results in blocking of activation and proliferation of t lymphocytes and eventually modulating innate and adaptive immune response . further , highly expressed er proteins such as canx , pdia4 , and erln2 were validated by performing rt - pcr using appropriate primers ( supplementary figure 2 and supplementary table 4 ) . proteins such as ribophorin ii ( rpn2 ) , transmembrane emp24 domain - containing protein 10 ( tmed10 ) , and phosphate disulphide isomerase 6 ( pdia6 ) were not detected after an infection with h37rv . rpn2 is a transmembrane glycoprotein and an essential subunit of the n - oligosaccharyltransferase complex that catalyzes the transfer of a high mannose oligosaccharide from lipid - linked oligosaccharide donor to an asparagines residue with an asn - x - ser / thr consensus motif in nascent polypeptide chains . here we can only speculate that h37rv employs this protein to gain entry into host macrophage cells . tmed10 is a member of emp24/gp25l / p24 family protein with gold domain and they bind to the copi and copii vesicles , thought to be involved in vesicle biosynthesis , cargo uptake , and maintaining the quality , but their actual function is still under debate . however , pdia6 is an er resident protein and its presence exhibited the induction of unfolded protein response ( upr ) . our results also indicated the downregulation of certain proteins after infection with h37ra such as inactive rhomboid protein 2 ( rhbdf2 ) , canopy-2 ( cnpy2 ) , hypoxia upregulated protein 1 ( hyou1 ) , and protein transport protein sec23a ( sc23a ) . cnpy2 and mir - interacting saposin - like protein ( msap ) act together with mylip / idol to degrade ldlr via protein ubiquitination . since , in our findings , cnpy2 is downregulated in h37ra infected macrophages it may upregulate the mylip / idol signaling by loss of gain function which in turn downregulates the ldlr thereby marginally increasing the cholesterol level . the marginal suppression of hyou1 was observed after an infection with h37ra of thp-1 cell . this is hypoxia - inducible stress protein , demonstrated to have a cytoprotective role , and its overexpression assisted the macrophage survival . thus again by loss of gain function the h37ra infected macrophages may undergo apoptosis readily . lastly , sc23a that is important for the transportation of newly synthesized secretory proteins from the endoplasmic reticulum to the golgi is also downregulated suggesting the activation of unfolded protein response . basically an intracellular pathogen , mtb , is amply equipped with successful strategies to overcome the host 's immune response and the rising rates of tuberculosis cases are a proof of its success as a pathogen . that averting of host macrophage apoptosis plays a key survival strategy of mtb and it is shown from previous studies that infection with susceptible strains of mtb such as h37ra or m. bovis bcg rapidly amplified the macrophage apoptosis . on the other hand virulent strains are successful in stalling the apoptotic response . furthermore , at some later phase , they favor to trigger necrosis so as to allow bacterial spread into the new uninfected host cells . an unanswered question that still continues , however , is the nature of perturbations that virulent mtb hold on the host cell , in order to offset the early activation of apoptotic pathways , but little is revealed on whether mtb also perturbs the er for the initiation of this process at a very early stage of infection . we , therefore , undertook the present study to explore the answer of this question . a comprehensive investigation of this issue clearly needs a thorough and in - depth analysis . therefore , the current work was constructed more as an illustrative investigation , to provide whether the differential thp-1 cellular perturbations to either h37ra or h37rv infection could be drawn at the level of early modulations in er function . thus , er in cells infected with either h37ra or h37rv showed distinct alterations in ultrastructure . of particular interest though was the fact that these distinct outcomes were indeed marked soon after infection , at the level of architectural and functional perturbations in the er of the host cell . in addition , h37ra induced the transformation of er to ser and in reverse h37rv promoted the induction of rer . these results , therefore , suggested investigating whether the observed variations could be explained through regulation in er proteome and lipidome . although our present endoplasmic reticulum proteomic study was somewhat limited in scope the results of our quantitative analysis , nonetheless , yield a ready rationalization for the distinct consequences of h37ra versus h37rv infection . thus , for example , the increased cytosolic calcium level by endoplasmic reticulum in h37ra infected cells was observed , at the functional level , by the specific reduction of atp2a2 in these cells . as indicated , changes in the pc / pe balance in a cellular setting can significantly perturb serca function . further , the perturbation in thp-1 cells after an infection with h37ra was diagrammatically depicted in figure 4 . notably , our results also provided a preliminary insight into the mechanisms entailed in the h37rv - dependent suppression of cellular apoptosis . earlier studies suggest a very distinct cell death mode induced by virulent strains of mtb from the one induced by avirulent one . similarly , from our ultrastructural observations , it is quite clear that the ultrastructural features of er in case of h37rv infected cells are very different from those observed in h37ra infected cell . further to support our ultrastructural findings some of the proteins like ppib , enpl , canx , pdia4 , and pdia6 regulate cell death directly or indirectly . these proteins have antiapoptotic activity which is markedly upregulated and would at least partly be responsible for this consequence . such an interpretation is backed by the fact that these proteins were not induced after an infection with h37ra . on the contrary , significantly induced level of the sm supports the ultrastructural state of er seen in cells infected with h37ra . sm has been implicated to function as an inducer of apoptosis by increasing the expression of tnf- and caspase 3 mrna level . in accordance with the observed er morphology , it is tempting to speculate that cells upon h37ra infection self - induce destabilization of er eventually leading to death . the host lipids metabolic pathway related to mtb virulence specific distinction is also of particular interest . interestingly , we have recently shown that only virulent mtb strains can induce lbs in the thp-1 cell , and that both h37ra and m. smegmatis strains lack this capability . in our present study , expression of lrpap1 protein was downregulated in cells infected with h37rv which helps in folding the ldl receptor family proteins . since est1 is responsible for the total cholesterol ester hydrolytic activity , it is likely that macrophages maintain the cholesterol in its esterified form in the foam cells and the finding of est1 that is an endoplasmic reticulum protein would certainly highlight the role of endoplasmic reticulum in making the foamy macrophage . in addition to these proteins , our lipid results indicated the upregulation of pip2 in cells infected with h37rv . previously , pip2 has been demonstrated to be an inhibitor of caspase 8 and caspase 9 . but surprisingly , our result indicated the reduction of only caspase 8 but not caspase 9 which remains unperturbed . thus again , figure 5 is a representation of modulations brought in human macrophages by h37rv . it is therefore evident from the above results that an analysis of the endoplasmic reticulum proteome and lipidome indeed provided some glimpse into the molecular mechanisms between the host and the mtb pathogen . importantly , our approach of correlating the h37ra versus h37rv infection also proved useful as it yielded an insight into the mechanisms by which virulent mtb modulates host cellular consequences . however , indeed still of a preliminary in nature , our results also highlight the combined nature of the effects that h37rv exercises on the host macrophage . thus , the early inhibition of apoptosis and the support in activation of lb , collectively ensure the substantial persistence of the virulent bacilli in the macrophages . therefore , this study features utility of performing a more comprehensive examination of the mtb - induced changes in the host cell endoplasmic reticulum proteome . significantly , a time - course functional analysis for the entire duration of infection can be expected to yield new and vital information on the mechanisms that equips the adaptive immune response of virulent mtb within the macrophage . in conclusion , interrogation of ultrastructural changes in er observed through tem and then er proteome and lipidome of mtb infected macrophages provided a preliminary glimpse of the underlying changes that occur at the protein and lipids level , thereby also rationalizing the detected alterations in functional properties . the studies described here yield new insights into the host - pathogen interplay that occurs in mtb infected macrophages . importantly , these studies provide an integrated perspective on the strategies adopted by the virulent pathogen , in order to successfully adapt within the hostile intracellular milieu of the macrophage . more detailed studies in this direction are likely to further enhance our understanding of these processes and can then aid in the development of novel approaches for the treatment of tb .

even though endoplasmic reticulum ( er ) stress associated with mycobacterial infection has been well studied , the molecular basis of er as a crucial organelle to determine the fate of mtb is yet to be established . here , we have studied the ability of mtb to manipulate the ultrastructural architecture of macrophage er and found that the er - phenotypes associated with virulent ( h37rv ) and avirulent ( h37ra ) strains were different : a rough er ( rer ) with the former against a smooth er ( ser ) with the later . further , the functional attributes of these changes were probed by ms - based quantitative proteomics ( 133 er proteins ) and lipidomics ( 8 phospholipids ) . our omics approaches not only revealed the host pathogen cross - talk but also emphasized how precisely mtb uses proteins and lipids in combination to give rise to characteristic er - phenotypes . h37ra - infected macrophages increased the cytosolic ca2 + levels by attenuating the atp2a2 protein and simultaneous induction of pc / pe expression to facilitate apoptosis . however , h37rv inhibited apoptosis and further controlled the expression of est-1 and amrp proteins to disturb cholesterol homeostasis resulting in sustained infection . this approach offers the potential to decipher the specific roles of er in understanding the cell biology of mycobacterial infection with special reference to the impact of host response .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

tuberculosis ( tb ) continues to haunt as a major health concern , and its spread has been aggravated by the onset of multidrug - resistant strains of mycobacterium tuberculosis ( mtb ) . tb , one of the oldest reemerging diseases , remains to be eliminated . however , in spite of this endemic occurrence , the underlying mechanisms of pathogenesis of tb are not fully delineated . although apoptosis in microbial infections leads to tissue damage , the initiation of apoptosis may be advantageous to the host , as it also ends up eliminating the microorganisms . however , virulent mtb does not elicit macrophage apoptosis and instead initiates necrosis at excessive intracellular bacterial cargoes . have shown that the endoplasmic reticulum ( er ) stress response may be linked to mtb - induced apoptosis , and this process has an important role in controlling the survival of intracellular mtb . in addition to this critical role in protein folding , quality control , and targeting , the er is also involved in the synthesis of a wide range of cellular lipids [ 8 , 9 ] along with regulation of ca homeostasis . any malfunction in er can lead to cell death by activating a series of er chaperones that participate in the regulation of protein folding and the induction of cell death . even though apoptosis favoring the host protection is inferred and normally considered to be responsible for the modulation of intracellular mtb survival , the biological significance of this event remains to be clarified . showed esat-6 antigen stimulates er stress - mediated apoptosis in mtb infected macrophages that induce growth arrest by dna damage - induced gene-153 ( gadd153 ) production . these proteins were capable of differentially influencing several biochemical pathways such as atp production ( atp50 subunit ) , citric acid cycle ( uqcrh and dld ) and associated electron transport chain , voltage - dependent anion channels ( vdac2 ) , ros ( prdx1 ) , no production ( oas2 , sqrdl ) , phospholipid synthesis ( acsl1 , acsl4 , and acat1 ) , and fatty acid metabolism ( hadha ) , and lipid bodies ( lbs ) synthesis eventually leading to foamy macrophages formation in h37rv infected macrophages . the present study is designed to monitor the changes brought about by mtb at the er organelle level by observing the ultrastructural features of the host cell employing transmission electron microscopy to probe for alterations in morphology of the er , as a function of the duration of infection . er is the primary site for maintaining cellular calcium homeostasis , decisions on cell survival versus death , and all processes involved in maintaining homeostasis of the various molecular constituents of the cell . here again it was decided to compare between the effects of a virulent versus an avirulent infection so as to distinguish those effects that specifically relate to the virulence properties of the pathogen . it is expected that such a comparison would provide mechanistic information on the nature of crosstalk between the host and the pathogen . two strains of mtb , namely , h37ra and h37rv , were used for the study . at 24 hrs after infection , the uninfected or infected thp-1 cells were used to monitor the ultrastructural changes with the assistance of transmission electron microscope ( technai 12 biotwin tm , fei co. , the netherlands ) . at 24 hrs after infection , equal numbers ( 10 10 ) of uninfected , this cell pool was then employed for isolation of endoplasmic reticulum by using the er isolation kit adhering to the laid down protocol recommended by the manufacturer . each fraction was reconstituted in 15 l of 1a buffer ( 98% water , 2% acn and 0.1% tfa ) and 12 l was picked up by auto sampler and directly loaded on to lc tempo column ( chromolith monolithic capillary , rp c18 , 150 0.1 mm ) and separated using 50 min gradient : 5% acn to 50% acn . protein identification was performed on an ab sciex maldi toftof 5800 analyzer ( ab sciex , foster city , ca ) with the mass tolerance kept at 50 ppm . all automatic data analysis ( ms and ms / ms ) and database searching were conducted against the uniprot database ( version 02 - 15 - 2014 ) using the proteinpilot software ( version 4.0 , revision 148085 , applied biosystems ) with the paragon method utilizing the following search parameters : homo sapiens as species , trypsin as enzyme ( one missed cleavage allowed ) , with fixed modification of methyl methanethiosulfonate ( mmts ) labeled cysteine parameter enabled , silac - lys6 , and lys8 labeled as sample type and the search effort parameter thorough i d , which provides a broad search of various protein modifications , were chosen . samples from two biological replicates were analyzed together and identified a total of 133 unique human er proteins ( supplementary table 1 ) . we used the uniprot knowledgebase ( uniprotkb ) and gene ontology ( go ) database information of the identified proteins to fix its er localization , molecular function , and biological processes . briefly , 10 10 thp-1 cells were cultured and infected with a virulent or avirulent strains of mtb . samples were introduced with the help of model 11 plus syringe pump ( harvard apparatus , holliston , usa ) at the flow rate of 10 l / min . the mean fluorescence intensity / cell was determined with the help of density sum and area tools in the software . after obtaining mean fluorescence intensity / cell , the values fold increase in calcium content refers to the ratio of the averaged intensity of infected thp-1 cells versus that of uninfected thp-1 cells . at 24 hrs after infection , the cells were homogenized in 400 l of ice - cold lysis buffer ( 150 mm nacl , 50 mm hepes ph 7.4 , 1% np-40 , and 25 g / ml digitonin ) and incubated on ice for 30 min . the functional relevance of the 24 hrs time point of observation is also supported by recent findings , as evidenced from a study of the perturbations induced in mitochondrial function . we could observe prominent modifications induced in the morphology of er on infection with avirulent h37ra . with respect to the er , evidence for hyperproliferation of smooth form of er ( ser ) could be detected ( figure 1(a ) ) . in contrast to the observation made with h37ra , examination of over 100 cells profiles by tem showed significant morphological changes in er in response to infection with virulent h37rv ( figure 1(b ) ) which had shown the proliferation of rough form of endoplasmic reticulum ( rer ) . this suggests that virulent mtb induces an increase in protein synthesis and trafficking in the host cell soon after its entry . previously , it has been shown that er has the ability to modify according to the differentiation and functional state of the cells . consequently , this helps in adaptation of the er to its various synthetic and metabolic functions . for comparison , an ultrastructure of er ( microsome ) of uninfected thp-1 cells was shown ( figure 1(c ) ) . further to examine the adaptability of er , we chose the proteomics and lipidomics approach to check as to whether virulence is having any impact on the host cell er functionality . consistent with our tem interpretation , proteomics analysis performed on two replicate samples identified a total of 133 proteins that were known to be associated with er ( supplementary table 1 ) . when a cut - off difference of at least 10-fold was applied for up- or downregulated proteins as compared to that of in uninfected cells , the relative levels of 18 proteins ( table 1 ) were significantly altered in response to infection and differential effects induced by h37ra versus h37rv were clearly evident . similarly , lipids ( phospholipids ) which were differentially perturbed were enlisted in table 2 and among them phosphatidic acid ( pa ) , phosphatidylinositol bisphosphate ( pip2 ) , sphingomyelin ( sm ) , and phosphatidyl choline / phosphatidyl ethanolamine ( pc / pe ) were most prominent . after extensive literature survey these sets of proteins and lipids it is evident that serca has been shown to be involved in ca transport and maintaining the intracellular calcium levels . we found that the expression levels of atp2a2 protein ( table 1 ) were strongly attenuated in host cells infected with h37ra . however , atp2a2 is responsible for reducing the accumulation of cytosolic calcium by transferring it to er lumen from cytosol . to examine the relative expression of atp2a2 in thp-1 cells after an infection with either h37ra or h37rv , western blot analysis was employed using anti - atp2a2 primary antibody to estimate the abundance of er resident atp2a2 proteins in corresponding cell lysates . the representative western blot of the atp2a2 proteins expressed in thp-1 cells infected with either h37ra or h37rv was shown in figure 2(a)(i ) , which is consistent with our omics results . it is evident from figure 2(a)(ii ) ; the relative abundance of atp2a2 protein in thp-1 cells infected with h37ra was markedly reduced ( 0.6 0.14 ) when compared with the h37rv infected thp-1 . further , consistency seen with the perturbation at the level of protein , our lipids , specifically pc / pe ratio ( table 2 ) was found to be more than sixfold in thp-1 cells infected with h37ra compared to h37rv ( figure 2(b ) ) . so , the obvious next step was the quantitative estimation of cytosolic calcium level after an infection with either case of virulent or avirulent strain of mtb . it was observed that the cytosolic calcium level was significantly induced in the h37ra infected thp-1 cells as shown in figure 2(c ) . thus , from the relative intensity of the fluo-4 dye which irreversibly binds to intracellular calcium it was found to be twofold higher in thp-1 cells after infection with h37ra ( figure 2(d ) ) . our lipidomics results showed that the relative levels of pip2 were found to be more than fivefold ( 5.5 1.8 ) in case of thp-1 cells infected with h37rv ( figure 3(a ) ) . next , the activities of caspase 8 and caspase 9 on infection with either h37ra or h37rv were monitored at 24 hrs of infection , using calorimetric assay , and we found that the expression level of caspase 8 was reduced to 50% ( 0.8 0.3 ) in host cells infected with h37rv . here , we can only speculate that h37rv strain of mtb might use some other pathway to counteract the host cell apoptotic challenge . further , our proteomics results exhibited the differential perturbations of est1 and amrp on the onset of infection with h37ra and h37rv in thp-1 cell ( table 1 ) . figure 3(c ) shows the western blot image of the er resident est1 and amrp proteins . the representative western blot for visual comparison the relative intensity values of est1 and amrp proteins were quantitatively measured from three independent experiments . as it is evident from figure 3(d)(ii ) , while the relative expression of est1 protein was reduced significantly ( 0.8 0.3 ) , the relative expression of amrp protein was down to half - fold ( 0.6 0.2 ) in h37rv infected thp-1 cells . these results point to the concurrent efforts of h37rv , the virulent strain of mtb to avoid the apoptosis and prepare host cells for their latent infection . it was found that several proteins ( table 1 ) were either directly or indirectly modulated various cellular functions which impart a possible role for host cell and bacterial cross - talk . virulent strain of mtb , that is , h37rv , upregulated the proteins like endoplasmin ( enpl ) , protein disulphide - isomerase a4 ( pdia4 ) , calnexin ( canx ) , peptide - prolyl cis - trans isomerase b ( ppib ) , glucosidase ii subunit beta ( glu2b ) , erlin-2 ( erln2 ) , and cathepsin z ( ctsz ) . observed increase in pdia4 ( er resident chaperone ) levels can be associated with the termination of the classical mitochondrial apoptotic pathway . changes in calcium concentration in the lumen of the er play a crucial role in modulating cell sensitivity to apoptosis . thus , to counteract the higher activity of glu2b and maintain intracellular ca levels , h37rv infected macrophages upregulated the erln2 protein also which facilitates the er - associated degradation of inactivated inositol 1,4,5-trisphosphate receptor ( ip3r ) . further , highly expressed er proteins such as canx , pdia4 , and erln2 were validated by performing rt - pcr using appropriate primers ( supplementary figure 2 and supplementary table 4 ) . tmed10 is a member of emp24/gp25l / p24 family protein with gold domain and they bind to the copi and copii vesicles , thought to be involved in vesicle biosynthesis , cargo uptake , and maintaining the quality , but their actual function is still under debate . however , pdia6 is an er resident protein and its presence exhibited the induction of unfolded protein response ( upr ) . our results also indicated the downregulation of certain proteins after infection with h37ra such as inactive rhomboid protein 2 ( rhbdf2 ) , canopy-2 ( cnpy2 ) , hypoxia upregulated protein 1 ( hyou1 ) , and protein transport protein sec23a ( sc23a ) . since , in our findings , cnpy2 is downregulated in h37ra infected macrophages it may upregulate the mylip / idol signaling by loss of gain function which in turn downregulates the ldlr thereby marginally increasing the cholesterol level . the marginal suppression of hyou1 was observed after an infection with h37ra of thp-1 cell . lastly , sc23a that is important for the transportation of newly synthesized secretory proteins from the endoplasmic reticulum to the golgi is also downregulated suggesting the activation of unfolded protein response . basically an intracellular pathogen , mtb , is amply equipped with successful strategies to overcome the host 's immune response and the rising rates of tuberculosis cases are a proof of its success as a pathogen . that averting of host macrophage apoptosis plays a key survival strategy of mtb and it is shown from previous studies that infection with susceptible strains of mtb such as h37ra or m. bovis bcg rapidly amplified the macrophage apoptosis . an unanswered question that still continues , however , is the nature of perturbations that virulent mtb hold on the host cell , in order to offset the early activation of apoptotic pathways , but little is revealed on whether mtb also perturbs the er for the initiation of this process at a very early stage of infection . thus , er in cells infected with either h37ra or h37rv showed distinct alterations in ultrastructure . of particular interest though was the fact that these distinct outcomes were indeed marked soon after infection , at the level of architectural and functional perturbations in the er of the host cell . in addition , h37ra induced the transformation of er to ser and in reverse h37rv promoted the induction of rer . thus , for example , the increased cytosolic calcium level by endoplasmic reticulum in h37ra infected cells was observed , at the functional level , by the specific reduction of atp2a2 in these cells . as indicated , changes in the pc / pe balance in a cellular setting can significantly perturb serca function . further , the perturbation in thp-1 cells after an infection with h37ra was diagrammatically depicted in figure 4 . similarly , from our ultrastructural observations , it is quite clear that the ultrastructural features of er in case of h37rv infected cells are very different from those observed in h37ra infected cell . on the contrary , significantly induced level of the sm supports the ultrastructural state of er seen in cells infected with h37ra . sm has been implicated to function as an inducer of apoptosis by increasing the expression of tnf- and caspase 3 mrna level . in accordance with the observed er morphology , it is tempting to speculate that cells upon h37ra infection self - induce destabilization of er eventually leading to death . the host lipids metabolic pathway related to mtb virulence specific distinction is also of particular interest . interestingly , we have recently shown that only virulent mtb strains can induce lbs in the thp-1 cell , and that both h37ra and m. smegmatis strains lack this capability . since est1 is responsible for the total cholesterol ester hydrolytic activity , it is likely that macrophages maintain the cholesterol in its esterified form in the foam cells and the finding of est1 that is an endoplasmic reticulum protein would certainly highlight the role of endoplasmic reticulum in making the foamy macrophage . previously , pip2 has been demonstrated to be an inhibitor of caspase 8 and caspase 9 . it is therefore evident from the above results that an analysis of the endoplasmic reticulum proteome and lipidome indeed provided some glimpse into the molecular mechanisms between the host and the mtb pathogen . however , indeed still of a preliminary in nature , our results also highlight the combined nature of the effects that h37rv exercises on the host macrophage . thus , the early inhibition of apoptosis and the support in activation of lb , collectively ensure the substantial persistence of the virulent bacilli in the macrophages . therefore , this study features utility of performing a more comprehensive examination of the mtb - induced changes in the host cell endoplasmic reticulum proteome . in conclusion , interrogation of ultrastructural changes in er observed through tem and then er proteome and lipidome of mtb infected macrophages provided a preliminary glimpse of the underlying changes that occur at the protein and lipids level , thereby also rationalizing the detected alterations in functional properties . the studies described here yield new insights into the host - pathogen interplay that occurs in mtb infected macrophages . more detailed studies in this direction are likely to further enhance our understanding of these processes and can then aid in the development of novel approaches for the treatment of tb .

haemophilus influenzae , along with streptococcus pneumoniae , is a major pathogen in respiratory tract infection and invasive infection in children . previously , h. influenzae developed resistance to ampicillin ( abpc ) by producing -lactamase ; however , since the beginning of the 2000s , there has been a rapid increase in the prevalence of ampicillin - resistant strains that do not produce -lactamase , that is , -lactamase - nonproducing abpc - resistant ( blnar ) strains . with regard to the blnar strains , their sensitivity to cephems and carbapenems , as well as to abpc , decreases as a result of mutations in the ftsi genes that encode penicillin - binding protein ( pbp ) 3 , causing major problems in the development of treatment strategies for pediatric infection such as meningitis . the drug - resistant pathogen surveillance group in pediatric infectious disease reported that from phase 1 ( 20002001 ) to phase 2 ( 2004 ) of nationwide surveillance there was a rapid increase in the distribution of blnar strains and a decrease in -lactamase - nonproducing abpc - sensitive ( blnas ) strains . it is very important to maintain an understanding of the trends in development of drug resistance in h. influenzae to be able to choose the proper antimicrobial agent in situations where there are significant changes in the prevalence of drug - resistant strains . we therefore conducted phase 3 ( 2007 ) and phase 4 ( 2010 ) surveillance studies , following the first two phases . we collected h. influenzae isolated from clinical specimens taken from pediatric patients at 27 institutions nationwide , all of which participated in the drug - resistant pathogen surveillance group in pediatric infectious disease , and used the 386 strains accumulated from january to june in 2007 for phase 3 and the 484 strains accumulated from january to june in 2010 for phase 4 . the sources of the isolates were as follows in phase 3 : nasopharynx , 299 strains ; pharynx , 51 strains ; sputum , 29 strains ; blood , 3 strains ; nasal discharge and pus , 1 strain for each ; and unknown origin , 2 strains . sources of isolates were as follows in phase 4 : nasopharynx , 396 strains ; sputum , 40 strains ; pharynx , 30 strains ; blood , 8 strains ; cerebrospinal fluid , 4 strains ; and unknown origin , 6 strains . for antimicrobial susceptibility testing , we measured the minimum inhibitory concentration ( mic ) by the broth microdilution method , complying with the clinical and laboratory standards institute ( clsi ) standards . sensitivity to the following 21 drugs was tested during phase 3 : abpc , clavulanic acid / amoxicillin ( cva / ampc ) , piperacillin ( pipc ) , tazobactam / piperacillin ( taz / pipc ) , cefaclor ( ccl ) , cefditoren ( cdtr ) , cefcapene ( cfpn ) , cefpodoxime ( cpdx ) , cefdinir ( cfdn ) , cefotaxime ( ctx ) , cefteram ( cftm ) , cefotiam ( ctm ) , ceftriaxone ( ctrx ) , faropenem ( frpm ) , panipenem ( papm ) , meropenem ( mepm ) , azithromycin ( azm ) , clarithromycin ( cam ) , rokitamycin ( rkm ) , telithromycin ( tel ) , and levofloxacin ( lvfx ) . during phase 4 , sensitivity was tested against a total of 23 drugs including those tested during phase 3 ( other than tel ) , with the following additional drugs : tebipenem ( tbpm ) , doripenem ( drpm ) , and tosufloxacin ( tflx ) . the strains were classified according to the clsi criteria : that is , -lactamase - nonproducing strains were classified into blnas strains , for which the mic for abpc was 1 g / ml or less ; -lactamase - nonproducing abpc - intermediately resistant ( blnai ) strains , with mic for abpc of 2 g / ml ; and blnar strains , with mic for abpc of 4 g / ml or more . -lactamase - producing strains were classified into -lactamase - producing abpc - resistant ( blpar ) strains , with mic for cva / ampc of 4 g / ml or less ; and -lactamase - producing cva / ampc - resistant ( blpacr ) strains , with mic for cva / ampc of 8 g / ml or more . additionally , all the strains were tested for h. influenzae serotype b ( hib ) by the polymerase chain reaction ( pcr ) method . fourteen strains ( 3.6 % ) in phase 3 and 23 strains ( 4.8 % ) in phase 4 were detected as hib . the sources of hib strains in phase 3 were as follows : nasopharynx , 9 strains ; blood , 3 strains ; and pharynx and pus , 1 strain for each . sources of hib strains in phase 4 : nasopharynx , 9 strains ; blood , 8 strains ; cerebrospinal fluid , 4 strains ; and sputum and pharynx , 1 strain each . to examine the relationship between background factors and the development of drug resistance , the frequency of isolation was compared between drug - resistant strains and blnas strains in relationship to six background factors , including sex , age , presence or absence of siblings , attendance or nonattendance at a daycare center , siblings attendance or nonattendance at a daycare center , and prior administration of antimicrobial agents within 1 month . the standard for drug - resistant strains was the same as that used in phase 1 and phase 2 and was defined as blnai + blnar . the test was used to identify whether a significant difference exists , using two - sided testing at a 5 % level of significance . we collected h. influenzae isolated from clinical specimens taken from pediatric patients at 27 institutions nationwide , all of which participated in the drug - resistant pathogen surveillance group in pediatric infectious disease , and used the 386 strains accumulated from january to june in 2007 for phase 3 and the 484 strains accumulated from january to june in 2010 for phase 4 . the sources of the isolates were as follows in phase 3 : nasopharynx , 299 strains ; pharynx , 51 strains ; sputum , 29 strains ; blood , 3 strains ; nasal discharge and pus , 1 strain for each ; and unknown origin , 2 strains . sources of isolates were as follows in phase 4 : nasopharynx , 396 strains ; sputum , 40 strains ; pharynx , 30 strains ; blood , 8 strains ; cerebrospinal fluid , 4 strains ; and unknown origin , 6 strains . for antimicrobial susceptibility testing , we measured the minimum inhibitory concentration ( mic ) by the broth microdilution method , complying with the clinical and laboratory standards institute ( clsi ) standards . sensitivity to the following 21 drugs was tested during phase 3 : abpc , clavulanic acid / amoxicillin ( cva / ampc ) , piperacillin ( pipc ) , tazobactam / piperacillin ( taz / pipc ) , cefaclor ( ccl ) , cefditoren ( cdtr ) , cefcapene ( cfpn ) , cefpodoxime ( cpdx ) , cefdinir ( cfdn ) , cefotaxime ( ctx ) , cefteram ( cftm ) , cefotiam ( ctm ) , ceftriaxone ( ctrx ) , faropenem ( frpm ) , panipenem ( papm ) , meropenem ( mepm ) , azithromycin ( azm ) , clarithromycin ( cam ) , rokitamycin ( rkm ) , telithromycin ( tel ) , and levofloxacin ( lvfx ) . during phase 4 , sensitivity was tested against a total of 23 drugs including those tested during phase 3 ( other than tel ) , with the following additional drugs : tebipenem ( tbpm ) , doripenem ( drpm ) , and tosufloxacin ( tflx ) . the strains were classified according to the clsi criteria : that is , -lactamase - nonproducing strains were classified into blnas strains , for which the mic for abpc was 1 g / ml or less ; -lactamase - nonproducing abpc - intermediately resistant ( blnai ) strains , with mic for abpc of 2 g / ml ; and blnar strains , with mic for abpc of 4 g / ml or more . -lactamase - producing strains were classified into -lactamase - producing abpc - resistant ( blpar ) strains , with mic for cva / ampc of 4 g / ml or less ; and -lactamase - producing cva / ampc - resistant ( blpacr ) strains , with mic for cva / ampc of 8 g / ml or more . additionally , all the strains were tested for h. influenzae serotype b ( hib ) by the polymerase chain reaction ( pcr ) method . fourteen strains ( 3.6 % ) in phase 3 and 23 strains ( 4.8 % ) in phase 4 were detected as hib . the sources of hib strains in phase 3 were as follows : nasopharynx , 9 strains ; blood , 3 strains ; and pharynx and pus , 1 strain for each . sources of hib strains in phase 4 : nasopharynx , 9 strains ; blood , 8 strains ; cerebrospinal fluid , 4 strains ; and sputum and pharynx , 1 strain each . to examine the relationship between background factors and the development of drug resistance , the frequency of isolation was compared between drug - resistant strains and blnas strains in relationship to six background factors , including sex , age , presence or absence of siblings , attendance or nonattendance at a daycare center , siblings attendance or nonattendance at a daycare center , and prior administration of antimicrobial agents within 1 month . the standard for drug - resistant strains was the same as that used in phase 1 and phase 2 and was defined as blnai + blnar . the test was used to identify whether a significant difference exists , using two - sided testing at a 5 % level of significance . figure 1 shows the number of strains by degrees of resistance . in phase 3 , there were 133 strains of blnas ( 34.5 % ) , 88 strains of blnai ( 22.8 % ) , 148 strains of blnar ( 38.3 % ) , 11 strains of blpar ( 2.8 % ) , and 6 strains of blpacr ( 1.6 % ) . in phase 4 , there were 161 strains of blnas ( 33.3 % ) , 98 strains of blnai ( 20.2 % ) , 183 strains of blnar ( 37.8 % ) , 19 strains of blpar ( 3.9 % ) , and 23 strains of blpacr ( 4.8 % ) .fig . blnas , -lactamase - nonproducing abpc - sensitive strain ; blnai , -lactamase - nonproducing abpc - intermediately resistant strain ; blnar , -lactamase - nonproducing abpc - resistant strain ; blpar , -lactamase - producing abpc - resistant strain ; blpacr , -lactamase - producing cva / ampc - resistant strain distribution of haemophilus influenzae strains classified by ampicillin ( abpc ) or clavulanic acid / amoxicillin ( cva / ampc ) resistance in phases 3 and 4 . blnas , -lactamase - nonproducing abpc - sensitive strain ; blnai , -lactamase - nonproducing abpc - intermediately resistant strain ; blnar , -lactamase - nonproducing abpc - resistant strain ; blpar , -lactamase - producing abpc - resistant strain ; blpacr , -lactamase - producing cva / ampc - resistant strain figure 2 shows the number of hib strains by degrees of resistance . in phase 3 , there were 9 strains of blnas ( 64.3 % ) , 3 strains of blnai ( 21.4 % ) , 1 strain of blnar ( 7.1 % ) , 1 strain of blpar ( 7.1 % ) , and no blpacr strain . in phase 4 , there were 13 strains of blnas ( 56.5 % ) , 4 strains of blnai ( 17.4 % ) , 6 strains of blpar ( 26.1 % ) , and no blnar or blpacr strains.fig . 2distribution of haemophilus influenzae serotype b strains classified by ampicillin ( abpc ) or clavulanic acid / amoxicillin ( cva / ampc ) resistance in phases 3 and 4 . blnas , -lactamase - nonproducing abpc - sensitive strain ; blnai , -lactamase - nonproducing abpc - intermediately resistant strain ; blnar , -lactamase - nonproducing abpc - resistant strain ; blpar , -lactamase - producing abpc - resistant strain ; blpacr , -lactamase - producing cva / ampc - resistant strain distribution of haemophilus influenzae serotype b strains classified by ampicillin ( abpc ) or clavulanic acid / amoxicillin ( cva / ampc ) resistance in phases 3 and 4 . blnas , -lactamase - nonproducing abpc - sensitive strain ; blnai , -lactamase - nonproducing abpc - intermediately resistant strain ; blnar , -lactamase - nonproducing abpc - resistant strain ; blpar , -lactamase - producing abpc - resistant strain ; blpacr , -lactamase - producing cva / ampc - resistant strain table 1 shows the mic50 , mic90 , and mic range of antimicrobial agents for h. influenzae . in phase 3 with a total of 386 strains , the oral antimicrobial agent with the lowest mic90 was lvfx ( 0.063 g / ml ) , followed by cdtr ( 0.25 g / ml ) . the intravenous antimicrobial agent with the lowest mic90 was taz / pipc ( 0.125 g / ml ) , followed by pipc , ctrx , and mepm ( 0.25 g / ml ) . in phase 4 with a total of 484 strains , the oral antimicrobial agent with the lowest mic90 was lvfx and tflx ( 0.063 g / ml ) , followed by cdtr ( 0.25 g / ml ) . the intravenous antimicrobial agent with the lowest mic90 was taz / pipc ( 0.125 g / ml ) , followed by pipc and ctrx ( 0.25 g / ml ) . between phase 3 and phase 4 , there was a twofold increase in the mic90 values of papm ( 24 g / ml ) and mepm ( 0.250.5 g / ml ) , but there were no changes in those of other drugs.table 1susceptibilities for haemophilus influenzae in phase 3 and phase 4phase 3phase 4number of strains386484micmic50mic90mic rangemic50mic90mic rangeabpc280.12>128280.063>128cva / ampc480.2532480.12516pipc0.0630.250.063>1280.0630.250.063>128taz / pipc0.0630.1250.06310.0630.1250.0630.25ccl16640.25>12816640.25128cdtr0.1250.250.0630.50.1250.250.0631cfpn120.0634120.0638cpdx240.0638240.0638cfdn280.06316280.06316cftm0.510.06320.510.0632ctm8640.0631288640.125128ctrx0.1250.250.0630.50.1250.250.0630.5ctx0.510.06340.510.0634azm120.12564120.0634cam481>64480.532rkm8161328160.06332tel120.258frpm240.0634240.0638tbpm0.2510.0632papm120.0634140.0638mepm0.1250.250.06310.1250.50.0631drpm0.520.0634lvfx0.0630.0630.0630.50.0630.0630.0630.5tflx0.0630.0630.0632abpc ampicillin , cva / ampc clavulanic acid / amoxicillin , pipc piperacillin , taz / pipc tazobactam / piperacillin , ccl cefaclor , cdtr cefditoren , cfpn cefcapene , cpdx cefpodoxime , cfdn cefdinir , cftm cefteram , ctm cefotiam , ctrx ceftriaxone , ctx cefotaxime , azm azithromycin , cam clarithromycin , rkm rokitamycin , tel telithromycin , frpm faropenem , tbpm tebipenem , papm panipenem , mepm meropenem , drpm doripenem , lvfx levofloxacin , tflx tosufloxacin susceptibilities for haemophilus influenzae in phase 3 and phase 4 abpc ampicillin , cva / ampc clavulanic acid / amoxicillin , pipc piperacillin , taz / pipc tazobactam / piperacillin , ccl cefaclor , cdtr cefditoren , cfpn cefcapene , cpdx cefpodoxime , cfdn cefdinir , cftm cefteram , ctm cefotiam , ctrx ceftriaxone , ctx cefotaxime , azm azithromycin , cam clarithromycin , rkm rokitamycin , tel telithromycin , frpm faropenem , tbpm tebipenem , papm panipenem , mepm meropenem , drpm doripenem , lvfx levofloxacin , tflx tosufloxacin table 2 shows the mic50 , mic90 , and mic range of antimicrobial agents for h. influenzae divided into the following five groups by degrees of resistance , the blnas , blnai , blnar , blpar , and blpacr groups , respectively . in the blnar group , the mic50 values of the -lactams excluding pipc and taz / pipc were 4- to 64 fold higher , and the mic90 values of all the agents were 2- to 4 fold higher than the values in the blnas group in phase 3 . in phase 4 , in the blnar group , the mic50 values of the -lactams excluding pipc and taz / pipc were 4- to 32 fold higher , and the mic90 values of all the agents were 2- to 8 fold higher than the values in the blnas group . in both phase 3 and phase 4 , the mic50 values of pipc and taz / pipc were the same in the blnas and blnar groups ( 0.063 g / ml).table 2suscepsibilities for haemophilus influenzae ( divided into five groups ) in phase 3 and phase 4classblnasblnaiblnarphasephase 3phase 4phase 3phase 4phase 3phase 4no . of strains ( % ) 133 ( 34.5)161(33.3)88 ( 22.8)98 ( 20.2)148 ( 38.3)183 ( 37.8)micmic50mic90mic rangemic50mic90mic rangemic50mic90mic rangemic50mic90mic rangemic50mic90mic rangemic50mic90mic rangeabpc0.2510.12510.510.063122222222484164848cva / ampc0.520.2540.520.1254481844188823248216pipc0.0630.1250.0630.50.0630.1250.0630.50.0630.250.0630.50.0630.1250.0630.250.0630.250.06310.0630.250.0630.5taz / pipc0.0630.0630.0630.50.0630.0630.0630.250.0630.1250.0630.250.0630.1250.0630.250.0630.250.06310.0630.1250.0630.25ccl4160.25644160.256432644>12816642128641284to 12832642128cdtr0.0630.0630.0630.50.0630.1250.0630.50.1250.250.0630.50.250.250.0630.50.250.250.0630.50.250.50.0631cfpn0.0630.50.06320.0630.50.0631120.0634120.0634220.254240.0638cpdx0.06310.06380.06310.0634280.258240.12584418240.258cfdn0.2520.06380.520.0634480.516240.58480.516480.516cftm0.0630.50.06310.0630.50.06310.510.06310.510.06320.510.1252110.0631ctm280.06364280.1253216642648321643264212832641128ctrx0.0630.1250.0630.250.0630.1250.0630.250.1250.250.0630.50.1250.250.0630.50.250.250.0630.50.250.250.0630.5ctx0.0630.50.06310.0630.50.0630.50.510.06320.510.06320.520.1254120.0634azm120.1254110.0634120.254120.1254120.254120.1252cam48116480.532481164161168811688216rkm8161324160.063328162168161328162168160.2516tel120.258120.58220.54frpm0.510.06320.520.0634220.254120.254240.254240.58tbpm0.1250.250.06310.2510.06310.510.0632papm0.510.06320.520.0634120.1254140.0638220.1254240.258mepm0.0630.1250.0630.1250.0630.1250.0630.50.1250.250.0630.50.250.50.0630.50.250.50.06310.250.50.0631drpm0.1250.50.06310.510.0632120.0634lvfx0.0630.0630.0630.1250.0630.0630.0630.50.0630.0630.0630.1250.0630.0630.0630.50.0630.0630.0630.50.0630.0630.0630.125tflx0.0630.0630.06320.0630.0630.0630.0630.0630.0630.0630.125classblparblpacrphasephase 3phase 4phase 3phase 4no . of strains ( % ) 11 ( 2.8)19 ( 3.9)6 ( 1.6)23 ( 4.8)micmic50mic90mic rangemic50mic90mic rangemic50mic90mic rangemic50mic90mic rangeabpc128>1282 to > 12832>12816 to > 128>128>128>128 to > 128>128>12864 to > 128cva / ampc140.254240.54816816816816pipc128>1280.25 to > 12816>1282 to > 128>128>128>128 to > 12864>1288 to > 128taz / pipc0.0630.0630.0630.0630.0630.1250.0630.1250.0630.1250.0630.1250.0630.0630.0630.125ccl832264832232641284128326416128cdtr0.0630.1250.0630.250.0630.250.0630.50.1250.250.1250.250.250.250.0630.25cfpn0.06310.06320.510.0631220.52220.52cpdx0.06320.0634120.063244242424cfdn0.2520.1254140.25448284828cftm0.0630.50.0630.50.50.50.0630.50.510.510.510.251ctm2320.5644160.51664128812832648128ctrx0.0630.250.0630.250.0630.250.0630.250.250.250.1250.250.250.250.0630.5ctx0.0630.50.06310.250.50.0631110.51110.251azm120.5640.510.2511640.564120.54cam882 to > 6448284>644 to > 64816416rkm8164164818282888216tel24141818frpm0.520.12520.520.063422222414tbpm0.0630.50.0630.50.510.0632papm0.520.06320.2540.0634140.54280.258mepm0.0630.250.0630.50.0630.250.0630.250.1250.250.0630.250.250.50.0631drpm0.12510.0631120.0632lvfx0.0630.0630.0630.0630.0630.0630.0630.0630.0630.250.0630.250.0630.0630.0630.063tflx0.0630.0630.0630.0630.0630.0630.0630.063abpc ampicillin , cva / ampc clavulanic acid / amoxicillin , pipc piperacillin , taz / pipc tazobactam / piperacillin , ccl cefaclor , cdtr cefditoren , cfpn cefcapene , cpdx cefpodoxime , cfdn cefdinir , cftm cefteram , ctm cefotiam , ctrx ceftriaxone , ctx cefotaxime , azm azithromycin , cam clarithromycin , rkm rokitamycin , tel telithromycin , frpm faropenem , tbpm tebipenem , papm panipenem , mepm meropenem , drpm doripenem , lvfx levofloxacin , tflx tosufloxacin suscepsibilities for haemophilus influenzae ( divided into five groups ) in phase 3 and phase 4 abpc ampicillin , cva / ampc clavulanic acid / amoxicillin , pipc piperacillin , taz / pipc tazobactam / piperacillin , ccl cefaclor , cdtr cefditoren , cfpn cefcapene , cpdx cefpodoxime , cfdn cefdinir , cftm cefteram , ctm cefotiam , ctrx ceftriaxone , ctx cefotaxime , azm azithromycin , cam clarithromycin , rkm rokitamycin , tel telithromycin , frpm faropenem , tbpm tebipenem , papm panipenem , mepm meropenem , drpm doripenem , lvfx levofloxacin , tflx tosufloxacin the mic90 values of antimicrobial agents for h. influenzae , categorized by degrees of resistance , are as follows : in the blnas group , the oral antimicrobial agents with the lowest mic90 were cdtr and lvfx ( 0.063 g / ml ) in phase 3 , and lvfx and tflx ( 0.063 g / ml ) in phase 4 , whereas the intravenous agent with the lowest mic90 was taz / pipc ( 0.063 g / ml ) in both phase 3 and phase 4 . in the blnai , blnar , and blpar groups , the oral antimicrobial agent with the lowest mic90 was lvfx ( 0.063 g / ml ) in phase 3 , and lvfx and tflx ( 0.063 g / ml ) in phase 4 . in the blnai group , the intravenous antimicrobial agent with the lowest mic90 was taz / pipc ( 0.125 g / ml ) in phase 3 , and pipc and taz / pipc ( 0.125 g / ml ) in phase 4 . in the blnar group , the intravenous antimicrobial agents with the lowest mic90 were pipc , taz / pipc , and ctrx ( 0.25 g / ml ) in phase 3 , and taz / pipc ( 0.125 g / ml ) in phase 4 . in the blpar group , the intravenous antimicrobial agent with the lowest mic90 was taz / pipc in both phase 3 and phase 4 ( phase 3 , 0.063 g / ml ; phase 4 , 0.125 g / ml ) . in the blpacr group , the oral antimicrobial agent with the lowest mic90 was cdtr and lvfx ( 0.25 g / ml ) in phase 3 , and lvfx and tflx ( 0.063 g / ml ) in phase 4 , whereas the intravenous antimicrobial agent with the lowest mic90 was taz / pipc in both phase 3 and phase 4 ( phase 3 , 0.125 g / ml ; phase 4 , 0.063 g / ml ) . the frequency of isolation was compared between drug - resistant strains and blnas strains in relationship to six background factors : sex , age , presence or absence of siblings , attendance or nonattendance at a daycare center , siblings attendance or nonattendance at a daycare center , and prior administration of antimicrobial agents within 1 month ( table 3 ) . in phase 3 , the isolation rate of drug - resistant strains was higher ( p = 0.005 ) in cases with prior administration of antimicrobial agents.table 3number of cases of -lactamase - nonproducing abpc - sensitive strain ( blnas ) or -lactamase - nonproducing abpc - intermediately resistant strain ( blnai ) + -lactamase - nonproducing abpc - resistant strain ( blnar ) according to background factorbackground factorphase 3phase 4number of casesstatisticsnumber of casesstatisticsblnasblnai + blnarblnasblnai + blnarsex boy7812391152 girl46111p = 0.060770128p = 0.6495age category infant26402874 toddler91177p = 0.4703122196p = 0.0546 schoolchild13171111sibling / siblings yes8014494179 no5191p = 0.968867102p = 0.2684group daycare yes7814188147 no4883p = 0.846770108p = 0.6972group daycare ( siblings ) yes6211470122 no816p = 0.85522137p = 0.9722previous use of antimicrobial agents yes5813987176 no7597p = 0.004774105p = 0.0765 penicillins8292046 cephems29853693 macrolides21554074 -lactam3610353123 macrolides2155p = 0.78324074p = 0.3754abpc ampicillin number of cases of -lactamase - nonproducing abpc - sensitive strain ( blnas ) or -lactamase - nonproducing abpc - intermediately resistant strain ( blnai ) + -lactamase - nonproducing abpc - resistant strain ( blnar ) according to background factor the drug - resistant pathogen surveillance group in pediatric infectious disease has continued to conduct national surveillance for the antimicrobial susceptibility of h. influenzae since 2000 . the previous study reported that the frequency of blnar strains dramatically increased from 28.8 % in phase 1 ( 20002001 ) to 59.3 % in phase 2 ( 2004 ) . an abpc mic of 2 g / ml or more was used as a criterion for the identification of blnar strains in phase 1 and phase 2 . had an abpc mic of 4 g / ml or more been used as in the current study , the frequency of blnar strains would have decreased to 11.4 % and 43.4 % in phases 1 and 2 , respectively . nonetheless , the distribution of blnar strains still increased dramatically between phase 1 and phase 2 . in this study , the frequency of blnar strains was 38.3 % in phase 3 and 37.8 % in phase 4 , indicating a downward tendency from its peak in phase 2 . according to a report on the national surveillance conducted in spain , the frequency of blnar strains gradually decreased from 13.5 % in 19961997 to 0.7 % in 20062007 . possible reasons for the decline in the number of blnar strains are changes in the number of prescriptions and clonal spread of sensitive strains . a positive correlation between the dosage of antimicrobial agents and the development of drug resistance has been observed with the use of population genetic methods . owing to the widespread use of guidelines in the pediatric field [ 9 , 10 ] , the proper use of antimicrobial agents may have been promoted in japan . in contrast to the decline in the number of blnar strains , the number of -lactamase - producing strains , which had continued to decrease , from 8.3 % in phase 1 to 6.4 % in phase 2 , and to 4.4 % in phase 3 , increased to 8.7 % in phase 4 . this difference was greatly influenced by the blpacr strains , whose number increased from 1.6 % in phase 3 to 4.8 % in phase 4 . the blpacr strains , which have two mechanisms of antimicrobial resistance that comprise -lactamase production and mutations in ftsi genes , show resistance to many -lactams . high detection rates of blpacr strains have been reported in france ( 13.9 % ) from 1999 to 2000 and in spain ( 22.4 % ) from 2005 to 2007 , and in some cases clonal dissemination of the blpacr strains has been observed [ 12 , 13 ] . a similar phenomenon may have happened in japanese children ; analysis of clonality using multilocus sequence typing or pulsed - field gel electrophoresis method will thus be required to explain the increase of blpacr strains . the blnar strains , which have mutations in the ftsi genes that make pbp3 with poor affinity for antimicrobial agents , showed reduced susceptibility to various types of -lactams including cephems . when the number of blnar strains rapidly increased between phase 1 and phase 2 , the mic90 values of most -lactams showed a corresponding 2- to 4 fold increase . on the other hand , the blnar strains showed a trend toward improved susceptibility after phase 2 , when the number of blnar strains decreased and the mic90 value of each -lactam antimicrobial agent decreased from one half to one fourth between phase 2 and phase 3 . there were no increases in mic90 values of -lactams between phase 3 and phase 4 with the exception of cdtr , cfpn , and papm , which showed a 2-fold increase . however , the mic90 values of -lactams in blnar strains were 2- to 4 fold higher in phase 3 and 2- to 8 fold higher in phase 4 , when compared with blnas strains . as blnar strains show lower susceptibility to most oral -lactams , it is considered that limited treatment options will continue to be a particular problem . in this study , the antimicrobial agents with the lowest mic90 were lvfx in phase 3 and lvfx and tflx in phase 4 . fluoroquinolones , which are not affected by mutations in ftsi genes , showed lower mic50 and mic90 values of 0.063 g / ml for the blnar strains . although the mic of lvfx was continuously measured in phases 3 and 4 , no reduced susceptibility was observed . however , the emergence of fluoroquinolone - resistant h. influenzae strains has been detected in adult patients who use fluoroquinolones frequently . treatment with tflx in pediatric patients with otitis media or pneumonia has been covered by insurance since 2009 ; the use of fluoroquinolones in pediatric patients can thus be expected to increase steadily . the proper use of fluoroquinolones is extremely important if we are to prevent fluoroquinolone - resistant strains from spreading in pediatric patients . in japan , hib vaccine was introduced as a voluntary vaccination in december 2008 , between phases 3 and 4 . two years after its introduction , at the end of 2010 , publicly subsidized vaccines became available , and since then the incidence of invasive hib infection has decreased because of an improvement in the vaccination rate . in our study , 14 ( 3.6 % ) and 23 hib strains ( 4.8 % ) were isolated in phase 3 and phase 4 , respectively , indicating no decrease in the number of hib cases after the introduction of the vaccine . however , the strains in phase 4 were acquired between january and june 2010 , before the initiation of the publicly subsidized vaccine program ; it is likely that the hib vaccine was not then widely available . we will have to wait for the next phase of surveillance to confirm the effect of vaccination on hib detection rates . during phase 1 and phase 2 surveillance , the relationship between three background factors ( age , prior administration of antimicrobial agents , and attendance at a daycare center ) and the isolation rate of drug - resistant strains was examined , and there were significant differences in age and prior administration of antimicrobial agents in phase 2 . in this study , in relationship to a total of six background factors , including the three factors from the previous study and three additional factors ( sex , siblings , and siblings attendance at a daycare center ) , there were significant differences as regards prior administration of antimicrobial agents in phase 3 . in phase 3 , the isolation rate of drug - resistant strains was high in those cases with prior administration of antimicrobial agents . in phase 2 , there were differences related to the types of antimicrobial agents ; the frequency of drug - resistant strains in the cases treated with -lactams was higher than that in the cases treated with macrolides , while no differences were observed in phase 3 . although the mutations in pbp3 in the blnar strains are considered to be easily induced by the abuse of the oral cephems , no significant difference was found among penicillins , cephems , and macrolides . it is likely that each type of antimicrobial agent was prescribed in a balanced manner . in relationship to other background factors , no significant difference was found in the frequency of drug - resistant strains . in phase 2 , the isolation rate of drug - resistant strains was significantly higher in children under 3 years of age , while no such difference was found in phases 3 and 4 ( data not shown ) . as there was also an apparent decrease in the number of the blnar strains in infants , similar future transitions deserve attention . we would like to continue conducting surveillance so that we can provide useful information on the drug resistance of h. influenzae for use in clinical practice .

the drug - resistant pathogen surveillance group in pediatric infectious disease conducted national surveillance for haemophilus influenzae in 2007 ( phase 3 ) and 2010 ( phase 4 ) , following the previous surveillance conducted from 2000 to 2001 ( phase 1 ) and in 2004 ( phase 2 ) . we examined the antimicrobial susceptibility for h. influenzae derived from clinical specimens of pediatric patients collected nationwide from 27 institutions during phases 3 ( 386 strains ) and 4 ( 484 strains ) . the frequency of -lactamase - nonproducing ampicillin ( abpc)-resistant ( blnar ) strains , which rapidly increased from 11.4 % in phase 1 to 43.4 % in phase 2 , has gradually decreased from 38.3 % in phase 3 to 37.8 % in phase 4 . in contrast , on the other hand , the frequency of -lactamase - producing strains , which continuously decreased from 8.3 % in phase 1 to 4.4 % in phase 3 , has increased to 8.7 % in phase 4 . prevalence of -lactamase - producing clavulanic acid / amoxicillin - resistant ( blpacr ) strains , especially , has increased from 1.6 % in phase 3 to 4.8 % in phase 4 . the oral antimicrobial agents with the lowest mic90 were levofloxacin in both phases , and tosufloxacin in phase 4 ( 0.063 g / ml ) , whereas for intravenous use the corresponding agent was tazobactam / piperacillin in both phases ( 0.125 g / ml ) . there was no increase in the mic90 of most -lactams between phase 3 and phase 4 . in relationship to sex , age , presence of siblings , attendance at a daycare center , siblings attendance at a daycare center , and prior administration of antimicrobial agents within 1 month , the frequency of -lactamase - nonproducing abpc - intermediately resistant ( blnai ) strains + blnar strains was high ( p = 0.005 ) in cases with prior administration of antimicrobial agents in phase 3 .

Introduction Materials and methods Strains, antimicrobial susceptibility testing, and capsular typing for serotype b strains Background factors and statistical analysis Results Discussion

the drug - resistant pathogen surveillance group in pediatric infectious disease reported that from phase 1 ( 20002001 ) to phase 2 ( 2004 ) of nationwide surveillance there was a rapid increase in the distribution of blnar strains and a decrease in -lactamase - nonproducing abpc - sensitive ( blnas ) strains . we collected h. influenzae isolated from clinical specimens taken from pediatric patients at 27 institutions nationwide , all of which participated in the drug - resistant pathogen surveillance group in pediatric infectious disease , and used the 386 strains accumulated from january to june in 2007 for phase 3 and the 484 strains accumulated from january to june in 2010 for phase 4 . the strains were classified according to the clsi criteria : that is , -lactamase - nonproducing strains were classified into blnas strains , for which the mic for abpc was 1 g / ml or less ; -lactamase - nonproducing abpc - intermediately resistant ( blnai ) strains , with mic for abpc of 2 g / ml ; and blnar strains , with mic for abpc of 4 g / ml or more . to examine the relationship between background factors and the development of drug resistance , the frequency of isolation was compared between drug - resistant strains and blnas strains in relationship to six background factors , including sex , age , presence or absence of siblings , attendance or nonattendance at a daycare center , siblings attendance or nonattendance at a daycare center , and prior administration of antimicrobial agents within 1 month . we collected h. influenzae isolated from clinical specimens taken from pediatric patients at 27 institutions nationwide , all of which participated in the drug - resistant pathogen surveillance group in pediatric infectious disease , and used the 386 strains accumulated from january to june in 2007 for phase 3 and the 484 strains accumulated from january to june in 2010 for phase 4 . to examine the relationship between background factors and the development of drug resistance , the frequency of isolation was compared between drug - resistant strains and blnas strains in relationship to six background factors , including sex , age , presence or absence of siblings , attendance or nonattendance at a daycare center , siblings attendance or nonattendance at a daycare center , and prior administration of antimicrobial agents within 1 month . blnas , -lactamase - nonproducing abpc - sensitive strain ; blnai , -lactamase - nonproducing abpc - intermediately resistant strain ; blnar , -lactamase - nonproducing abpc - resistant strain ; blpar , -lactamase - producing abpc - resistant strain ; blpacr , -lactamase - producing cva / ampc - resistant strain distribution of haemophilus influenzae strains classified by ampicillin ( abpc ) or clavulanic acid / amoxicillin ( cva / ampc ) resistance in phases 3 and 4 . blnas , -lactamase - nonproducing abpc - sensitive strain ; blnai , -lactamase - nonproducing abpc - intermediately resistant strain ; blnar , -lactamase - nonproducing abpc - resistant strain ; blpar , -lactamase - producing abpc - resistant strain ; blpacr , -lactamase - producing cva / ampc - resistant strain distribution of haemophilus influenzae serotype b strains classified by ampicillin ( abpc ) or clavulanic acid / amoxicillin ( cva / ampc ) resistance in phases 3 and 4 . in phase 3 with a total of 386 strains , the oral antimicrobial agent with the lowest mic90 was lvfx ( 0.063 g / ml ) , followed by cdtr ( 0.25 g / ml ) . in phase 4 with a total of 484 strains , the oral antimicrobial agent with the lowest mic90 was lvfx and tflx ( 0.063 g / ml ) , followed by cdtr ( 0.25 g / ml ) . between phase 3 and phase 4 , there was a twofold increase in the mic90 values of papm ( 24 g / ml ) and mepm ( 0.250.5 g / ml ) , but there were no changes in those of other drugs.table 1susceptibilities for haemophilus influenzae in phase 3 and phase 4phase 3phase 4number of strains386484micmic50mic90mic rangemic50mic90mic rangeabpc280.12>128280.063>128cva / ampc480.2532480.12516pipc0.0630.250.063>1280.0630.250.063>128taz / pipc0.0630.1250.06310.0630.1250.0630.25ccl16640.25>12816640.25128cdtr0.1250.250.0630.50.1250.250.0631cfpn120.0634120.0638cpdx240.0638240.0638cfdn280.06316280.06316cftm0.510.06320.510.0632ctm8640.0631288640.125128ctrx0.1250.250.0630.50.1250.250.0630.5ctx0.510.06340.510.0634azm120.12564120.0634cam481>64480.532rkm8161328160.06332tel120.258frpm240.0634240.0638tbpm0.2510.0632papm120.0634140.0638mepm0.1250.250.06310.1250.50.0631drpm0.520.0634lvfx0.0630.0630.0630.50.0630.0630.0630.5tflx0.0630.0630.0632abpc ampicillin , cva / ampc clavulanic acid / amoxicillin , pipc piperacillin , taz / pipc tazobactam / piperacillin , ccl cefaclor , cdtr cefditoren , cfpn cefcapene , cpdx cefpodoxime , cfdn cefdinir , cftm cefteram , ctm cefotiam , ctrx ceftriaxone , ctx cefotaxime , azm azithromycin , cam clarithromycin , rkm rokitamycin , tel telithromycin , frpm faropenem , tbpm tebipenem , papm panipenem , mepm meropenem , drpm doripenem , lvfx levofloxacin , tflx tosufloxacin susceptibilities for haemophilus influenzae in phase 3 and phase 4 abpc ampicillin , cva / ampc clavulanic acid / amoxicillin , pipc piperacillin , taz / pipc tazobactam / piperacillin , ccl cefaclor , cdtr cefditoren , cfpn cefcapene , cpdx cefpodoxime , cfdn cefdinir , cftm cefteram , ctm cefotiam , ctrx ceftriaxone , ctx cefotaxime , azm azithromycin , cam clarithromycin , rkm rokitamycin , tel telithromycin , frpm faropenem , tbpm tebipenem , papm panipenem , mepm meropenem , drpm doripenem , lvfx levofloxacin , tflx tosufloxacin table 2 shows the mic50 , mic90 , and mic range of antimicrobial agents for h. influenzae divided into the following five groups by degrees of resistance , the blnas , blnai , blnar , blpar , and blpacr groups , respectively . in both phase 3 and phase 4 , the mic50 values of pipc and taz / pipc were the same in the blnas and blnar groups ( 0.063 g / ml).table 2suscepsibilities for haemophilus influenzae ( divided into five groups ) in phase 3 and phase 4classblnasblnaiblnarphasephase 3phase 4phase 3phase 4phase 3phase 4no . of strains ( % ) 11 ( 2.8)19 ( 3.9)6 ( 1.6)23 ( 4.8)micmic50mic90mic rangemic50mic90mic rangemic50mic90mic rangemic50mic90mic rangeabpc128>1282 to > 12832>12816 to > 128>128>128>128 to > 128>128>12864 to > 128cva / ampc140.254240.54816816816816pipc128>1280.25 to > 12816>1282 to > 128>128>128>128 to > 12864>1288 to > 128taz / pipc0.0630.0630.0630.0630.0630.1250.0630.1250.0630.1250.0630.1250.0630.0630.0630.125ccl832264832232641284128326416128cdtr0.0630.1250.0630.250.0630.250.0630.50.1250.250.1250.250.250.250.0630.25cfpn0.06310.06320.510.0631220.52220.52cpdx0.06320.0634120.063244242424cfdn0.2520.1254140.25448284828cftm0.0630.50.0630.50.50.50.0630.50.510.510.510.251ctm2320.5644160.51664128812832648128ctrx0.0630.250.0630.250.0630.250.0630.250.250.250.1250.250.250.250.0630.5ctx0.0630.50.06310.250.50.0631110.51110.251azm120.5640.510.2511640.564120.54cam882 to > 6448284>644 to > 64816416rkm8164164818282888216tel24141818frpm0.520.12520.520.063422222414tbpm0.0630.50.0630.50.510.0632papm0.520.06320.2540.0634140.54280.258mepm0.0630.250.0630.50.0630.250.0630.250.1250.250.0630.250.250.50.0631drpm0.12510.0631120.0632lvfx0.0630.0630.0630.0630.0630.0630.0630.0630.0630.250.0630.250.0630.0630.0630.063tflx0.0630.0630.0630.0630.0630.0630.0630.063abpc ampicillin , cva / ampc clavulanic acid / amoxicillin , pipc piperacillin , taz / pipc tazobactam / piperacillin , ccl cefaclor , cdtr cefditoren , cfpn cefcapene , cpdx cefpodoxime , cfdn cefdinir , cftm cefteram , ctm cefotiam , ctrx ceftriaxone , ctx cefotaxime , azm azithromycin , cam clarithromycin , rkm rokitamycin , tel telithromycin , frpm faropenem , tbpm tebipenem , papm panipenem , mepm meropenem , drpm doripenem , lvfx levofloxacin , tflx tosufloxacin suscepsibilities for haemophilus influenzae ( divided into five groups ) in phase 3 and phase 4 abpc ampicillin , cva / ampc clavulanic acid / amoxicillin , pipc piperacillin , taz / pipc tazobactam / piperacillin , ccl cefaclor , cdtr cefditoren , cfpn cefcapene , cpdx cefpodoxime , cfdn cefdinir , cftm cefteram , ctm cefotiam , ctrx ceftriaxone , ctx cefotaxime , azm azithromycin , cam clarithromycin , rkm rokitamycin , tel telithromycin , frpm faropenem , tbpm tebipenem , papm panipenem , mepm meropenem , drpm doripenem , lvfx levofloxacin , tflx tosufloxacin the mic90 values of antimicrobial agents for h. influenzae , categorized by degrees of resistance , are as follows : in the blnas group , the oral antimicrobial agents with the lowest mic90 were cdtr and lvfx ( 0.063 g / ml ) in phase 3 , and lvfx and tflx ( 0.063 g / ml ) in phase 4 , whereas the intravenous agent with the lowest mic90 was taz / pipc ( 0.063 g / ml ) in both phase 3 and phase 4 . in the blnai , blnar , and blpar groups , the oral antimicrobial agent with the lowest mic90 was lvfx ( 0.063 g / ml ) in phase 3 , and lvfx and tflx ( 0.063 g / ml ) in phase 4 . in the blnai group , the intravenous antimicrobial agent with the lowest mic90 was taz / pipc ( 0.125 g / ml ) in phase 3 , and pipc and taz / pipc ( 0.125 g / ml ) in phase 4 . in the blnar group , the intravenous antimicrobial agents with the lowest mic90 were pipc , taz / pipc , and ctrx ( 0.25 g / ml ) in phase 3 , and taz / pipc ( 0.125 g / ml ) in phase 4 . in the blpar group , the intravenous antimicrobial agent with the lowest mic90 was taz / pipc in both phase 3 and phase 4 ( phase 3 , 0.063 g / ml ; phase 4 , 0.125 g / ml ) . in the blpacr group , the oral antimicrobial agent with the lowest mic90 was cdtr and lvfx ( 0.25 g / ml ) in phase 3 , and lvfx and tflx ( 0.063 g / ml ) in phase 4 , whereas the intravenous antimicrobial agent with the lowest mic90 was taz / pipc in both phase 3 and phase 4 ( phase 3 , 0.125 g / ml ; phase 4 , 0.063 g / ml ) . the frequency of isolation was compared between drug - resistant strains and blnas strains in relationship to six background factors : sex , age , presence or absence of siblings , attendance or nonattendance at a daycare center , siblings attendance or nonattendance at a daycare center , and prior administration of antimicrobial agents within 1 month ( table 3 ) . in phase 3 , the isolation rate of drug - resistant strains was higher ( p = 0.005 ) in cases with prior administration of antimicrobial agents.table 3number of cases of -lactamase - nonproducing abpc - sensitive strain ( blnas ) or -lactamase - nonproducing abpc - intermediately resistant strain ( blnai ) + -lactamase - nonproducing abpc - resistant strain ( blnar ) according to background factorbackground factorphase 3phase 4number of casesstatisticsnumber of casesstatisticsblnasblnai + blnarblnasblnai + blnarsex boy7812391152 girl46111p = 0.060770128p = 0.6495age category infant26402874 toddler91177p = 0.4703122196p = 0.0546 schoolchild13171111sibling / siblings yes8014494179 no5191p = 0.968867102p = 0.2684group daycare yes7814188147 no4883p = 0.846770108p = 0.6972group daycare ( siblings ) yes6211470122 no816p = 0.85522137p = 0.9722previous use of antimicrobial agents yes5813987176 no7597p = 0.004774105p = 0.0765 penicillins8292046 cephems29853693 macrolides21554074 -lactam3610353123 macrolides2155p = 0.78324074p = 0.3754abpc ampicillin number of cases of -lactamase - nonproducing abpc - sensitive strain ( blnas ) or -lactamase - nonproducing abpc - intermediately resistant strain ( blnai ) + -lactamase - nonproducing abpc - resistant strain ( blnar ) according to background factor the drug - resistant pathogen surveillance group in pediatric infectious disease has continued to conduct national surveillance for the antimicrobial susceptibility of h. influenzae since 2000 . in this study , the frequency of blnar strains was 38.3 % in phase 3 and 37.8 % in phase 4 , indicating a downward tendency from its peak in phase 2 . in contrast to the decline in the number of blnar strains , the number of -lactamase - producing strains , which had continued to decrease , from 8.3 % in phase 1 to 6.4 % in phase 2 , and to 4.4 % in phase 3 , increased to 8.7 % in phase 4 . during phase 1 and phase 2 surveillance , the relationship between three background factors ( age , prior administration of antimicrobial agents , and attendance at a daycare center ) and the isolation rate of drug - resistant strains was examined , and there were significant differences in age and prior administration of antimicrobial agents in phase 2 . in this study , in relationship to a total of six background factors , including the three factors from the previous study and three additional factors ( sex , siblings , and siblings attendance at a daycare center ) , there were significant differences as regards prior administration of antimicrobial agents in phase 3 .

many cancers metastasize to bone , with the most common sites of origin of primary disease being breast , lung , thyroid , kidney , prostate , and malignant melanoma of the skin . the presence of tumor in the bone can lead to local symptoms such as pain , spinal cord compression , and pathologic fracture , as well as systemic effects caused by hypercalcemia . the work - up and treatment of bone metastases requires input and interventions from many medical disciplines , including radiologists , orthopedic surgeons or neurosurgeons , radiation oncologists , medical oncologists , pain medicine specialists , physical medicine and rehabilitation physicians , and palliative care professionals . the delivery of radiation therapy to these patients requires communication and coordination of scheduling with these other specialists . furthermore , the aggressiveness of treatment must take into account patient factors such as performance status and co - morbidities , tumor factors such as stage and histology , and treatment factors such as sequencing and risks of concurrent therapy , , . as a palliative intervention , radiotherapy is effective and efficient at treating painful bone metastases , and the side effects associated with its use are manageable and usually self - limiting in nature . between 50% and 80% of patients gain at least partial relief of their pain following external beam radiotherapy ( ebrt ) , and complete relief may be seen in up to one - third . external beam radiotherapy may be delivered to the same anatomic site of affected bone in the case of recurrent pain . technological advances have created interest in the possibility that highly conformal therapies may improve either the rates of pain relief or the duration of the results of treatment , especially in cases of tumors located in bones of the spine . these treatments are termed stereotactic body radiation therapy ( sbrt ) , or stereotactic ablative body radiotherapy ( sabr ) , and are given by machines that deliver intensity modulated radiation therapy ( imrt ) , cyberknife therapy , tomotherapy , or proton therapy . patients with spinal cord compression may receive ebrt primarily or as an adjuvant treatment after surgical decompression . kyphoplasty or vertebroplasty may be used in cases where there is no spinal cord compression , but where spinal instability is noted and contributes to metastatic bone pain . furthermore , injectable radiopharmaceuticals such as strontium 89 , samarium 153 , and radium 223 may be delivered to patients with widespread tumors whose histologies are osteoblastic and therefore easily visualized on a technetium 99 bone scan . finally , the addition of osteoclast inhibiting agents may be considered concurrently or sequentially with ebrt . the driving forces behind the emergence of radiotherapy guidelines include a desire to maximize pain relief and functional capabilities of an individual patient while minimizing the risks of treatment toxicity . the treatment guidelines are also meant to serve as a means by which to guarantee a minimum standard of care across geographic locales and different practice settings . and , given some areas of incomplete data regarding the proper choice for treatment , one goal of the guidelines is to acknowledge and address the controversies that arise due to that lack of complete data . from a societal standpoint , the guidelines provide a means to assess the best practice patterns as developed countries face an increased number of cancer patients with resource constraints and many developing countries struggle with throughput limitations on antiquated machinery . the prelude to many of the questions posed and answers offered by the existing bone metastases treatment guidelines was contained in previous international consensus conference bone metastases treatment recommendation publications . the first international consensus workshop on radiation in the treatment of metastatic and locally advanced cancer convened in the united states in 1990 . a group of 116 experts evaluated the available palliative radiotherapy data and generated consensus statements for the treatment of bone metastases , amongst other clinical circumstances . those statements included treatment pathway recommendations , an assessment of international variations in treatment approach , the effects of successful treatment on quality of life , and the role of economic factors in the management of this patient group . the second workshop on palliative radiotherapy and symptom control convened in london in 2000 and confirmed the efficacy of ebrt in controlling pain caused by metastatic bone disease . that group reviewed the efficacy of a single 8 gy fraction , they better defined the proper use of radiopharmaceuticals for patients with widespread painful disease , and they recommended the standardization of response measurement that led to the development of the international consensus on palliative radiotherapy endpoints document . finally , the third international consensus conference workshop was held in conjunction with the astro meeting with representatives from astro , european society for therapeutic radiology and oncology ( estro ) , trans - tasman radiation oncology group ( trog ) and canadian association of radiation oncology ( caro ) in san diego , california , in 2010 and called for both formal treatment guidelines and a means by which to enhance palliative radiotherapy efforts in developing countries around the world . the american college of radiology ( acr ) appropriateness criteria format employs common clinical circumstances , or variants , which serve as a means for an expert panel to vote upon the most appropriate interventions for that scenario ( table 1 ) . the panel members collectively base their assessments upon the results of published literature , though the clinical experience of those experts may influence their decision - making , especially in situations where the available data set is incomplete . the bone metastases treatment panel consists of representatives from radiation oncology , nuclear medicine , orthopedic surgery , and medical oncology . the clinical case scenarios allow for recommendations about the best combination of interventions as well as an assessment of the proper radiotherapy treatment set - ups and fractionation schemes . while previous acr publications have included all types of bone metastases situations in a single manuscript , the increasing complexity of treatment of spine metastases and spinal cord compression led to the division of spine and non - spine topics . the most recent update of the non - spine topic has just been published , while the spine topic update is still being formulated . the first variant in the non - spine topic describes a patient with an excellent performance status , a favorable life expectancy , and an asymptomatic femur lesion which does not pose an obvious risk for pathologic fracture . while the authors acknowledge that research has begun to determine whether patients with bone - only metastatic disease and otherwise favorable findings may be treated aggressively , they stop short of endorsing curative - intent therapy for patients with oligometastases because the available data do not yet prove the usefulness of such an approach . their recommendations therefore call for an osteoclast inhibitor and a hormone blocking agent , with radiotherapy reserved for an oligometastatic treatment trial . the results of ongoing research may well come to indicate that patients in this most favorable clinical circumstance of metastatic disease should be treated more aggressively than others with less favorable prognostic indicators . the second variant describes a patient with a good performance status who has a painful lesion in a weight - bearing bone . the task force defined the need for quickly establishing a pain medicine regimen while concurrently consulting an orthopedic surgeon to assess the need for surgical pinning to prevent pathologic fracture , , . given a low risk of fracture determined by the surgeon , the team recommended external beam radiotherapy ( ebrt ) based upon ct , fluoroscopic , or clinical simulation , with radiation delivery through anterior and posterior fields sparing a skin strip to minimize the risk of long term lymphedema of the extremity . while the panel detailed the pain relief equivalency between a single 8 gy fraction and multi - fraction schedules , they pointed out the data which suggests that the use of fractionated regimens might minimize the risk of subsequent pathologic fracture in this setting . the group essentially declared that pain relief equivalency has been conclusively determined for either single fraction or multi - fraction regimens , obviating the need for further research to examine that question . finally , the existence of a fairly significant tumor burden in that patient led to recommendations for considering systemic chemotherapy and osteoclast inhibitors . in the third variant , the patient has suffered a pathologic fracture from a lytic metastasis in a weight - bearing bone that required surgical stabilization . the panel recommended postoperative radiotherapy with 30 gy in 10 fractions planned by ct , fluoroscopic or clinical simulation , with anterior and posterior opposed fields and a skin strip spared to once again minimize the risk of long term lymphedema . the vignette is valuable in its ability to highlight the need for orthopedic consultation to assess and provide surgical stabilization as well as the need for communication for the patient to receive the necessary post - operative adjuvant radiotherapy . given a good performance status and significant tumor burden , recommendations were made for considerations of systemic chemotherapy , hormonal ablation treatment , and an osteoclast inhibitor . the patient in variant number 4 has previously received palliative radiotherapy for a site of painful bony disease with a good initial response , though their pain has recurred and the panel was tasked with evaluating the safety and efficacy of ebrt re - treatment to the same painful site . the panel described the available re - treatment data as being of low quality because it was mostly retrospective , single - institutional , and dated , , , , , . given those limitations , the group recommended caution when treating volumes containing normal tissue structures which might suffer side effects from the combined palliative doses . the brachial plexus was found to be one normal tissue at risk in this particular case , and the team also reminded the reader to re - evaluate the affected long bone for risk of pathologic fracture before offering re - irradiation . the panel therefore recommended treatment planned by ct , fluoroscopic , or clinical simulation , with anterior and posterior opposed fields sparing a skin strip to minimize the risk for upper extremity edema . given limited data regarding the best dose to use in this setting , the panel recommended placing the patient on an available re - treatment protocol . when completed , the results of that trial will need to serve as a template for the appropriateness of re - treatment to the same painful site , given the lack of prospective data available at this time . while the use of systemic chemotherapy was not thought to be wholly inappropriate , the patient 's poor performance status and prognosis led the group to suggest that the patient be seen by a palliative care team and be given the option to choose hospice care . the final variant deals with a patient who has a poor prognosis due to visceral metastases and who suffers from a single site of painful bone disease . the purpose of the case was to evaluate the panel 's views of supportive care with analgesic medications plus or minus ebrt . the group did recommend ebrt for the patient , but they were specific in their belief that the dose should be limited to a single 8 gy fraction in an effort to decrease time spent in treatment and discomfort from being transferred on and off the treatment table . the likelihood of an increased need for re - treatment to the same site in this patient is diminished by his short expected lifespan . it was recommended that ct , fluoroscopic , or clinical simulation may be used while preparing treatment through ap and pa directions while sparing a skin strip . anti - inflammatory medicines were described as the best method to manage any temporary flare reaction that might occur after the single fraction . finally , direct placement to hospice was thought to be a reasonable directive , either before or after the completion of the single fraction ebrt dosing . in its text , the acr appropriateness criteria bone metastases group suggested several general statements applicable to most or all five variants . first , ebrt was re - defined as an effective means to palliate the pain caused by metastatic bone disease , with rates of relief of 50%80% and equivalence for fractionation schemes including 30 gy in 10 fractions , 24 gy in 6 fractions , 20 gy in 5 fractions , and a single 8 gy fraction , , . the preferred treatment set - up and prescription points should follow those defined in the international consensus on palliative radiotherapy endpoints . the group determined that the use of highly conformal therapy with intensity - modulated radiation therapy ( imrt ) , stereotactic body radiation therapy ( sbrt ) , or proton therapy have not been proven for this subset of patients without spine disease . lastly , the need for concurrent pain medicine dosing and palliative care was seen to be imperative , with hospice admission not viewed as being mutually exclusive with the delivery of palliative radiotherapy for bone pain . the american society for radiation oncology ( astro ) has only recently begun creating clinical treatment guidelines , especially when compared to the longstanding existence of the acr appropriateness criteria . in 2009 the astro board of directors the astro group consisted of a neurosurgeon and palliative medicine expert as well as radiation oncologists from academic , private practice , and residency settings . the group was asked to create guidelines that were applicable to patients as well as healthcare providers , with one of the main themes being the integration of radiotherapy with other treatment modalities useful in the care of patients with painful bone metastases . the original literature search covered the most recent ten years of citations in the national library of medicine 's pubmed database and yielded over 4000 publications . within that group of papers were found 25 randomized clinical trials , 20 prospective single - arm studies , and 4 meta - analyses or systematic reviews . given the complexity of the clinical situations involved in the care of these patients , the task force was divided into subgroups to concentrate on those issues that fit each individual 's own expertise . the results of the subgroups ' work were subsequently presented to the entire group , made available online for public comment , and approved by the astro board of directors prior to publication . the format of the astro guidelines was based upon the task force answers to several questions posed by the board of directors . the first several questions dealt with the most appropriate external beam radiation therapy ( ebrt ) fractionation scheme to use for the treatment of painful bone metastases . while the rates of pain relief following ebrt appear to be similar across a wide array of dose fractionation schemes , one recent worldwide survey revealing that more than 101 fractionation schemes are used for this one clinical circumstance . the first goal of the task force was therefore to narrow the list of acceptable fractionation schemes to those which have been sufficiently studied in adequately - powered trials . similar to the acr group , they documented that several prospective , randomized trials have evaluated different dose - fractionation schedules , with the results suggesting equivalence in pain relief after schedules including 30 gy in 10 fractions , 24 gy in 6 fractions , 20 gy in 5 fractions , and a single 8 gy fraction , . the advantage of single fraction radiotherapy was seen to be increased convenience and decreased expense for the patient and their caregivers , while multiple fraction therapy was advantageous because it is associated with a lower incidence of re - treatment to the same painful site than in single fraction treatment ( 8% versus 20% , respectively ) . in answer to concerns raised about the safety of a single 8 gy fraction to anatomic sites historically considered to be sensitive to hypofractionated doses , the task force evaluated the literature but could not find any long term side effect risks that should deter clinicians from using a single dose to spine fields that contain the spinal cord or cauda equina . the re - treatment of metastatic bone disease causing recurrent pain after an initial course of ebrt was seen to be feasible with a reasonable rate of symptom relief , , , , , , . in echoing the acr findings , the task force noted that the available data was derived from studies where re - treatment was not the primary endpoint studied , and that many of the descriptions of re - treatment were based upon small numbers of patients . additionally , the authors cautioned that re - treatment may only be considered when taking into account the normal tissue tolerance of structures included in the treated volumes . the spinal cord and cauda equina were specifically mentioned as structures whose tolerance to the combined dosing must be taken into account when delivering a second course of ebrt to the spine . given significant interest in newer technologies amongst radiation oncologists and neurosurgeons , the astro task force enthusiastically recognized the promise for improvements in care with highly conformal therapy which includes all technologies that can deliver higher doses to metastatic bone disease with a steep dose gradient to spare adjacent normal structures . the team focused their analysis on the potential benefits of stereotactic body radiation therapy ( sbrt ) for metastases in spine bones , though they described that the available data for this intervention has to this point been accrued in single institutional studies with small numbers of patients whose responses have been measured with novel treatment outcomes . as such , the task force suggested that patients who receive sbrt should strongly be considered for the available treatment protocols to better accrue data about efficacy and toxicity measures . the theoretical advantage of sbrt for sparing spinal cord or cauda equina in the re - treatment of recurrent , painful spine lesions was documented in much greater detail than was true in the acr appropriateness criteria , , , , , . in an attempt to clarify confusion regarding the use of radiotherapy with other available interventions for painful metastatic bone disease , the astro task force clearly stated that ebrt is still necessary in situations where patients receive surgery for spinal cord compression or long bone stabilization , intravenous radiopharmaceuticals for widespread bone disease , osteoclast inhibitors , or kyphoplasty or vertebroplasty for lytic lesions causing spinal instability , , , . surgery was only recommended for patients with spinal cord compression who have a favorable prognosis and sufficient performance status to warrant the surgical risks and post - operative rehabilitation required for that degree of intervention . radiopharmaceuticals were deemed most appropriate in patients with widespread , painful osteoblastic metastases that are apparent on a technetium-99 bone scan . while the use of osteoclast inhibitors was seen as being a reasonable means by which to palliate bone pain and promote re - ossification , the task force pointed out that there are no data to suggest that the palliation of a single site of metastatic bone pain is superior with osteoclast inhibitors plus ebrt versus ebrt alone . finally , the task force described the theoretical advantage of using kyphoplasty or vertebroplasty for spinal instability caused by lytic metastases , though they shared the belief that the data proving those assumptions was mostly derived from retrospective , single institutional studies . in its conclusions , the astro group suggested that future bone metastases treatment trials should be made uniform by the measurement of consistent variables as defined by the international consensus on palliative radiotherapy endpoints while also assessing functional domains and quality of life with validated instruments such as the european organization for research and treatment of cancer bone metastases quality - of - life questionnaire , . the national comprehensive cancer network is made up of experts from cancer centers of excellence around the united states who designate representatives to committees that evaluate data and provide treatment options for most common cancers . while there is no specific nccn group designated to evaluate the use of radiotherapy for bone metastases , the topic is dealt with to varying degrees in the publications which deal with primary diagnoses that are most likely to metastasize to bone . the nccn guidelines also include a wider variety of author specialty types for each clinical site than do the acr and astro guidelines . furthermore , while radiation oncologists make up the majority of panel members on the acr and astro committees , radiation oncologists generally make up a distinct minority , or are a singular member , of the nccn committees . as such , the nccn guideline recommendations regarding radiotherapy for bone metastases are likely to result from less vigorous conversations and voting criteria than might be true for those offered by the acr and astro groups . the authors of the nccn non - small cell lung cancer guidelines provide the most guidance about radiotherapy dosing for painful bone metastases of any of the primary sites evaluated . bone metastases due to non - small cell lung cancer are separated into those with soft tissue mass versus those without soft tissue mass , with recommendations for 2030 gy in 510 fractions for the former circumstance and 830 gy in 110 fractions for the latter . the small cell lung cancer guidelines go so far as to only state that radiotherapy may provide excellent palliation of painful bone lesions . the kidney cancer guidelines hint at the notion of radiotherapy for oligometastases , describing that long - term progression - free survival has been noted in patients treated with radiotherapy for a single bone metastases and controlled primary disease . the multiple myeloma guidelines recommend a low dose of radiation therapy to between 10 and 30 gy for bone pain , impending pathologic fracture , or impending spinal cord compression . the multiple myeloma authors caution clinicians to limit the volume of irradiated fields to minimize the impact upon bone marrow given the potential for additional chemotherapy or stem cell harvest . the prostate cancer guidelines suggest that a single 8 gy dose should be used to treat painful bone disease , though in contradistinction to the acr and astro guidelines , the nccn prostate cancer authors suggest that vertebral metastases should receive a fractionated rather than single fraction dose . they also offer a recommendation to use radiopharmaceuticals such as strontium 89 or samarium 153 for widespread bony metastases . similarly , the thyroid cancer group only mentions ebrt in the setting of optimization of dosimetry for iodine 131 for treatment of painful bone metastases . the nccn adult cancer pain guidelines suggest that radiotherapy be considered for painful lesions which are likely to respond to antineoplastic therapies . lastly , neither the breast cancer treatment guidelines nor the palliative care guidelines mention the use of radiotherapy for painful bone disease , . the publication of treatment guidelines may cause angst for practitioners , given justifiable concerns that their decision - making autonomy may be threatened by a need to pigeon - hole clinical circumstances into pre - determined bins . it is certainly true that third party payers and the centers for medicare and medicaid services are interested in using guidelines to reward literature - based patterns of care while questioning treatment patterns that deviate sharply from those data . still , the advantages for the use of treatment guidelines include the provision of a minimum standard of care and a delineation of those topics which remain controversial enough to spur additional clinical trials to reach consensus on outcomes . the interest for the bone metastases treatment guidelines has been high , as is evidenced by the fact that the astro bone metastases treatment guidelines were the most frequently downloaded articles in 2011 from the international journal of radiation oncology , biology , and physics website . one might foresee that ongoing interest in guidelines , in general , will spur more formal comparisons of formatting and content that will aid in standardization across the publications from different societies . this convergence of guidelines would most certainly decrease any discrepancies that currently exist in recommendations offered by the task force groups . the national quality forum ( nqf ) has been tasked with measuring quality of care for specific clinical circumstances , to analyze reports of how frequently those quality measures are employed , and to provide guidance that improves patterns of care . the affordable care act requires that the nqf provide annual input to the department of health and human services regarding a national quality strategy that provides measures and tracks progress toward fulfilling those goals . the nqf cancer endorsement 2011 group will evaluate ebrt dose fractionation schemes for bone metastases treatment as its first potential measure of radiation oncology quality . if the nqf cancer committee and board of directors accept that measure , then the full implications of bone metastases guidelines will be ascertained . finally , virtually all of the guidelines products provide a disclaimer stating that the ultimate appropriateness of therapy relies on the judgment of treating physician while taking into account their experiences , the available clinical data , and the specific circumstances of the patient who is undergoing that care . similarly , the details of the guidelines evaluated in this manuscript are available online and should be read in detail prior to making conclusive comments about their content and recommendations .

bone metastases are a common manifestation of malignancy , and external beam radiotherapy ( ebrt ) effectively and safely palliates the pain caused by this clinical circumstance . the myriad of ebrt dosing schemes and complexities involved with coordinating radiotherapy with other interventions necessitated the need for bone metastases treatment guidelines . here we compare and contrast the bone metastases radiotherapy treatment guidelines recently published by the american society for radiation oncology ( astro ) and the american college of radiology ( acr ) . these evaluations acknowledge current controversies in treatment approaches , they evaluate the nuances of astro and acr task force decision - making regarding standard approaches to care , and they project the upcoming research results that may clarify approaches to palliative radiotherapy for bone metastases . the results of these two dedicated radiotherapy guidelines are compared to the brief mentions of radiotherapy for bone metastases in the national comprehensive cancer network ( nccn ) guidelines . finally , the paper describes how treatment guidelines may influence patterns of care and reimbursement by their use as quality measures by groups such as the national quality forum ( nqf ) .

Bone metastases as a clinical problem Radiotherapy for bone metastases Emergence of radiotherapy guidelines International Consensus Conference Bone Metastases treatment recommendations Formal radiotherapy bone metastases treatment guidelines American Society for Radiation Oncology (ASTRO) bone metastases guidelines National Comprehensive Cancer Network (NCCN) and bone metastases treatment recommendations Implications of bone metastases guidelines

many cancers metastasize to bone , with the most common sites of origin of primary disease being breast , lung , thyroid , kidney , prostate , and malignant melanoma of the skin . the presence of tumor in the bone can lead to local symptoms such as pain , spinal cord compression , and pathologic fracture , as well as systemic effects caused by hypercalcemia . the work - up and treatment of bone metastases requires input and interventions from many medical disciplines , including radiologists , orthopedic surgeons or neurosurgeons , radiation oncologists , medical oncologists , pain medicine specialists , physical medicine and rehabilitation physicians , and palliative care professionals . furthermore , the aggressiveness of treatment must take into account patient factors such as performance status and co - morbidities , tumor factors such as stage and histology , and treatment factors such as sequencing and risks of concurrent therapy , , . as a palliative intervention , radiotherapy is effective and efficient at treating painful bone metastases , and the side effects associated with its use are manageable and usually self - limiting in nature . between 50% and 80% of patients gain at least partial relief of their pain following external beam radiotherapy ( ebrt ) , and complete relief may be seen in up to one - third . external beam radiotherapy may be delivered to the same anatomic site of affected bone in the case of recurrent pain . technological advances have created interest in the possibility that highly conformal therapies may improve either the rates of pain relief or the duration of the results of treatment , especially in cases of tumors located in bones of the spine . these treatments are termed stereotactic body radiation therapy ( sbrt ) , or stereotactic ablative body radiotherapy ( sabr ) , and are given by machines that deliver intensity modulated radiation therapy ( imrt ) , cyberknife therapy , tomotherapy , or proton therapy . furthermore , injectable radiopharmaceuticals such as strontium 89 , samarium 153 , and radium 223 may be delivered to patients with widespread tumors whose histologies are osteoblastic and therefore easily visualized on a technetium 99 bone scan . finally , the addition of osteoclast inhibiting agents may be considered concurrently or sequentially with ebrt . the driving forces behind the emergence of radiotherapy guidelines include a desire to maximize pain relief and functional capabilities of an individual patient while minimizing the risks of treatment toxicity . the treatment guidelines are also meant to serve as a means by which to guarantee a minimum standard of care across geographic locales and different practice settings . from a societal standpoint , the guidelines provide a means to assess the best practice patterns as developed countries face an increased number of cancer patients with resource constraints and many developing countries struggle with throughput limitations on antiquated machinery . the prelude to many of the questions posed and answers offered by the existing bone metastases treatment guidelines was contained in previous international consensus conference bone metastases treatment recommendation publications . a group of 116 experts evaluated the available palliative radiotherapy data and generated consensus statements for the treatment of bone metastases , amongst other clinical circumstances . those statements included treatment pathway recommendations , an assessment of international variations in treatment approach , the effects of successful treatment on quality of life , and the role of economic factors in the management of this patient group . the second workshop on palliative radiotherapy and symptom control convened in london in 2000 and confirmed the efficacy of ebrt in controlling pain caused by metastatic bone disease . that group reviewed the efficacy of a single 8 gy fraction , they better defined the proper use of radiopharmaceuticals for patients with widespread painful disease , and they recommended the standardization of response measurement that led to the development of the international consensus on palliative radiotherapy endpoints document . finally , the third international consensus conference workshop was held in conjunction with the astro meeting with representatives from astro , european society for therapeutic radiology and oncology ( estro ) , trans - tasman radiation oncology group ( trog ) and canadian association of radiation oncology ( caro ) in san diego , california , in 2010 and called for both formal treatment guidelines and a means by which to enhance palliative radiotherapy efforts in developing countries around the world . the american college of radiology ( acr ) appropriateness criteria format employs common clinical circumstances , or variants , which serve as a means for an expert panel to vote upon the most appropriate interventions for that scenario ( table 1 ) . the panel members collectively base their assessments upon the results of published literature , though the clinical experience of those experts may influence their decision - making , especially in situations where the available data set is incomplete . the bone metastases treatment panel consists of representatives from radiation oncology , nuclear medicine , orthopedic surgery , and medical oncology . the clinical case scenarios allow for recommendations about the best combination of interventions as well as an assessment of the proper radiotherapy treatment set - ups and fractionation schemes . while previous acr publications have included all types of bone metastases situations in a single manuscript , the increasing complexity of treatment of spine metastases and spinal cord compression led to the division of spine and non - spine topics . the first variant in the non - spine topic describes a patient with an excellent performance status , a favorable life expectancy , and an asymptomatic femur lesion which does not pose an obvious risk for pathologic fracture . the results of ongoing research may well come to indicate that patients in this most favorable clinical circumstance of metastatic disease should be treated more aggressively than others with less favorable prognostic indicators . the task force defined the need for quickly establishing a pain medicine regimen while concurrently consulting an orthopedic surgeon to assess the need for surgical pinning to prevent pathologic fracture , , . given a low risk of fracture determined by the surgeon , the team recommended external beam radiotherapy ( ebrt ) based upon ct , fluoroscopic , or clinical simulation , with radiation delivery through anterior and posterior fields sparing a skin strip to minimize the risk of long term lymphedema of the extremity . while the panel detailed the pain relief equivalency between a single 8 gy fraction and multi - fraction schedules , they pointed out the data which suggests that the use of fractionated regimens might minimize the risk of subsequent pathologic fracture in this setting . the group essentially declared that pain relief equivalency has been conclusively determined for either single fraction or multi - fraction regimens , obviating the need for further research to examine that question . finally , the existence of a fairly significant tumor burden in that patient led to recommendations for considering systemic chemotherapy and osteoclast inhibitors . in the third variant , the patient has suffered a pathologic fracture from a lytic metastasis in a weight - bearing bone that required surgical stabilization . the panel recommended postoperative radiotherapy with 30 gy in 10 fractions planned by ct , fluoroscopic or clinical simulation , with anterior and posterior opposed fields and a skin strip spared to once again minimize the risk of long term lymphedema . the vignette is valuable in its ability to highlight the need for orthopedic consultation to assess and provide surgical stabilization as well as the need for communication for the patient to receive the necessary post - operative adjuvant radiotherapy . given a good performance status and significant tumor burden , recommendations were made for considerations of systemic chemotherapy , hormonal ablation treatment , and an osteoclast inhibitor . the patient in variant number 4 has previously received palliative radiotherapy for a site of painful bony disease with a good initial response , though their pain has recurred and the panel was tasked with evaluating the safety and efficacy of ebrt re - treatment to the same painful site . given those limitations , the group recommended caution when treating volumes containing normal tissue structures which might suffer side effects from the combined palliative doses . the brachial plexus was found to be one normal tissue at risk in this particular case , and the team also reminded the reader to re - evaluate the affected long bone for risk of pathologic fracture before offering re - irradiation . given limited data regarding the best dose to use in this setting , the panel recommended placing the patient on an available re - treatment protocol . when completed , the results of that trial will need to serve as a template for the appropriateness of re - treatment to the same painful site , given the lack of prospective data available at this time . while the use of systemic chemotherapy was not thought to be wholly inappropriate , the patient 's poor performance status and prognosis led the group to suggest that the patient be seen by a palliative care team and be given the option to choose hospice care . the group did recommend ebrt for the patient , but they were specific in their belief that the dose should be limited to a single 8 gy fraction in an effort to decrease time spent in treatment and discomfort from being transferred on and off the treatment table . the likelihood of an increased need for re - treatment to the same site in this patient is diminished by his short expected lifespan . finally , direct placement to hospice was thought to be a reasonable directive , either before or after the completion of the single fraction ebrt dosing . in its text , the acr appropriateness criteria bone metastases group suggested several general statements applicable to most or all five variants . first , ebrt was re - defined as an effective means to palliate the pain caused by metastatic bone disease , with rates of relief of 50%80% and equivalence for fractionation schemes including 30 gy in 10 fractions , 24 gy in 6 fractions , 20 gy in 5 fractions , and a single 8 gy fraction , , . the preferred treatment set - up and prescription points should follow those defined in the international consensus on palliative radiotherapy endpoints . lastly , the need for concurrent pain medicine dosing and palliative care was seen to be imperative , with hospice admission not viewed as being mutually exclusive with the delivery of palliative radiotherapy for bone pain . the american society for radiation oncology ( astro ) has only recently begun creating clinical treatment guidelines , especially when compared to the longstanding existence of the acr appropriateness criteria . the group was asked to create guidelines that were applicable to patients as well as healthcare providers , with one of the main themes being the integration of radiotherapy with other treatment modalities useful in the care of patients with painful bone metastases . the original literature search covered the most recent ten years of citations in the national library of medicine 's pubmed database and yielded over 4000 publications . within that group of papers were found 25 randomized clinical trials , 20 prospective single - arm studies , and 4 meta - analyses or systematic reviews . given the complexity of the clinical situations involved in the care of these patients , the task force was divided into subgroups to concentrate on those issues that fit each individual 's own expertise . the results of the subgroups ' work were subsequently presented to the entire group , made available online for public comment , and approved by the astro board of directors prior to publication . the format of the astro guidelines was based upon the task force answers to several questions posed by the board of directors . the first several questions dealt with the most appropriate external beam radiation therapy ( ebrt ) fractionation scheme to use for the treatment of painful bone metastases . while the rates of pain relief following ebrt appear to be similar across a wide array of dose fractionation schemes , one recent worldwide survey revealing that more than 101 fractionation schemes are used for this one clinical circumstance . the first goal of the task force was therefore to narrow the list of acceptable fractionation schemes to those which have been sufficiently studied in adequately - powered trials . similar to the acr group , they documented that several prospective , randomized trials have evaluated different dose - fractionation schedules , with the results suggesting equivalence in pain relief after schedules including 30 gy in 10 fractions , 24 gy in 6 fractions , 20 gy in 5 fractions , and a single 8 gy fraction , . the advantage of single fraction radiotherapy was seen to be increased convenience and decreased expense for the patient and their caregivers , while multiple fraction therapy was advantageous because it is associated with a lower incidence of re - treatment to the same painful site than in single fraction treatment ( 8% versus 20% , respectively ) . in answer to concerns raised about the safety of a single 8 gy fraction to anatomic sites historically considered to be sensitive to hypofractionated doses , the task force evaluated the literature but could not find any long term side effect risks that should deter clinicians from using a single dose to spine fields that contain the spinal cord or cauda equina . the re - treatment of metastatic bone disease causing recurrent pain after an initial course of ebrt was seen to be feasible with a reasonable rate of symptom relief , , , , , , . in echoing the acr findings , the task force noted that the available data was derived from studies where re - treatment was not the primary endpoint studied , and that many of the descriptions of re - treatment were based upon small numbers of patients . additionally , the authors cautioned that re - treatment may only be considered when taking into account the normal tissue tolerance of structures included in the treated volumes . the spinal cord and cauda equina were specifically mentioned as structures whose tolerance to the combined dosing must be taken into account when delivering a second course of ebrt to the spine . given significant interest in newer technologies amongst radiation oncologists and neurosurgeons , the astro task force enthusiastically recognized the promise for improvements in care with highly conformal therapy which includes all technologies that can deliver higher doses to metastatic bone disease with a steep dose gradient to spare adjacent normal structures . as such , the task force suggested that patients who receive sbrt should strongly be considered for the available treatment protocols to better accrue data about efficacy and toxicity measures . the theoretical advantage of sbrt for sparing spinal cord or cauda equina in the re - treatment of recurrent , painful spine lesions was documented in much greater detail than was true in the acr appropriateness criteria , , , , , . in an attempt to clarify confusion regarding the use of radiotherapy with other available interventions for painful metastatic bone disease , the astro task force clearly stated that ebrt is still necessary in situations where patients receive surgery for spinal cord compression or long bone stabilization , intravenous radiopharmaceuticals for widespread bone disease , osteoclast inhibitors , or kyphoplasty or vertebroplasty for lytic lesions causing spinal instability , , , . while the use of osteoclast inhibitors was seen as being a reasonable means by which to palliate bone pain and promote re - ossification , the task force pointed out that there are no data to suggest that the palliation of a single site of metastatic bone pain is superior with osteoclast inhibitors plus ebrt versus ebrt alone . finally , the task force described the theoretical advantage of using kyphoplasty or vertebroplasty for spinal instability caused by lytic metastases , though they shared the belief that the data proving those assumptions was mostly derived from retrospective , single institutional studies . in its conclusions , the astro group suggested that future bone metastases treatment trials should be made uniform by the measurement of consistent variables as defined by the international consensus on palliative radiotherapy endpoints while also assessing functional domains and quality of life with validated instruments such as the european organization for research and treatment of cancer bone metastases quality - of - life questionnaire , . the national comprehensive cancer network is made up of experts from cancer centers of excellence around the united states who designate representatives to committees that evaluate data and provide treatment options for most common cancers . while there is no specific nccn group designated to evaluate the use of radiotherapy for bone metastases , the topic is dealt with to varying degrees in the publications which deal with primary diagnoses that are most likely to metastasize to bone . furthermore , while radiation oncologists make up the majority of panel members on the acr and astro committees , radiation oncologists generally make up a distinct minority , or are a singular member , of the nccn committees . as such , the nccn guideline recommendations regarding radiotherapy for bone metastases are likely to result from less vigorous conversations and voting criteria than might be true for those offered by the acr and astro groups . the authors of the nccn non - small cell lung cancer guidelines provide the most guidance about radiotherapy dosing for painful bone metastases of any of the primary sites evaluated . bone metastases due to non - small cell lung cancer are separated into those with soft tissue mass versus those without soft tissue mass , with recommendations for 2030 gy in 510 fractions for the former circumstance and 830 gy in 110 fractions for the latter . the kidney cancer guidelines hint at the notion of radiotherapy for oligometastases , describing that long - term progression - free survival has been noted in patients treated with radiotherapy for a single bone metastases and controlled primary disease . the multiple myeloma guidelines recommend a low dose of radiation therapy to between 10 and 30 gy for bone pain , impending pathologic fracture , or impending spinal cord compression . the prostate cancer guidelines suggest that a single 8 gy dose should be used to treat painful bone disease , though in contradistinction to the acr and astro guidelines , the nccn prostate cancer authors suggest that vertebral metastases should receive a fractionated rather than single fraction dose . they also offer a recommendation to use radiopharmaceuticals such as strontium 89 or samarium 153 for widespread bony metastases . similarly , the thyroid cancer group only mentions ebrt in the setting of optimization of dosimetry for iodine 131 for treatment of painful bone metastases . lastly , neither the breast cancer treatment guidelines nor the palliative care guidelines mention the use of radiotherapy for painful bone disease , . the publication of treatment guidelines may cause angst for practitioners , given justifiable concerns that their decision - making autonomy may be threatened by a need to pigeon - hole clinical circumstances into pre - determined bins . it is certainly true that third party payers and the centers for medicare and medicaid services are interested in using guidelines to reward literature - based patterns of care while questioning treatment patterns that deviate sharply from those data . still , the advantages for the use of treatment guidelines include the provision of a minimum standard of care and a delineation of those topics which remain controversial enough to spur additional clinical trials to reach consensus on outcomes . the interest for the bone metastases treatment guidelines has been high , as is evidenced by the fact that the astro bone metastases treatment guidelines were the most frequently downloaded articles in 2011 from the international journal of radiation oncology , biology , and physics website . this convergence of guidelines would most certainly decrease any discrepancies that currently exist in recommendations offered by the task force groups . the national quality forum ( nqf ) has been tasked with measuring quality of care for specific clinical circumstances , to analyze reports of how frequently those quality measures are employed , and to provide guidance that improves patterns of care . the affordable care act requires that the nqf provide annual input to the department of health and human services regarding a national quality strategy that provides measures and tracks progress toward fulfilling those goals . the nqf cancer endorsement 2011 group will evaluate ebrt dose fractionation schemes for bone metastases treatment as its first potential measure of radiation oncology quality . finally , virtually all of the guidelines products provide a disclaimer stating that the ultimate appropriateness of therapy relies on the judgment of treating physician while taking into account their experiences , the available clinical data , and the specific circumstances of the patient who is undergoing that care . similarly , the details of the guidelines evaluated in this manuscript are available online and should be read in detail prior to making conclusive comments about their content and recommendations .

all experiments were performed in compliance with the national institutes of health guide for the care and use of laboratory animals and the arvo statement for the use ofanimals in ophthalmic and vision research . the protocol was approved by the institutional animal care and use committee of the university of miami ( miami , fl , usa ) . notch1 mice ( stock number 007181 ) and c57bl/6 j ( stock number 000664 ) mice were obtained from the jackson laboratory ( bar harbor , me , usa ) . mice were housed under standard conditions of humidity and temperature , with a 12-hour light / dark cycle and free access to food and water . we used 3-month - old mice and embryonic day ( e ) 15.5 and 16.5 fetuses . rna samples were extracted from adult ( 3-month old ) and embryonic ( e16.5 ) lacrimal glands using the absolutely rna microprep and nanoprep kits , respectively ( agilent technologies , santa clara , ca , usa ) . rna samples were sent to ocean ridge biosciences ( palm beach gardens , fl , usa ) for analysis using mouse exonic evidence - based oligonucleotide ( meebo ) microarrays . biotin - labeled complementary rna was synthesized from the total rna according to van gelder 's protocol . biotinylated complementary rna samples were fragmented , diluted in a formamide - containing hybridization buffer , and loaded onto the meebo microarray chips enclosed in custom hybridization chambers . the slides were hybridized for 16 to 18 hours in a model 400 hybridization oven ( scigene , sunnyvale , ca , usa ) . after hybridization , the microarray slides were washed under stringent conditions , stained with streptavidin - alexa-647 ( invitrogen , carlsbad , ca , usa ) , and scanned using an axon genepix 4000b scanner ( molecular devices , sunnyvale , ca , usa ) . spot intensities for each probe were calculated by subtracting median local background intensity from median local foreground intensity for each spot . the spot intensities were then normalized . after removing data for low - quality spots , the mouse probes ' intensities were filtered to identify all probes with intensity above a normalized threshold . for statistical analysis , microarray data were examined for differences by 1-way anova or student 's t - test . the data files have been uploaded in the national center for biotechnology information gene expression omnibus ( geo ; in the public domain , http://www.ncbi.nlm.nih.gov/geo/ ) and are available through geo series accession number gse78062 . quantitative rt - pcr analysis was performed as described previously using gene - specific primers ( table 1 ) . relative expression was calculated by comparison with a standard curve following normalization to expression of the housekeeping gene -actin ( actb ) , chosen as a control . list of rt - pcr primers and primary antibodies the lacrimal glands were lysed with t - per buffer ( thermo fisher scientific , waltham , ma , usa ) supplemented with complete protease inhibitor ( roche applied science , indianapolis , in , usa ) . an equal amount of total protein from each sample was resolved on sds - page gradient gels and transferred to a polyvinylidene difluoride ( pvdf ) membrane ( thermo fisher scientific ) . blots were blocked in 5% milk in tris - buffered saline ( tbs ; ph 7.6 ) ; probed overnight with primary antibodies ( listed in table 1 ) ; washed in 0.15% tween20 in tbs ; and incubated for 1 hour with a secondary antibody ( 1:10,000 ; ge healthcare bio - sciences , pittsburgh , pa , usa ) diluted in tbs . adult mice ( the source of adult lacrimal glands ) or pregnant mothers ( the source of embryonic lacrimal glands ) were transcardially perfused with 4% paraformaldehyde in pbs . while whole embryonic lacrimal glands were immunostained , adult lgs were cryoprotected with 30% sucrose , embedded in optimum cutting temperature ( oct ) medium , and cryosectioned at 25-m thickness . prior to immunostaining , whole embryonic lacrimal glands or sections of adult lacrimal glands were permeabilized in 0.3% triton x-100 in pbs for 30 minutes . tissues were blocked with pbs containing 0.15% tween 20 , 2% bsa , and 5% serum at room temperature for 30 minutes . the samples were then incubated with primary antibodies ( table 1 ) for 16 hours in blocking solution , followed by species - specific secondary antibodies ( alexafluor ; thermo fisher scientific ) . imaging was performed with a leica tsl aobs sp5 confocal microscope ( leica microsystems , exton , pa , usa ) . the lacrimal gland explant ex vivo cultures were prepared from e15.5 embryos prior to the onset of branching morphogenesis . to this end , the lacrimal glands were aseptically removed , washed in pbs , and plated on culture inserts ( transwell filters , costar ; sigma - aldrich corp . , st . louis , mo , usa ) pretreated with matrigel ( 160 l of a 1:30 dilution of matrigel in dulbecco 's modified eagle 's medium [ dmem]/f12 ; bd biosciences , bedford , ma , usa ) . the inserts ( each containing a lacrimal gland in dmem / f12 supplemented with 10% fbs and 3.3% matrigel ) were placed into the culture wells of a 12-well plate ( containing the same medium , without matrigel ) . subsequently , the desired concentration of dapt ( d5942 ; sigma - aldrich corp . ) was added to the media . to transduce lacrimal gland explants with ad - cmv - icre ( vector biolabs , malvern , pa , usa ) , the virus was added ( 5 10 genome copies per well ) to the media . the 12-well plates were then transferred into a co2 incubator , where they were kept for 72 hours at 37c with 5% co2 . all experiments were performed in compliance with the national institutes of health guide for the care and use of laboratory animals and the arvo statement for the use ofanimals in ophthalmic and vision research . the protocol was approved by the institutional animal care and use committee of the university of miami ( miami , fl , usa ) . notch1 mice ( stock number 007181 ) and c57bl/6 j ( stock number 000664 ) mice were obtained from the jackson laboratory ( bar harbor , me , usa ) . mice were housed under standard conditions of humidity and temperature , with a 12-hour light / dark cycle and free access to food and water . we used 3-month - old mice and embryonic day ( e ) 15.5 and 16.5 fetuses . rna samples were extracted from adult ( 3-month old ) and embryonic ( e16.5 ) lacrimal glands using the absolutely rna microprep and nanoprep kits , respectively ( agilent technologies , santa clara , ca , usa ) . rna samples were sent to ocean ridge biosciences ( palm beach gardens , fl , usa ) for analysis using mouse exonic evidence - based oligonucleotide ( meebo ) microarrays . biotin - labeled complementary rna was synthesized from the total rna according to van gelder 's protocol . biotinylated complementary rna samples were fragmented , diluted in a formamide - containing hybridization buffer , and loaded onto the meebo microarray chips enclosed in custom hybridization chambers . the slides were hybridized for 16 to 18 hours in a model 400 hybridization oven ( scigene , sunnyvale , ca , usa ) . after hybridization , the microarray slides were washed under stringent conditions , stained with streptavidin - alexa-647 ( invitrogen , carlsbad , ca , usa ) , and scanned using an axon genepix 4000b scanner ( molecular devices , sunnyvale , ca , usa ) . spot intensities for each probe were calculated by subtracting median local background intensity from median local foreground intensity for each spot . the spot intensities were then normalized . after removing data for low - quality spots , the mouse probes ' intensities were filtered to identify all probes with intensity above a normalized threshold . for statistical analysis , microarray data were examined for differences by 1-way anova or student 's t - test . the data files have been uploaded in the national center for biotechnology information gene expression omnibus ( geo ; in the public domain , http://www.ncbi.nlm.nih.gov/geo/ ) and are available through geo series accession number gse78062 . quantitative rt - pcr analysis was performed as described previously using gene - specific primers ( table 1 ) . relative expression was calculated by comparison with a standard curve following normalization to expression of the housekeeping gene -actin ( actb ) , chosen as a control . the lacrimal glands were lysed with t - per buffer ( thermo fisher scientific , waltham , ma , usa ) supplemented with complete protease inhibitor ( roche applied science , indianapolis , in , usa ) . an equal amount of total protein from each sample was resolved on sds - page gradient gels and transferred to a polyvinylidene difluoride ( pvdf ) membrane ( thermo fisher scientific ) . blots were blocked in 5% milk in tris - buffered saline ( tbs ; ph 7.6 ) ; probed overnight with primary antibodies ( listed in table 1 ) ; washed in 0.15% tween20 in tbs ; and incubated for 1 hour with a secondary antibody ( 1:10,000 ; ge healthcare bio - sciences , pittsburgh , pa , usa ) diluted in tbs . adult mice ( the source of adult lacrimal glands ) or pregnant mothers ( the source of embryonic lacrimal glands ) were transcardially perfused with 4% paraformaldehyde in pbs . while whole embryonic lacrimal glands were immunostained , adult lgs were cryoprotected with 30% sucrose , embedded in optimum cutting temperature ( oct ) medium , and cryosectioned at 25-m thickness . prior to immunostaining , whole embryonic lacrimal glands or sections of adult lacrimal glands were permeabilized in 0.3% triton x-100 in pbs for 30 minutes . tissues were blocked with pbs containing 0.15% tween 20 , 2% bsa , and 5% serum at room temperature for 30 minutes . the samples were then incubated with primary antibodies ( table 1 ) for 16 hours in blocking solution , followed by species - specific secondary antibodies ( alexafluor ; thermo fisher scientific ) . imaging was performed with a leica tsl aobs sp5 confocal microscope ( leica microsystems , exton , pa , usa ) . the lacrimal gland explant ex vivo cultures were prepared from e15.5 embryos prior to the onset of branching morphogenesis . to this end , the lacrimal glands were aseptically removed , washed in pbs , and plated on culture inserts ( transwell filters , costar ; sigma - aldrich corp . , st . louis , mo , usa ) pretreated with matrigel ( 160 l of a 1:30 dilution of matrigel in dulbecco 's modified eagle 's medium [ dmem]/f12 ; bd biosciences , bedford , ma , usa ) . the inserts ( each containing a lacrimal gland in dmem / f12 supplemented with 10% fbs and 3.3% matrigel ) were placed into the culture wells of a 12-well plate ( containing the same medium , without matrigel ) . subsequently , the desired concentration of dapt ( d5942 ; sigma - aldrich corp . ) was added to the media . to transduce lacrimal gland explants with ad - cmv - icre ( vector biolabs , malvern , pa , usa ) , the virus was added ( 5 10 genome copies per well ) to the media . the 12-well plates were then transferred into a co2 incubator , where they were kept for 72 hours at 37c with 5% co2 . statistical analysis was conducted with a 1-way anova for multiple comparisons . for single comparisons , the student t - test was applied . to characterize and compare lacrimal glands isolated from embryonic and adult mice , rna extracted from lacrimal glands of 3-month - old ( adult ) mice and e16.5 fetuses was used for microarray analysis . a total of three independent biological replicates of e16.5 lacrimal glands and three independent biological replicates of adult lacrimal glands were obtained for comparative profiling . the results of our microarray analysis showed that a total of 8188 genes passed the quality control criteria and were differentially expressed ( p < 0.05 ) between the two studied groups ( table 2 ; supplementary table s1 ) . the changes in gene expression detected by microarrays were further confirmed by quantitative rt - pcr , western blot analysis , and immunohistochemistry for a group of selected genes ( figs . importantly , we detected expression of all genes that had been previously reported to be involved in lacrimal gland development ( table 3 ) . surprisingly , while expression of fgf10 , fgfr2 , sox10 , igsf3 , and egfr was significantly upregulated in embryonic lacrimal glands , expression of pax6 , sox9 , and egf was significantly upregulated in adult lacrimal glands ( table 3 ) . the randomly selected genes expressed differently between the two studied groups differential gene expression between the embryonic and adult lacrimal glands revealed by microarray analysis was confirmed for a select group of genes by quantitative rt - pcr ( a ) and western blot analysis ( b ) ( * p < 0.05 , * * p < 0.01 , em16.5 and ad microarray data ) . the notch1 receptor is present in the embryonic and adult lacrimal gland at the protein level . while notch1 was highly expressed in the epithelium and mesenchyme of e16.5 lacrimal glands ( a ) , notch1 was expressed only in single , isolated cells in adult lacrimal glands ( b , c ) . ( a ) significantly high dlk1 expression was detected predominantly in mesenchymal cells of e16.5 lgs . ngfr was highly expressed in mesenchymal cells of embryonic lacrimal glands ( a ) , but only in single , isolated cells in adult lacrimal glands ( b , c ) . genes known to be expressed ( before the study ) by lacrimal gland tissue the significant differences in gene expression between embryonic and adult lacrimal glands created challenges in pathway analysis of our microarray data . because our study focused predominantly on pathways involved in glandular development , we first analyzed the gene ontology ( go ) database associated with gland development , and extracted a list of genes that passed the threshold level in our microarray data ( supplementary table s2 ) . we used david functional annotation bioinformatic analysis tool to pinpoint enriched kegg and panther pathways ( supplementary table s3 ) . kegg and panther pathway mapping demonstrated that genes of the notch , wnt , tgf , and hedgehog signaling cascades were highly represented in our microarray data ( supplementary table s3 ) . this finding prompted us to investigate whether other genes in these pathways were also highly expressed . using the kegg pathway database , we found that a significant number of genes from the notch , wnt , tgf , and hedgehog signaling pathways are expressed in both embryonic and adult lacrimal glands ( supplementary table s4 ) . some of these genes were significantly upregulated in e16.5 lacrimal glands ( notch1 , notch3 , jag1 , etc . ) , while others were significantly upregulated in adult lacrimal glands ( rfng , psenen , lefty2 , etc . ) , suggesting that these signaling pathways may play a role in both lacrimal gland development and in the maintenance of adult lacrimal gland homeostasis ( supplementary table s4 ) . because the notch , wnt , tgf , and hedgehog pathways all participate in the regulation of epithelial - to - mesenchymal transition ( emt ) , it is plausible that this biological process may play active roles in both embryonic and adult lacrimal glands . our findings support this hypothesis , as genes from the go database 's emt list were significantly represented in our microarray data ( supplementary table s5 ) . in addition , we tested for the presence of genes from the go database 's mesenchymal to epithelial transition ( met ) list and found that these genes were also represented in our microarray data ( supplementary table s5 ) . finally , we used these observations to generate the most comprehensive list of lacrimal gland two techniques the use of adult stem cells in lacrimal gland regenerative therapy , and the transplantation of lacrimal gland specific stem cells / progenitors harbor the potential to restore lacrimal gland function and confer tremendous clinical benefits . unfortunately , at the present time only c - kit ( kit ) , aldh1 ( aldh1a1 , aldh1a2 , and aldh1a3 ) , and abcg2 ( abcg2 ) have been identified as putative lacrimal gland our data indicate that these markers are expressed in both embryonic and adult lacrimal gland tissue . all of them except aldh1a1 were upregulated in e16.5 lacrimal glands compared with adult lacrimal glands ( table 4 ) . to identify new potential lacrimal gland specific stem cell / progenitor markers , we compared the results of our microarray data with a list of genes and proteins commonly associated with stem / progenitor cell states ( table 4 ) . we found that expression of some of these genes was significantly upregulated in the e16.5 lacrimal gland compared with the adult lacrimal gland ( table 4 ) . in addition , we employed immunohistochemistry assays to evaluate the distribution of two of the gene products , ngfr / p75 and notch1 , in fixed embryonic and adult lacrimal glands . expression of ngfr and notch1 was significantly higher in embryonic lacrimal glands compared with adult lacrimal glands ( table 4 ) . our immunohistochemistry data were consistent with these expression data ( figs . 2 and 4 ) . in e16.5 lacrimal glands , 2 ) , while substantial ngfr - specific immunostaining was predominantly localized to the mesenchymal cells ( fig . , we found that ngfr and notch1 were expressed only in single , isolated cells ( figs . 2 , 4 ) . this observation led us to reason that these single cells may represent an adult lacrimal gland stem cell/ progenitors . known and previously undescribed putative lacrimal gland stem cell / progenitor markers our data uncovered several signaling pathways that may play important roles in lacrimal gland development and morphogenesis . from the large list of potential candidates , we chose to focus on one the notch signaling pathway in order to directly evaluate its role in lacrimal gland development . we selected notch because , as noted above , many genes of the notch signaling pathway , including notch1 , notch3 , hes1 , dlk1 , jag1 , jag2 , and rbpj , were expressed at significantly higher levels in the embryonic lacrimal gland than in the adult lacrimal gland ( figs . we began our study of the notch pathway by treating embryonic lacrimal glands with dapt , a nonselective inhibitor of notch signaling . we cultured lacrimal glands from e15.5 mice in the presence of 10 , 50 , and 100 m of dapt . untreated lacrimal glands or lacrimal glands cultured in the presence of dimethyl sulfoxide ( dmso ) ( stock solutions of dapt were prepared in dmso ) were saved as controls . we found that while dapt treatment did not affect significantly the overall size of the cultured embryonic lacrimal glands , it did exert a strong effect on average lobule size and average number of lobules ( fig . dapt treatment led to a reduction in the average size of lobules ( 93 11 m [ dmso ] , 66 3 m [ 10-m dapt ] , 60 1 m [ 50-m dapt ] , 73 3 m [ 100-m dapt ] ) , but also to an increase in the average number of lobules ( 22 2 [ dmso ] , 53 2 [ 10-m dapt ] , 38 4 [ 50-m dapt ] , 31 1 [ 100-m dapt ] ; fig . we did not detect a difference between untreated and dmso - treated lacrimal glands ( fig . to this end , lacrimal glands isolated from notch1 mice and wild - type animals were treated with ad - cmv - icre , the adenovirus constitutively expressing cre recombinase that catalyzes site - specific recombination of dna between loxp sites . three days after ad - cmv - icre treatment , the glands were collected and morphometrically analyzed . in agreement with the dapt data above , we found that notch1 knockout in the embryonic lacrimal gland led to a reduction in the average size of lobules ( 90 3 m [ wild - type untreated ] , 82 1 m [ wild - type ad - cmv - icre treated ] , 81 3 m [ notch1 untreated ] , 56 2 m [ notch1 ad - cmv - icre treated ] ) , but also to an increase in the average number of lobules ( 29 3 [ wild - type untreated ] , 37 6 [ wild - type ad - cmv - icre treated ] , 40 2 [ notch1 untreated ] , 51 2 [ notch1 ad - cmv - icre treated ] ) , respectively ( fig . 6 ) . to verify that the observed effect was due to reduced notch1 expression , two groups of notch1 untreated and notch1 ad - cmv - icre treated lacrimal glands were used to test notch1 expression by quantitative rt - pcr . we found that notch1 expression was reduced in ad - cmv - icre treated lacrimal glands ( 100 9% [ notch1 untreated ] vs. 69 3% [ notch1 ad - cmv - icre treated ] , p value = 0.02 , n = 5 ) . notch signaling regulates the size and number of lobules in the developing lg : gamma secretase inhibition study . ( a ) representative confocal images of dapi - labeled developing lacrimal glands 3 days after treatment . the lacrimal glands were treated with a notch signaling inhibitor ( dapt ; 10 , 50 , and 100 m ) . ( b ) whole gland size , individual lobule size , and total number of lobules were measured and analyzed for each lacrimal gland ( * * p < 0.01 , * p < 0.05 , n = 57 ) . notch signaling regulates the size and number of lobules in the developing lacrimal gland : conditional notch1 knockout study . ( a ) representative confocal images of dapi - labeled developing lacrimal glands 3 days after respective treatment . the lacrimal glands were treated with cre - recombinase expressing adenovirus ( ad - cmv - icre / adeno - cre ) to induce notch1 knockout . controls included wild type ( wt ) , virus treated wild types ( wt adeno - cre ) , and nonvirus treated notch glands ( notch1_lox ) . ( b ) whole gland size , individual lobule size , and total number of lobules were measured and analyzed for each group of lacrimal glands ( * * p < 0.01 , * p < 0.05 , n = 57 ) . to characterize and compare lacrimal glands isolated from embryonic and adult mice , rna extracted from lacrimal glands of 3-month - old ( adult ) mice and e16.5 fetuses was used for microarray analysis . a total of three independent biological replicates of e16.5 lacrimal glands and three independent biological replicates of adult lacrimal glands were obtained for comparative profiling . the results of our microarray analysis showed that a total of 8188 genes passed the quality control criteria and were differentially expressed ( p < 0.05 ) between the two studied groups ( table 2 ; supplementary table s1 ) . the changes in gene expression detected by microarrays were further confirmed by quantitative rt - pcr , western blot analysis , and immunohistochemistry for a group of selected genes ( figs . importantly , we detected expression of all genes that had been previously reported to be involved in lacrimal gland development ( table 3 ) . surprisingly , while expression of fgf10 , fgfr2 , sox10 , igsf3 , and egfr was significantly upregulated in embryonic lacrimal glands , expression of pax6 , sox9 , and egf was significantly upregulated in adult lacrimal glands ( table 3 ) . the randomly selected genes expressed differently between the two studied groups differential gene expression between the embryonic and adult lacrimal glands revealed by microarray analysis was confirmed for a select group of genes by quantitative rt - pcr ( a ) and western blot analysis ( b ) ( * p < 0.05 , * * p < 0.01 , em16.5 and ad microarray data ) . the notch1 receptor is present in the embryonic and adult lacrimal gland at the protein level . while notch1 was highly expressed in the epithelium and mesenchyme of e16.5 lacrimal glands ( a ) , notch1 was expressed only in single , isolated cells in adult lacrimal glands ( b , c ) . ( a ) significantly high dlk1 expression was detected predominantly in mesenchymal cells of e16.5 lgs . ngfr was highly expressed in mesenchymal cells of embryonic lacrimal glands ( a ) , but only in single , isolated cells in adult lacrimal glands ( b , c ) . genes known to be expressed ( before the study ) by lacrimal gland tissue the significant differences in gene expression between embryonic and adult lacrimal glands created challenges in pathway analysis of our microarray data . because our study focused predominantly on pathways involved in glandular development , we first analyzed the gene ontology ( go ) database associated with gland development , and extracted a list of genes that passed the threshold level in our microarray data ( supplementary table s2 ) . we used david functional annotation bioinformatic analysis tool to pinpoint enriched kegg and panther pathways ( supplementary table s3 ) . kegg and panther pathway mapping demonstrated that genes of the notch , wnt , tgf , and hedgehog signaling cascades were highly represented in our microarray data ( supplementary table s3 ) . this finding prompted us to investigate whether other genes in these pathways were also highly expressed . using the kegg pathway database , we found that a significant number of genes from the notch , wnt , tgf , and hedgehog signaling pathways are expressed in both embryonic and adult lacrimal glands ( supplementary table s4 ) . some of these genes were significantly upregulated in e16.5 lacrimal glands ( notch1 , notch3 , jag1 , etc . ) , while others were significantly upregulated in adult lacrimal glands ( rfng , psenen , lefty2 , etc . ) , suggesting that these signaling pathways may play a role in both lacrimal gland development and in the maintenance of adult lacrimal gland homeostasis ( supplementary table s4 ) . because the notch , wnt , tgf , and hedgehog pathways all participate in the regulation of epithelial - to - mesenchymal transition ( emt ) , it is plausible that this biological process may play active roles in both embryonic and adult lacrimal glands . our findings support this hypothesis , as genes from the go database 's emt list were significantly represented in our microarray data ( supplementary table s5 ) . in addition , we tested for the presence of genes from the go database 's mesenchymal to epithelial transition ( met ) list and found that these genes were also represented in our microarray data ( supplementary table s5 ) . finally , we used these observations to generate the most comprehensive list of lacrimal gland two techniques the use of adult stem cells in lacrimal gland regenerative therapy , and the transplantation of lacrimal gland specific stem cells / progenitors harbor the potential to restore lacrimal gland function and confer tremendous clinical benefits . unfortunately , at the present time only c - kit ( kit ) , aldh1 ( aldh1a1 , aldh1a2 , and aldh1a3 ) , and abcg2 ( abcg2 ) have been identified as putative lacrimal gland our data indicate that these markers are expressed in both embryonic and adult lacrimal gland tissue . all of them except aldh1a1 were upregulated in e16.5 lacrimal glands compared with adult lacrimal glands ( table 4 ) . to identify new potential lacrimal gland specific stem cell / progenitor markers , we compared the results of our microarray data with a list of genes and proteins commonly associated with stem / progenitor cell states ( table 4 ) . we found that expression of some of these genes was significantly upregulated in the e16.5 lacrimal gland compared with the adult lacrimal gland ( table 4 ) . in addition , we employed immunohistochemistry assays to evaluate the distribution of two of the gene products , ngfr / p75 and notch1 , in fixed embryonic and adult lacrimal glands . expression of ngfr and notch1 was significantly higher in embryonic lacrimal glands compared with adult lacrimal glands ( table 4 ) . our immunohistochemistry data were consistent with these expression data ( figs . 2 and 4 ) . in e16.5 lacrimal glands , notch1-specific immunostaining was evident in both the epithelium and mesenchyme ( fig . 2 ) , while substantial ngfr - specific immunostaining was predominantly localized to the mesenchymal cells ( fig . , we found that ngfr and notch1 were expressed only in single , isolated cells ( figs . 2 , 4 ) . this observation led us to reason that these single cells may represent an adult lacrimal gland stem cell/ progenitors . our data uncovered several signaling pathways that may play important roles in lacrimal gland development and morphogenesis . from the large list of potential candidates , we chose to focus on one the notch signaling pathway in order to directly evaluate its role in lacrimal gland development . we selected notch because , as noted above , many genes of the notch signaling pathway , including notch1 , notch3 , hes1 , dlk1 , jag1 , jag2 , and rbpj , were expressed at significantly higher levels in the embryonic lacrimal gland than in the adult lacrimal gland ( figs . we began our study of the notch pathway by treating embryonic lacrimal glands with dapt , a nonselective inhibitor of notch signaling . we cultured lacrimal glands from e15.5 mice in the presence of 10 , 50 , and 100 m of dapt . untreated lacrimal glands or lacrimal glands cultured in the presence of dimethyl sulfoxide ( dmso ) ( stock solutions of dapt were prepared in dmso ) were saved as controls . we found that while dapt treatment did not affect significantly the overall size of the cultured embryonic lacrimal glands , it did exert a strong effect on average lobule size and average number of lobules ( fig . dapt treatment led to a reduction in the average size of lobules ( 93 11 m [ dmso ] , 66 3 m [ 10-m dapt ] , 60 1 m [ 50-m dapt ] , 73 3 m [ 100-m dapt ] ) , but also to an increase in the average number of lobules ( 22 2 [ dmso ] , 53 2 [ 10-m dapt ] , 38 4 [ 50-m dapt ] , 31 1 [ 100-m dapt ] ; fig . we did not detect a difference between untreated and dmso - treated lacrimal glands ( fig . lacrimal glands isolated from notch1 mice and wild - type animals were treated with ad - cmv - icre , the adenovirus constitutively expressing cre recombinase that catalyzes site - specific recombination of dna between loxp sites . three days after ad - cmv - icre treatment , the glands were collected and morphometrically analyzed . in agreement with the dapt data above , we found that notch1 knockout in the embryonic lacrimal gland led to a reduction in the average size of lobules ( 90 3 m [ wild - type untreated ] , 82 1 m [ wild - type ad - cmv - icre treated ] , 81 3 m [ notch1 untreated ] , 56 2 m [ notch1 ad - cmv - icre treated ] ) , but also to an increase in the average number of lobules ( 29 3 [ wild - type untreated ] , 37 6 [ wild - type ad - cmv - icre treated ] , 40 2 [ notch1 untreated ] , 51 2 [ notch1 ad - cmv - icre treated ] ) , respectively ( fig . 6 ) . to verify that the observed effect was due to reduced notch1 expression , two groups of notch1 untreated and notch1 ad - cmv - icre treated lacrimal glands were used to test notch1 expression by quantitative rt - pcr . we found that notch1 expression was reduced in ad - cmv - icre treated lacrimal glands ( 100 9% [ notch1 untreated ] vs. 69 3% [ notch1 ad - cmv - icre treated ] , p value = 0.02 , n = 5 ) . notch signaling regulates the size and number of lobules in the developing lg : gamma secretase inhibition study . ( a ) representative confocal images of dapi - labeled developing lacrimal glands 3 days after treatment . the lacrimal glands were treated with a notch signaling inhibitor ( dapt ; 10 , 50 , and 100 m ) . ( b ) whole gland size , individual lobule size , and total number of lobules were measured and analyzed for each lacrimal gland ( * * p < 0.01 , * p < 0.05 , n = 57 ) . notch signaling regulates the size and number of lobules in the developing lacrimal gland : conditional notch1 knockout study . ( a ) representative confocal images of dapi - labeled developing lacrimal glands 3 days after respective treatment . the lacrimal glands were treated with cre - recombinase expressing adenovirus ( ad - cmv - icre / adeno - cre ) to induce notch1 knockout . controls included wild type ( wt ) , virus treated wild types ( wt adeno - cre ) , and nonvirus treated notch glands ( notch1_lox ) . ( b ) whole gland size , individual lobule size , and total number of lobules were measured and analyzed for each group of lacrimal glands ( * * p < 0.01 , * p < 0.05 , n = 57 ) . although the structure and function of the lacrimal gland have been studied extensively , the mechanisms behind lacrimal gland development , morphogenesis , and differentiation remain rather poorly understood . enhancing our knowledge of these mechanisms will be critically important for the design of new treatment paradigms , such as postnatal lacrimal gland regeneration or lacrimal gland replacement therapies . in this study , comparing embryonic and adult lacrimal glands , we collected extensive data on lacrimal gland developmental biology , and further confirmed these findings in vitro . we leveraged this expression data to help identify new putative markers of a lacrimal gland stem / progenitor cell phenotype . wnt- , tgf- , and hedgehog pathway - regulated emt should be a critical mechanism in lacrimal gland development . in addition , our data indicate that the notch signaling exert a strong effect on average lobule size and average number of lobules in the developing lacrimal gland . this study has shed new light on the mechanisms of the lacrimal gland 's development and morphology . our data indicate that the majority of genes involved in the notch- , wnt- , tgf- , and hedgehog - signaling pathways are expressed in the lacrimal gland during both embryonic and adult stages . such sustained expression lends credence to the hypothesis that the notch- , wnt- , tgf- , and hedgehog - signaling cascades play roles both in lacrimal gland development and in the maintenance of adult lacrimal gland homeostasis . importantly , these cascades do not act independently , but instead maintain steady crosstalk between each other . we noted that the notch , wnt , tgf , and hedgehog pathways can activate regulators of emt , and may therefore be involved in morphologic modulation of the developing gland . as expected , we found that many of these genes are indeed highly expressed in the lacrimal gland . some of them ( e.g. , zfhx1a , twist1 , snai2 , etc . ) were highly upregulated in embryonic glands , while others ( e.g. , snai1 , foxa1 , tcf4 , etc . ) were more highly expressed in the adult glands , suggesting that emt and met are involved in lacrimal gland development and in the maintenance of adult lacrimal gland homeostasis . given the notch pathway 's essential role in preventing progenitor differentiation in the mammary gland ( and many other tissues types ) , we suggest that the canonical notch ligands jag1/jag2 and the noncanonical dlk1 may activate notch1/notch3/rbpj , preventing differentiation of all lacrimal gland cells expressing notch receptors . meanwhile , because elf5 mediates mammary gland progenitor cell differentiation by inhibiting notch signaling , we can suggest that a similar process may occur during lacrimal gland development , as elf5 expression while present in the embryonic gland was significantly upregulated in the adult lacrimal gland . in order to directly evaluate the role of the identified pathways in lacrimal gland development , we chose to focus on the notch signaling pathway . our study allows us to link notch signaling with lacrimal gland branching because inhibition of noth signaling significantly increased the average number of lobules and significantly reduced average lobule size observed . branching processes are critical for the normal development of a number of organs , including the lung , kidney , pancreas , prostate , salivary gland , and lacrimal gland . the branching process in glands ( including the lacrimal gland ) begins with the formation of clefts within a single epithelial bud . as these clefts invaginate , they cleave the single bud into smaller buds , each of which grows to become a lobule . recursive iteration of this process leads to the formation of an increasingly intricate glandular structure . thus , our findings suggest that notch signaling may regulate lacrimal gland branching by inhibiting cleft formation . we suspect that the observed results are associated with notch signaling activity 's control of progenitor cell cycle exit ( into a postmitotic , differentiated phenotype ) . it was shown previously that extracellular matrix ( ecm ) and metalloproteases ( mmps ; enzymes that degrade ecm ) regulate the branching process in other glands . the ecm forms a barrier to gland growth , and if sufficiently high levels of ecm accumulate , a cleft is formed ( as an analogy , imagine a balloon being inflated against a hard , narrow barrier ; the balloon will distend around the barrier , creating the appearance of two separate lobes of one continuous structure ) . metalloproteases activity degrades these ecm barriers , facilitating lobule formation and shaping the directionality of gland growth . if lacrimal gland progenitors highly express mmps , while differentiated lacrimal cells predominantly produce ecm then notch signaling inhibition should lead to an increased number of ecm - producing cells and , as a result , to an increased number of clefts ( fig . 7 ) . such a mechanism could explain how notch - mediated distribution of progenitor and differentiated cells in the developing lacrimal gland may influence the branching process ( fig . notch signaling may regulate lacrimal gland branching by controlling the transition from proliferative progenitor state into postmitotic differentiation state . ( a ) at the earliest stages of development , notch positive lacrimal gland progenitors ( yellow ) proliferate , growing radially outward like an expanding balloon . ( b ) cessation of notch signaling causes some progenitors to exit the cell cycle and become notch negative , differentiated cells ( red ) . ( c ) unlike notch positive proliferating progenitors , notch negative postmitotic cells secrete ecm ( blue ) , which accumulates between them to form physical barriers ( clefts ) . progenitors express mmps , enzymes that degrade ecm and allow the proliferating cells to continue their radial expansion . consequently , the glandular tissue expands outward like a balloon being inflated against a solid structure . ( d ) recursive iteration of this process gives rise to the complex branching and multilobular structure that is the mature lacrimal gland . in conclusion , we have assembled here the most comprehensive list of putative lacrimal gland stem / progenitor cell markers to date ( table 4 ) . in addition , available evidence appears to indicate important roles for the notch- , wnt- , tgf- , and hedgehog - signaling cascades in lacrimal gland development , morphogenic features , maintenance of phenotypes , and homeostasis . our findings suggest a role of emt and met in both embryonic and adult lacrimal gland , possibly shedding new light on the biology of epithelial neoplasms in this tissue ( i.e. , adenoid cystic carcinoma ) . finally , our data further suggest that notch signaling may help regulate the lacrimal gland 's branching morphogenesis . thus , this study has allowed us to molecularly characterize , for the first time , the lacrimal gland as a whole , and to propose novel mechanisms that regulate its development .

purposealthough normal function of the lacrimal gland is essential for vision ( and thus for human well - being ) , the lacrimal gland remains rather poorly understood at a molecular level . the purpose of this study was to identify new genes and signaling cascades involved in lacrimal gland development.methodsto identify these genes , we used microarray analysis to compare the gene expression profiles of developing ( embryonic ) and adult lacrimal glands . differential data were validated by quantitative rt - pcr , and several corresponding proteins were confirmed by immunohistochemistry and western blot analysis . to evaluate the role of notch signaling in lacrimal gland ( lg ) development , we used the notch inhibitor dapt and conditional notch1 knockouts.resultsour microarray data and an in silico reconstruction of cellular networks revealed significant changes in the expression patterns of genes from the notch , wnt , tgf , and hedgehog pathways , all of which are involved in the regulation of epithelial - to - mesenchymal transition ( emt ) . our study also revealed new putative lacrimal gland stem cell / progenitor markers . we found that inhibiting notch signaling both increases the average number of lacrimal gland lobules and reduces the size of each lobule.conclusionsour findings suggest that notch- , wnt- , tgf- , and hedgehog - regulated emt transition are critical mechanisms in lacrimal gland development and morphogenesis . our data also supports the hypothesis that notch signaling regulates branching morphogenesis in the developing lacrimal gland by suppressing cleft formation .

Methods Animals RNA Extraction, Probe Preparation, and Array Hybridization Microarray Data Analysis Quantitative RT-PCR Analysis Western Blot Analysis Immunohistochemistry Lacrimal Gland Explant Ex Vivo Cultures, DAPT, and Ad-CMV-iCre Treatment Statistical Analysis Results An Putative Lacrimal Gland Stem Cell/Progenitor Markers Were Revealed by Microarray Analysis NOTCH Signaling Regulates Average Lobule Size and Average Number of Lobules in the Lacrimal Gland Discussion Supplementary Material

list of rt - pcr primers and primary antibodies the lacrimal glands were lysed with t - per buffer ( thermo fisher scientific , waltham , ma , usa ) supplemented with complete protease inhibitor ( roche applied science , indianapolis , in , usa ) . to this end , the lacrimal glands were aseptically removed , washed in pbs , and plated on culture inserts ( transwell filters , costar ; sigma - aldrich corp . to this end , the lacrimal glands were aseptically removed , washed in pbs , and plated on culture inserts ( transwell filters , costar ; sigma - aldrich corp . the changes in gene expression detected by microarrays were further confirmed by quantitative rt - pcr , western blot analysis , and immunohistochemistry for a group of selected genes ( figs . importantly , we detected expression of all genes that had been previously reported to be involved in lacrimal gland development ( table 3 ) . the randomly selected genes expressed differently between the two studied groups differential gene expression between the embryonic and adult lacrimal glands revealed by microarray analysis was confirmed for a select group of genes by quantitative rt - pcr ( a ) and western blot analysis ( b ) ( * p < 0.05 , * * p < 0.01 , em16.5 and ad microarray data ) . the notch1 receptor is present in the embryonic and adult lacrimal gland at the protein level . while notch1 was highly expressed in the epithelium and mesenchyme of e16.5 lacrimal glands ( a ) , notch1 was expressed only in single , isolated cells in adult lacrimal glands ( b , c ) . genes known to be expressed ( before the study ) by lacrimal gland tissue the significant differences in gene expression between embryonic and adult lacrimal glands created challenges in pathway analysis of our microarray data . because our study focused predominantly on pathways involved in glandular development , we first analyzed the gene ontology ( go ) database associated with gland development , and extracted a list of genes that passed the threshold level in our microarray data ( supplementary table s2 ) . kegg and panther pathway mapping demonstrated that genes of the notch , wnt , tgf , and hedgehog signaling cascades were highly represented in our microarray data ( supplementary table s3 ) . using the kegg pathway database , we found that a significant number of genes from the notch , wnt , tgf , and hedgehog signaling pathways are expressed in both embryonic and adult lacrimal glands ( supplementary table s4 ) . , suggesting that these signaling pathways may play a role in both lacrimal gland development and in the maintenance of adult lacrimal gland homeostasis ( supplementary table s4 ) . because the notch , wnt , tgf , and hedgehog pathways all participate in the regulation of epithelial - to - mesenchymal transition ( emt ) , it is plausible that this biological process may play active roles in both embryonic and adult lacrimal glands . in addition , we tested for the presence of genes from the go database 's mesenchymal to epithelial transition ( met ) list and found that these genes were also represented in our microarray data ( supplementary table s5 ) . finally , we used these observations to generate the most comprehensive list of lacrimal gland two techniques the use of adult stem cells in lacrimal gland regenerative therapy , and the transplantation of lacrimal gland specific stem cells / progenitors harbor the potential to restore lacrimal gland function and confer tremendous clinical benefits . unfortunately , at the present time only c - kit ( kit ) , aldh1 ( aldh1a1 , aldh1a2 , and aldh1a3 ) , and abcg2 ( abcg2 ) have been identified as putative lacrimal gland our data indicate that these markers are expressed in both embryonic and adult lacrimal gland tissue . to identify new potential lacrimal gland specific stem cell / progenitor markers , we compared the results of our microarray data with a list of genes and proteins commonly associated with stem / progenitor cell states ( table 4 ) . we found that expression of some of these genes was significantly upregulated in the e16.5 lacrimal gland compared with the adult lacrimal gland ( table 4 ) . in addition , we employed immunohistochemistry assays to evaluate the distribution of two of the gene products , ngfr / p75 and notch1 , in fixed embryonic and adult lacrimal glands . known and previously undescribed putative lacrimal gland stem cell / progenitor markers our data uncovered several signaling pathways that may play important roles in lacrimal gland development and morphogenesis . from the large list of potential candidates , we chose to focus on one the notch signaling pathway in order to directly evaluate its role in lacrimal gland development . we selected notch because , as noted above , many genes of the notch signaling pathway , including notch1 , notch3 , hes1 , dlk1 , jag1 , jag2 , and rbpj , were expressed at significantly higher levels in the embryonic lacrimal gland than in the adult lacrimal gland ( figs . we began our study of the notch pathway by treating embryonic lacrimal glands with dapt , a nonselective inhibitor of notch signaling . we found that while dapt treatment did not affect significantly the overall size of the cultured embryonic lacrimal glands , it did exert a strong effect on average lobule size and average number of lobules ( fig . dapt treatment led to a reduction in the average size of lobules ( 93 11 m [ dmso ] , 66 3 m [ 10-m dapt ] , 60 1 m [ 50-m dapt ] , 73 3 m [ 100-m dapt ] ) , but also to an increase in the average number of lobules ( 22 2 [ dmso ] , 53 2 [ 10-m dapt ] , 38 4 [ 50-m dapt ] , 31 1 [ 100-m dapt ] ; fig . in agreement with the dapt data above , we found that notch1 knockout in the embryonic lacrimal gland led to a reduction in the average size of lobules ( 90 3 m [ wild - type untreated ] , 82 1 m [ wild - type ad - cmv - icre treated ] , 81 3 m [ notch1 untreated ] , 56 2 m [ notch1 ad - cmv - icre treated ] ) , but also to an increase in the average number of lobules ( 29 3 [ wild - type untreated ] , 37 6 [ wild - type ad - cmv - icre treated ] , 40 2 [ notch1 untreated ] , 51 2 [ notch1 ad - cmv - icre treated ] ) , respectively ( fig . to verify that the observed effect was due to reduced notch1 expression , two groups of notch1 untreated and notch1 ad - cmv - icre treated lacrimal glands were used to test notch1 expression by quantitative rt - pcr . notch signaling regulates the size and number of lobules in the developing lg : gamma secretase inhibition study . the lacrimal glands were treated with a notch signaling inhibitor ( dapt ; 10 , 50 , and 100 m ) . ( b ) whole gland size , individual lobule size , and total number of lobules were measured and analyzed for each lacrimal gland ( * * p < 0.01 , * p < 0.05 , n = 57 ) . notch signaling regulates the size and number of lobules in the developing lacrimal gland : conditional notch1 knockout study . ( b ) whole gland size , individual lobule size , and total number of lobules were measured and analyzed for each group of lacrimal glands ( * * p < 0.01 , * p < 0.05 , n = 57 ) . the changes in gene expression detected by microarrays were further confirmed by quantitative rt - pcr , western blot analysis , and immunohistochemistry for a group of selected genes ( figs . importantly , we detected expression of all genes that had been previously reported to be involved in lacrimal gland development ( table 3 ) . the randomly selected genes expressed differently between the two studied groups differential gene expression between the embryonic and adult lacrimal glands revealed by microarray analysis was confirmed for a select group of genes by quantitative rt - pcr ( a ) and western blot analysis ( b ) ( * p < 0.05 , * * p < 0.01 , em16.5 and ad microarray data ) . the notch1 receptor is present in the embryonic and adult lacrimal gland at the protein level . while notch1 was highly expressed in the epithelium and mesenchyme of e16.5 lacrimal glands ( a ) , notch1 was expressed only in single , isolated cells in adult lacrimal glands ( b , c ) . genes known to be expressed ( before the study ) by lacrimal gland tissue the significant differences in gene expression between embryonic and adult lacrimal glands created challenges in pathway analysis of our microarray data . because our study focused predominantly on pathways involved in glandular development , we first analyzed the gene ontology ( go ) database associated with gland development , and extracted a list of genes that passed the threshold level in our microarray data ( supplementary table s2 ) . kegg and panther pathway mapping demonstrated that genes of the notch , wnt , tgf , and hedgehog signaling cascades were highly represented in our microarray data ( supplementary table s3 ) . using the kegg pathway database , we found that a significant number of genes from the notch , wnt , tgf , and hedgehog signaling pathways are expressed in both embryonic and adult lacrimal glands ( supplementary table s4 ) . , suggesting that these signaling pathways may play a role in both lacrimal gland development and in the maintenance of adult lacrimal gland homeostasis ( supplementary table s4 ) . because the notch , wnt , tgf , and hedgehog pathways all participate in the regulation of epithelial - to - mesenchymal transition ( emt ) , it is plausible that this biological process may play active roles in both embryonic and adult lacrimal glands . in addition , we tested for the presence of genes from the go database 's mesenchymal to epithelial transition ( met ) list and found that these genes were also represented in our microarray data ( supplementary table s5 ) . finally , we used these observations to generate the most comprehensive list of lacrimal gland two techniques the use of adult stem cells in lacrimal gland regenerative therapy , and the transplantation of lacrimal gland specific stem cells / progenitors harbor the potential to restore lacrimal gland function and confer tremendous clinical benefits . unfortunately , at the present time only c - kit ( kit ) , aldh1 ( aldh1a1 , aldh1a2 , and aldh1a3 ) , and abcg2 ( abcg2 ) have been identified as putative lacrimal gland our data indicate that these markers are expressed in both embryonic and adult lacrimal gland tissue . to identify new potential lacrimal gland specific stem cell / progenitor markers , we compared the results of our microarray data with a list of genes and proteins commonly associated with stem / progenitor cell states ( table 4 ) . we found that expression of some of these genes was significantly upregulated in the e16.5 lacrimal gland compared with the adult lacrimal gland ( table 4 ) . in addition , we employed immunohistochemistry assays to evaluate the distribution of two of the gene products , ngfr / p75 and notch1 , in fixed embryonic and adult lacrimal glands . our data uncovered several signaling pathways that may play important roles in lacrimal gland development and morphogenesis . from the large list of potential candidates , we chose to focus on one the notch signaling pathway in order to directly evaluate its role in lacrimal gland development . we selected notch because , as noted above , many genes of the notch signaling pathway , including notch1 , notch3 , hes1 , dlk1 , jag1 , jag2 , and rbpj , were expressed at significantly higher levels in the embryonic lacrimal gland than in the adult lacrimal gland ( figs . we began our study of the notch pathway by treating embryonic lacrimal glands with dapt , a nonselective inhibitor of notch signaling . we found that while dapt treatment did not affect significantly the overall size of the cultured embryonic lacrimal glands , it did exert a strong effect on average lobule size and average number of lobules ( fig . dapt treatment led to a reduction in the average size of lobules ( 93 11 m [ dmso ] , 66 3 m [ 10-m dapt ] , 60 1 m [ 50-m dapt ] , 73 3 m [ 100-m dapt ] ) , but also to an increase in the average number of lobules ( 22 2 [ dmso ] , 53 2 [ 10-m dapt ] , 38 4 [ 50-m dapt ] , 31 1 [ 100-m dapt ] ; fig . in agreement with the dapt data above , we found that notch1 knockout in the embryonic lacrimal gland led to a reduction in the average size of lobules ( 90 3 m [ wild - type untreated ] , 82 1 m [ wild - type ad - cmv - icre treated ] , 81 3 m [ notch1 untreated ] , 56 2 m [ notch1 ad - cmv - icre treated ] ) , but also to an increase in the average number of lobules ( 29 3 [ wild - type untreated ] , 37 6 [ wild - type ad - cmv - icre treated ] , 40 2 [ notch1 untreated ] , 51 2 [ notch1 ad - cmv - icre treated ] ) , respectively ( fig . to verify that the observed effect was due to reduced notch1 expression , two groups of notch1 untreated and notch1 ad - cmv - icre treated lacrimal glands were used to test notch1 expression by quantitative rt - pcr . notch signaling regulates the size and number of lobules in the developing lg : gamma secretase inhibition study . the lacrimal glands were treated with a notch signaling inhibitor ( dapt ; 10 , 50 , and 100 m ) . ( b ) whole gland size , individual lobule size , and total number of lobules were measured and analyzed for each lacrimal gland ( * * p < 0.01 , * p < 0.05 , n = 57 ) . notch signaling regulates the size and number of lobules in the developing lacrimal gland : conditional notch1 knockout study . ( b ) whole gland size , individual lobule size , and total number of lobules were measured and analyzed for each group of lacrimal glands ( * * p < 0.01 , * p < 0.05 , n = 57 ) . although the structure and function of the lacrimal gland have been studied extensively , the mechanisms behind lacrimal gland development , morphogenesis , and differentiation remain rather poorly understood . in this study , comparing embryonic and adult lacrimal glands , we collected extensive data on lacrimal gland developmental biology , and further confirmed these findings in vitro . we leveraged this expression data to help identify new putative markers of a lacrimal gland stem / progenitor cell phenotype . wnt- , tgf- , and hedgehog pathway - regulated emt should be a critical mechanism in lacrimal gland development . in addition , our data indicate that the notch signaling exert a strong effect on average lobule size and average number of lobules in the developing lacrimal gland . this study has shed new light on the mechanisms of the lacrimal gland 's development and morphology . our data indicate that the majority of genes involved in the notch- , wnt- , tgf- , and hedgehog - signaling pathways are expressed in the lacrimal gland during both embryonic and adult stages . such sustained expression lends credence to the hypothesis that the notch- , wnt- , tgf- , and hedgehog - signaling cascades play roles both in lacrimal gland development and in the maintenance of adult lacrimal gland homeostasis . we noted that the notch , wnt , tgf , and hedgehog pathways can activate regulators of emt , and may therefore be involved in morphologic modulation of the developing gland . as expected , we found that many of these genes are indeed highly expressed in the lacrimal gland . were more highly expressed in the adult glands , suggesting that emt and met are involved in lacrimal gland development and in the maintenance of adult lacrimal gland homeostasis . given the notch pathway 's essential role in preventing progenitor differentiation in the mammary gland ( and many other tissues types ) , we suggest that the canonical notch ligands jag1/jag2 and the noncanonical dlk1 may activate notch1/notch3/rbpj , preventing differentiation of all lacrimal gland cells expressing notch receptors . meanwhile , because elf5 mediates mammary gland progenitor cell differentiation by inhibiting notch signaling , we can suggest that a similar process may occur during lacrimal gland development , as elf5 expression while present in the embryonic gland was significantly upregulated in the adult lacrimal gland . in order to directly evaluate the role of the identified pathways in lacrimal gland development , we chose to focus on the notch signaling pathway . our study allows us to link notch signaling with lacrimal gland branching because inhibition of noth signaling significantly increased the average number of lobules and significantly reduced average lobule size observed . branching processes are critical for the normal development of a number of organs , including the lung , kidney , pancreas , prostate , salivary gland , and lacrimal gland . thus , our findings suggest that notch signaling may regulate lacrimal gland branching by inhibiting cleft formation . such a mechanism could explain how notch - mediated distribution of progenitor and differentiated cells in the developing lacrimal gland may influence the branching process ( fig . in conclusion , we have assembled here the most comprehensive list of putative lacrimal gland stem / progenitor cell markers to date ( table 4 ) . in addition , available evidence appears to indicate important roles for the notch- , wnt- , tgf- , and hedgehog - signaling cascades in lacrimal gland development , morphogenic features , maintenance of phenotypes , and homeostasis . our findings suggest a role of emt and met in both embryonic and adult lacrimal gland , possibly shedding new light on the biology of epithelial neoplasms in this tissue ( i.e. finally , our data further suggest that notch signaling may help regulate the lacrimal gland 's branching morphogenesis . thus , this study has allowed us to molecularly characterize , for the first time , the lacrimal gland as a whole , and to propose novel mechanisms that regulate its development .

from september 2002 to july 2005 , nodular ggos smaller than 3 cm were found on chest ct in 93 individuals ; follow - up thin - section ct ( 1 - 2.5 mm ) were performed in 64 individuals . six lesions in six individuals were confirmed as solid nodules on thin - section ct . three lesions in three individuals disappeared at the one - month follow - up with thin - section ct . the remaining 96 nodular ggos in 55 individuals ( m : f = 27:28 ; age range 44 - 80 ; median age 58 ; mean age 59 ) were included in this study . the lesions were followed by ct for at least one month from the initial chest ct ( 1 - 49 months , mean 7 months ) . forty nodular ggos in 30 individuals were pathologically confirmed to be : adenocarcinoma ( n = 15 ) , bac ( n = 11 ) , aah ( n = 8) , focal interstitial fibrosis ( n = 5 ) and aspergillosis ( n = 1 ) by lobectomy ( n = 30 ) , wedge resection ( n = 9 ) and cutting needle biopsy ( n = 1 ) . because of the retrospective nature of this study , the decision for surgical biopsy was not based on documented protocol . management decisions were made on a case by case basis with published data ( 1 ) and through conference with physicians and surgeons . thirty - eight individuals were non - smokers , 15 were current smokers and two were ex - smokers . the lesions were incidentally found in 53 individuals by regular screening or as part of the evaluation of other diseases , one was found by imaging work - up for hemoptysis , and another by ct imaging for persistent whitish sputum . eight individuals had a history of a previous malignancy in a variety of organs : stomach cancer ( n = 2 ) , bladder cancer ( n = 2 ) , breast cancer ( n = 1 ) , chondrosarcoma ( n = 1 ) , osteosarcoma ( n = 1 ) , and malignant fibrous histiocytoma ( n = 1 ) . three individuals had a concurrent adenocarcinoma with a solid mass in other lobes of the lung . in all individuals , a thin - section ct was performed at least one time during the follow - up . in 24 individuals , thin - section ct was performed with a lightspeed - ultra scanner ( ge medical systems , milwaukee , wi ) with 120 kvp , 400 mas , a pitch of 0.875 , and collimation of 1.25 mm . thin - section ct was performed with a sensation-16 scanner ( siemens medical solutions , erlangen , germany ) with 140 kvp , 250 effective mas , pitch of 1 , and collimation of 0.75 mm . thin - section ct was performed with a mx 8000 scanner ( phillips medical systems , netherlands ) with 120 kvp , 150 mas , pitch of 1.25 , and collimation of 2.5 mm . thin - section ct was performed with a somatom plus-4 ( siemens medical solutions ) with 120 kvp , 300 mas , pitch of 1 , and collimation of 1 - 2 mm . , the thin - section ct was performed at outside institutions with a variety of scanners with 120 - 140 kvp , 200 - 300 mas , pitch of 1 - 1.5 , collimation of 0.75 - 2.5 mm , and reconstruction thickness 0.75 - 2.5 mm . in all individuals , initial or follow - up chest ct was performed using a variety of scanners including : sensation-16 , lightspeed - ultra , mx-8000 , somatom plus 4 , or hispeed advantage ( ge medical systems ) . chest ct was performed with 120 kvp , 200 - 300 ma , pitch of 0.875 - 1.5 , and collimation of 0.75 - 8 mm . the ct images were read by three board - certified radiologists in consensus , each of whom had more than eight years of experience in chest image interpretation . the number of nodular ggos at the initial ct and the presence of mediastinal lymph node enlargement were initially evaluated . we classified each lesion as to its type - pure nodular ggo ( pnggo ) or mixed nodular ggo ( mnggo)-on the basis of the appearance on the thin - section ct scans . a lesion was classified as mnggo if it contained patches that completely obscured the lung parenchyma . a lesion was classified as pnggo if none of the lung parenchyma within it was totally obscured . in making the distinction between a pnggo and a mnggo , we did not regard blood vessels as solid components despite their solid appearance . the diameter of a nodule , as a measure of its size , was defined as the average of its length and width measured with electronic calipers on the image that showed the largest cross - sectional area of the nodule . the diameter was measured on the initial chest ct and on the final chest ct . subsequently , we categorized nodular ggos into four groups on the basis of the size on initial ct and type on the thin - section ct : pnggo 10 mm , pnggo > 10 mm , mnggo 10 mm , and mnggo > 10 mm . in each group , we evaluated the change in size during the follow - up period , and the pathological result . the rate of malignancy and rate of adenocarcinoma were calculated on a lesion by lesion basis . we graded the area of the solid portion in mnggos on the thin - section ct scans with a four - point scale . grade 1 was defined as a solid portion 25% , grade 2 as a solid portion 50% , grade 3 as a solid portion 75% and grade 4 as a solid portion > 75% . the change of size during the follow - up period and the pathological results according to the grade of solid portion was assessed . the rate of malignancy and the rate of adenocarcinoma according to the grade of solid portion were also evaluated . from september 2002 to july 2005 , nodular ggos smaller than 3 cm were found on chest ct in 93 individuals ; follow - up thin - section ct ( 1 - 2.5 mm ) were performed in 64 individuals . six lesions in six individuals were confirmed as solid nodules on thin - section ct . three lesions in three individuals disappeared at the one - month follow - up with thin - section ct . the remaining 96 nodular ggos in 55 individuals ( m : f = 27:28 ; age range 44 - 80 ; median age 58 ; mean age 59 ) were included in this study . the lesions were followed by ct for at least one month from the initial chest ct ( 1 - 49 months , mean 7 months ) . forty nodular ggos in 30 individuals were pathologically confirmed to be : adenocarcinoma ( n = 15 ) , bac ( n = 11 ) , aah ( n = 8) , focal interstitial fibrosis ( n = 5 ) and aspergillosis ( n = 1 ) by lobectomy ( n = 30 ) , wedge resection ( n = 9 ) and cutting needle biopsy ( n = 1 ) . because of the retrospective nature of this study , the decision for surgical biopsy was not based on documented protocol . management decisions were made on a case by case basis with published data ( 1 ) and through conference with physicians and surgeons . thirty - eight individuals were non - smokers , 15 were current smokers and two were ex - smokers . the lesions were incidentally found in 53 individuals by regular screening or as part of the evaluation of other diseases , one was found by imaging work - up for hemoptysis , and another by ct imaging for persistent whitish sputum . eight individuals had a history of a previous malignancy in a variety of organs : stomach cancer ( n = 2 ) , bladder cancer ( n = 2 ) , breast cancer ( n = 1 ) , chondrosarcoma ( n = 1 ) , osteosarcoma ( n = 1 ) , and malignant fibrous histiocytoma ( n = 1 ) . three individuals had a concurrent adenocarcinoma with a solid mass in other lobes of the lung . in all individuals , a thin - section ct was performed at least one time during the follow - up . in 24 individuals , thin - section ct was performed with a lightspeed - ultra scanner ( ge medical systems , milwaukee , wi ) with 120 kvp , 400 mas , a pitch of 0.875 , and collimation of 1.25 mm . thin - section ct was performed with a sensation-16 scanner ( siemens medical solutions , erlangen , germany ) with 140 kvp , 250 effective mas , pitch of 1 , and collimation of 0.75 mm . thin - section ct was performed with a mx 8000 scanner ( phillips medical systems , netherlands ) with 120 kvp , 150 mas , pitch of 1.25 , and collimation of 2.5 mm . thin - section ct was performed with a somatom plus-4 ( siemens medical solutions ) with 120 kvp , 300 mas , pitch of 1 , and collimation of 1 - 2 mm . images were reconstructed with 1 - 2 mm thickness . in the remaining four individuals , the thin - section ct was performed at outside institutions with a variety of scanners with 120 - 140 kvp , 200 - 300 mas , pitch of 1 - 1.5 , collimation of 0.75 - 2.5 mm , and reconstruction thickness 0.75 - 2.5 mm . in all individuals , initial or follow - up chest ct was performed using a variety of scanners including : sensation-16 , lightspeed - ultra , mx-8000 , somatom plus 4 , or hispeed advantage ( ge medical systems ) . chest ct was performed with 120 kvp , 200 - 300 ma , pitch of 0.875 - 1.5 , and collimation of 0.75 - 8 mm . the ct images were read by three board - certified radiologists in consensus , each of whom had more than eight years of experience in chest image interpretation . the number of nodular ggos at the initial ct and the presence of mediastinal lymph node enlargement were initially evaluated . we classified each lesion as to its type - pure nodular ggo ( pnggo ) or mixed nodular ggo ( mnggo)-on the basis of the appearance on the thin - section ct scans . a lesion was classified as mnggo if it contained patches that completely obscured the lung parenchyma . a lesion was classified as pnggo if none of the lung parenchyma within it was totally obscured . in making the distinction between a pnggo and a mnggo , we did not regard blood vessels as solid components despite their solid appearance . the diameter of a nodule , as a measure of its size , was defined as the average of its length and width measured with electronic calipers on the image that showed the largest cross - sectional area of the nodule . the diameter was measured on the initial chest ct and on the final chest ct . subsequently , we categorized nodular ggos into four groups on the basis of the size on initial ct and type on the thin - section ct : pnggo 10 mm , pnggo > 10 mm , mnggo 10 mm , and mnggo > 10 mm . in each group , we evaluated the change in size during the follow - up period , and the pathological result . the rate of malignancy and rate of adenocarcinoma were calculated on a lesion by lesion basis . we graded the area of the solid portion in mnggos on the thin - section ct scans with a four - point scale . grade 1 was defined as a solid portion 25% , grade 2 as a solid portion 50% , grade 3 as a solid portion 75% and grade 4 as a solid portion > 75% . the change of size during the follow - up period and the pathological results according to the grade of solid portion was assessed . the rate of malignancy and the rate of adenocarcinoma according to the grade of solid portion were also evaluated . forty - three individuals had a single lesion , five had two lesions , two had three lesions , two had four lesions , one had eight lesions , one had nine lesions and one had 12 lesions . in 12 individuals who had multiple lesions , nine had only pnggo and three had both types of ggos . the initial size of the 96 nodular ggos was 2 - 30 mm ( mean , 9.7 mm ) . fifty - five lesions in 23 individuals were pnggo 10 mm and 13 lesions in 13 individuals were pnggo > 10 mm . eleven lesions in eight individuals were mnggo 10 mm and 17 lesions in 16 individuals were mnggo > 10 mm . for the pnggos , a change of size was not noted in any lesion during follow - up ( fig . 1 ) . two lesions in the mnggos 10 mm and one lesion in the mnggos > 10 mm grew in size during the follow - up period . twelve lesions from the pnggos 10 mm , ten lesions from the pnggo > 10 mm , three lesions from the mnggo 10 mm and 15 lesions from the mnggo > 10 mm were resected . the histological results and change of size in each group are presented in table 2 . the resected pnggos 10 mm were aah ( n = 6 ) ( fig . 1 ) , bac ( n = 5 ) , and focal interstitial fibrosis ( n = 1 ) . the resected pnggos > 10 mm were focal aah ( n = 2 ) ( fig . 3 ) ( n = 2 ) and interstitial fibrosis ( n = 4 ) ( fig . the resected mnggos 10 mm were adenocarcinoma ( n = 2 ) and bac ( n = 1 ) . the resected mnggos > 10 mm were adenocarcinoma ( n = 11 ) ( fig . 5 ) , bac ( n = 3 ) and aspergillosis ( n = 1 ) ( fig . 6 ) . three lesions that had grown during the follow - up were histologically confirmed as adenocarcinoma with mixed acinar and bronchioloalveolar patterns . among 15 adenocarcinomas , 13 lesions were histologically confirmed as well - differentiated or moderately - differentiated adenocarcinoma with mixed acinar and bronchioloalveolar types and two were as well - differentiated adenocarcinoma with bronchioloalveolar type . in the resected lesions , the rate of malignancy on a lesion basis was 42% ( pnggos 10 mm ) , 40% ( pnggos > 10 mm ) , 100% ( mnggos 10 mm ) and 93% ( mnggos > 10 mm ) . the rate of adenocarcinoma was 0% ( pnggos 10 mm ) , 20% ( pnggos > 10 mm ) , 67% ( mnggos 10 mm ) and 73% ( mnggos > 10 mm ) . the pathology and change in size during the follow - up period according to the grade of the solid portion in the mnggos are presented in table 3 . one lesion that had grown during the follow - up was grade 2 , another was grade 3 , and a third was grade 4 . according to the grade of the solid portion of the mnggos , the rate of malignancy on a lesion by lesion basis was 83% ( grade 1 ) and 100% ( grades 2 - 4 ) . the rate of adenocarcinoma was 67% ( grade 1 ) , 100% ( grade 2 ) , 60% ( grade 3 ) and 67% ( grade 4 ) . the results of this study can be summarized as the following : a ) none of the pnggos showed considerable change in size during the follow - up period ; b ) most of the pnggos were histologically aah , bac , or focal interstitial fibrosis ; c ) we did not find adenocarcinoma among pnggos 10 mm but , there were several adenocarcinomas among pnggos > 10 mm ; d ) several mnggos grew during the follow - up period and they were histologically described as adenocarcinoma ; e ) in mnggos , the possibility of malignancy and the possibility of adenocarcinoma were high , regardless of the grade of the solid portion . in this study the term " ground - glass " was introduced to describe not only focal abnormalities but diffuse abnormalities as well . nodular ggos " is more appropriate than " focal ggos " or " localized ggos " because the latter also represents nonspecific irregular infiltration or mosaic attenuation of the lung . in the current study , pnggos remained stable over a considerable time . none of pnggo showed a change in size during the follow - up period regardless of size . a long doubling time for pnggos hasegawa et al . ( 3 ) reported a mean volume doubling time for pnggo tumors to be 813 days , which was significantly longer than for mnggo tumors with a solid central component ( 457 days ) or in entirely solid nodules ( 149 days ) . aoki et al . ( 4 ) reported that all adenocarcinomas with noguchi types a ( localized bac ) or b ( localized bac with foci of structural collapse of alveoli ) tumors with localized ggo ( 5 ) on high - resolution ct had a tumor doubling time of more than one year . ( 6 ) reported that , among 19 pnggos over a two - year follow - up , the size increased by more than 5 mm in five patients , increased up to 5 mm in six patients and showed no change in eight patients . the resected lesions were histologically bac , adenocarcinoma , aah , focal fibrosis and lymphoproliferative disease . kodama et al . did not describe the ct parameters and the method of measurement in their article . might be caused by the difference in the follow - up period , the method of measurement and ct parameters and the difference in the disease entities studied . ( 7 ) reported that eight pnggos over six - months changed during follow - up ; five increased in size , two decreased and one was stable in size with increasing density . by contrast , kakinuma et al . only enrolled the pnggos that were histologically confirmed as lung cancer and were larger than 5 mm in diameter at the time of the first thin - section ct . moreover , in most of the pnggos described by kakinuma et al . , the focal solid portion appeared during the follow - up , and growth occurred after the appearance of a focal solid portion . therefore the growth occurred when the nodule was a mnggo not a pnggo ( 7 ) . most of the pnggos in this study were histologically aah , bac or focal interstitial fibrosis . among pnggos smaller than 10 mm theoretically , pnggos on high resolution ct ( hrct ) should not possess a nonaerated component suggesting adenocarcinoma in their pathologic specimens , since nonaerated components usually appear as focal solid portions on hrct . this discrepancy may have developed from partial volume averaging due to relatively thick ct slices . one of two adenocarcinomas appeared as a pnggo on ct scanned with 2.5 mm slice thickness . the sizes of the nonaerated component of the resected adenocarcinomas were smaller than 3 mm . therefore , the detection of small nonaerated components , which enable aah or bac to be distinguished from adenocarcinoma , may be difficult with 2 - 2.5 mm slice ct . to discriminate between adenocarcinoma , bac and aah , a much higher resolution ct examination is needed . usually , a focal ggo due to acute inflammation or bleeding disappears during a three month follow - up period . however , pnggos due to focal interstitial fibrosis remained stable over a several months in our study . these lesions were histologically confirmed as focal interstitial fibrosis with fibroblast and type ii pneumocyte proliferation and collection of alveolar macrophages . previous investigators have suggested that a polygonal or flat shape of the lesions represent advanced scar tissue as a result of an inflammatory process from a variety of causes and that the possibility of malignancy or aah is substantially low ( 9 ) . in this study the pnggo smaller than 10 mm remained stable for a considerable period of time . this data suggest that most small pnggos might not progress to clinical disease and may be included in the category of over diagnosis bias . however , we still can not recommend follow - up thin - section ct without immediate diagnostic intervention because of the discrepancy with previous reports ( 6 , 7 ) . by contrast , when the pnggo is larger than 10 mm or shows an increase in size or density , wedge resection of the lesion should be recommended for the purpose of a diagnostic intervention as choice of management . although , transthoracic ct - guided biopsy has frequently offered an alternative to open surgical biopsy for preoperative diagnosis , there are drawbacks to transthoracic biopsy . for the differentiation of aah , bac and adenocarcinoma , an adequate tissue sample including the entire area of the lesion should be obtained . in addition , bac can not be diagnosed based on a small biopsy sample because the definition of bac requires that there be no evidence of invasive features within the entire lesion ( 10 ) . moreover , three different kinds of histological components could be mixed in a lesion with variable proportions and focal adenocarcinoma can be missed with transthoracic biopsy . these results suggest that mnggo may be a radiological manifestation of advanced cancer with a short doubling time compared to pnggo , which is supported by results of a previous study ( 3 ) . our study also showed that , regardless of the initial size of the lesion , the rate of malignancy and the rate of adenocarcinoma were high with the mnggos . they reported that , the frequency of malignancy in mnggo was much higher than in solid nodules , and that the distribution by malignancy type was strikingly different in the mnggos as compared with solid nodules . the malignancies in mnggos were typically bacs or adenocarcinomas with bronchioloalveolar features , whereas for the solid nodules the malignancies were typically other subtypes of adenocarcinoma . according to the 1999 who classification , bac is defined as a noninvasive tumor in adenocarcinoma ( 10 ) . aah has been suggested to be a possible precursor to bac , and many studies have been conducted to characterize aah ( 11 - 16 ) . both aah and bac without fibroblast proliferation display a replacement growth pattern . if this is true , hrct images could be used to distinguish precursor lesions and early ( in situ ) carcinomas from invasive carcinomas . among the histologically confirmed mnggo in our study , we could not find any aah lesions ; all lesions were bac or adenocarcinoma except for one aspergillosis . in our study , the separate classification of pnggo and mnggo was helpful in the differentiation between aah and adenocarcinoma . interestingly , the grading of the internal solid portion of the mnggo also did not contribute the differentiation of bac from adenocarcinoma . in the three cases of proven bac in our study , the grade of the internal solid portion on ct was 3 or 4 ; that is , the internal solid portion on ct exceeded 50% of the lesion . the potential for malignancy was very high in most mnggos regardless of size . to avoid resection of focal ggos due to acute inflammation or bleeding , a three - month follow - up ct should be recommended . if the lesion remains on follow - up ct , the patient should then undergo histological confirmation . for the lesions confirmed as bac or adenocarcinoma , the standard curative surgery such as lobectomy should be considered . several researchers have reported , although in retrospective studies , that limited resection may be an acceptable alternative in patients with t1 n0 m0 disease and insufficient pulmonary reserve ( 17 , 18 ) . recent reports have suggested that patients with no invasive lesions such as bac or pnggo on ct could be possible candidates for limited resection ( 19 - 21 ) . according to five - year follow - up results in a prospective limited resection clinical trial , lung tumors 2 cm or less in diameter with hrct findings of ggo , and without evident pleural indentations or vascular convergence may be safely managed with only limited resection ( 22 ) . however , considering the probable slow - growing nature of ggo lesions , five years of follow - up is not long enough to conclude that the disease is cured . moreover , a limited resection would be acceptable only if a bac without an invasive component is correctly diagnosed by frozen section of the tumor . this issue must be studied in further investigations . a critique of our study is required . first , our investigation is limited to the relatively small number of pathologically confirmed nodular ggos and relatively short period of follow - up . our study could be regarded as a preliminary report of long - term follow - up . long - term follow - up in a larger series with a prospective design will be required to elucidate the natural history of nodular ggo and to determine whether surgical intervention is acceptable or unnecessary , which may contribute to reduction of over diagnosis bias . . a recently developed computerized scheme ( 23 , 24 ) may help detect and characterize nodular ggos . fourth , serial ct scans in several individuals were performed with different reconstruction thickness in the same person . therefore a fine structural change could have been missed . in conclusion , mnggos had growth potential ; most of them were pathologically adenocarcinoma or bac . pnggos were stable for several months to years and most of them were aah , bac or focal interstitial fibrosis . none of pnggo showed a change in size during the follow - up period regardless of size . a long doubling time for pnggos hasegawa et al . ( 3 ) reported a mean volume doubling time for pnggo tumors to be 813 days , which was significantly longer than for mnggo tumors with a solid central component ( 457 days ) or in entirely solid nodules ( 149 days ) . ( 4 ) reported that all adenocarcinomas with noguchi types a ( localized bac ) or b ( localized bac with foci of structural collapse of alveoli ) tumors with localized ggo ( 5 ) on high - resolution ct had a tumor doubling time of more than one year . ( 6 ) reported that , among 19 pnggos over a two - year follow - up , the size increased by more than 5 mm in five patients , increased up to 5 mm in six patients and showed no change in eight patients . the resected lesions were histologically bac , adenocarcinoma , aah , focal fibrosis and lymphoproliferative disease . kodama et al . did not describe the ct parameters and the method of measurement in their article . might be caused by the difference in the follow - up period , the method of measurement and ct parameters and the difference in the disease entities studied . ( 7 ) reported that eight pnggos over six - months changed during follow - up ; five increased in size , two decreased and one was stable in size with increasing density . the discrepancy between our results and those of kakinuma et al . can be explained by the difference in patient selection . by contrast , kakinuma et al . only enrolled the pnggos that were histologically confirmed as lung cancer and were larger than 5 mm in diameter at the time of the first thin - section ct . moreover , in most of the pnggos described by kakinuma et al . , the focal solid portion appeared during the follow - up , and growth occurred after the appearance of a focal solid portion . therefore the growth occurred when the nodule was a mnggo not a pnggo ( 7 ) . most of the pnggos in this study were histologically aah , bac or focal interstitial fibrosis . among pnggos smaller than 10 mm theoretically , pnggos on high resolution ct ( hrct ) should not possess a nonaerated component suggesting adenocarcinoma in their pathologic specimens , since nonaerated components usually appear as focal solid portions on hrct . this discrepancy may have developed from partial volume averaging due to relatively thick ct slices . one of two adenocarcinomas appeared as a pnggo on ct scanned with 2.5 mm slice thickness . the sizes of the nonaerated component of the resected adenocarcinomas were smaller than 3 mm . nakajima et al . ( 8) reported that 10% of pnggo lesions were adenocarcinoma with stromal invasion . therefore , the detection of small nonaerated components , which enable aah or bac to be distinguished from adenocarcinoma , may be difficult with 2 - 2.5 mm slice ct . to discriminate between adenocarcinoma , bac and aah , usually , a focal ggo due to acute inflammation or bleeding disappears during a three month follow - up period . however , pnggos due to focal interstitial fibrosis remained stable over a several months in our study . these lesions were histologically confirmed as focal interstitial fibrosis with fibroblast and type ii pneumocyte proliferation and collection of alveolar macrophages . previous investigators have suggested that a polygonal or flat shape of the lesions represent advanced scar tissue as a result of an inflammatory process from a variety of causes and that the possibility of malignancy or aah is substantially low ( 9 ) . in this study the pnggo smaller than 10 mm remained stable for a considerable period of time . this data suggest that most small pnggos might not progress to clinical disease and may be included in the category of over diagnosis bias . however , we still can not recommend follow - up thin - section ct without immediate diagnostic intervention because of the discrepancy with previous reports ( 6 , 7 ) . by contrast , when the pnggo is larger than 10 mm or shows an increase in size or density , wedge resection of the lesion should be recommended for the purpose of a diagnostic intervention as choice of management . although , transthoracic ct - guided biopsy has frequently offered an alternative to open surgical biopsy for preoperative diagnosis , there are drawbacks to transthoracic biopsy . for the differentiation of aah , bac and adenocarcinoma , an adequate tissue sample including the entire area of the lesion should be obtained . in addition , bac can not be diagnosed based on a small biopsy sample because the definition of bac requires that there be no evidence of invasive features within the entire lesion ( 10 ) . moreover , three different kinds of histological components could be mixed in a lesion with variable proportions and focal adenocarcinoma can be missed with transthoracic biopsy . none of pnggo showed a change in size during the follow - up period regardless of size . a long doubling time for pnggos hasegawa et al . ( 3 ) reported a mean volume doubling time for pnggo tumors to be 813 days , which was significantly longer than for mnggo tumors with a solid central component ( 457 days ) or in entirely solid nodules ( 149 days ) . ( 4 ) reported that all adenocarcinomas with noguchi types a ( localized bac ) or b ( localized bac with foci of structural collapse of alveoli ) tumors with localized ggo ( 5 ) on high - resolution ct had a tumor doubling time of more than one year . ( 6 ) reported that , among 19 pnggos over a two - year follow - up , the size increased by more than 5 mm in five patients , increased up to 5 mm in six patients and showed no change in eight patients . the resected lesions were histologically bac , adenocarcinoma , aah , focal fibrosis and lymphoproliferative disease . kodama et al . did not describe the ct parameters and the method of measurement in their article . might be caused by the difference in the follow - up period , the method of measurement and ct parameters and the difference in the disease entities studied . ( 7 ) reported that eight pnggos over six - months changed during follow - up ; five increased in size , two decreased and one was stable in size with increasing density . the discrepancy between our results and those of kakinuma et al . can be explained by the difference in patient selection . by contrast , kakinuma et al . only enrolled the pnggos that were histologically confirmed as lung cancer and were larger than 5 mm in diameter at the time of the first thin - section ct . moreover , in most of the pnggos described by kakinuma et al . , the focal solid portion appeared during the follow - up , and growth occurred after the appearance of a focal solid portion . therefore the growth occurred when the nodule was a mnggo not a pnggo ( 7 ) . most of the pnggos in this study were histologically aah , bac or focal interstitial fibrosis . among pnggos smaller than 10 mm theoretically , pnggos on high resolution ct ( hrct ) should not possess a nonaerated component suggesting adenocarcinoma in their pathologic specimens , since nonaerated components usually appear as focal solid portions on hrct . this discrepancy may have developed from partial volume averaging due to relatively thick ct slices . one of two adenocarcinomas appeared as a pnggo on ct scanned with 2.5 mm slice thickness . the sizes of the nonaerated component of the resected adenocarcinomas were smaller than 3 mm . nakajima et al . ( 8) reported that 10% of pnggo lesions were adenocarcinoma with stromal invasion . therefore , the detection of small nonaerated components , which enable aah or bac to be distinguished from adenocarcinoma , may be difficult with 2 - 2.5 mm slice ct . to discriminate between adenocarcinoma , bac and aah , usually , a focal ggo due to acute inflammation or bleeding disappears during a three month follow - up period . however , pnggos due to focal interstitial fibrosis remained stable over a several months in our study . these lesions were histologically confirmed as focal interstitial fibrosis with fibroblast and type ii pneumocyte proliferation and collection of alveolar macrophages . previous investigators have suggested that a polygonal or flat shape of the lesions represent advanced scar tissue as a result of an inflammatory process from a variety of causes and that the possibility of malignancy or aah is substantially low ( 9 ) . in this study the pnggo smaller than 10 mm remained stable for a considerable period of time . this data suggest that most small pnggos might not progress to clinical disease and may be included in the category of over diagnosis bias . however , we still can not recommend follow - up thin - section ct without immediate diagnostic intervention because of the discrepancy with previous reports ( 6 , 7 ) . by contrast , when the pnggo is larger than 10 mm or shows an increase in size or density , wedge resection of the lesion should be recommended for the purpose of a diagnostic intervention as choice of management . although , transthoracic ct - guided biopsy has frequently offered an alternative to open surgical biopsy for preoperative diagnosis , there are drawbacks to transthoracic biopsy . for the differentiation of aah , bac and adenocarcinoma , an adequate tissue sample including the entire area of the lesion should be obtained . in addition , bac can not be diagnosed based on a small biopsy sample because the definition of bac requires that there be no evidence of invasive features within the entire lesion ( 10 ) . moreover , three different kinds of histological components could be mixed in a lesion with variable proportions and focal adenocarcinoma can be missed with transthoracic biopsy . these results suggest that mnggo may be a radiological manifestation of advanced cancer with a short doubling time compared to pnggo , which is supported by results of a previous study ( 3 ) . our study also showed that , regardless of the initial size of the lesion , the rate of malignancy and the rate of adenocarcinoma were high with the mnggos . they reported that , the frequency of malignancy in mnggo was much higher than in solid nodules , and that the distribution by malignancy type was strikingly different in the mnggos as compared with solid nodules . the malignancies in mnggos were typically bacs or adenocarcinomas with bronchioloalveolar features , whereas for the solid nodules the malignancies were typically other subtypes of adenocarcinoma . according to the 1999 who classification , bac is defined as a noninvasive tumor in adenocarcinoma ( 10 ) . aah has been suggested to be a possible precursor to bac , and many studies have been conducted to characterize aah ( 11 - 16 ) . both aah and bac without fibroblast proliferation display a replacement growth pattern . if this is true , hrct images could be used to distinguish precursor lesions and early ( in situ ) carcinomas from invasive carcinomas . among the histologically confirmed mnggo in our study , we could not find any aah lesions ; all lesions were bac or adenocarcinoma except for one aspergillosis . in our study , the separate classification of pnggo and mnggo was helpful in the differentiation between aah and adenocarcinoma . interestingly , the grading of the internal solid portion of the mnggo also did not contribute the differentiation of bac from adenocarcinoma . in the three cases of proven bac in our study , the grade of the internal solid portion on ct was 3 or 4 ; that is , the internal solid portion on ct exceeded 50% of the lesion . the potential for malignancy was very high in most mnggos regardless of size . to avoid resection of focal ggos due to acute inflammation or bleeding , a three - month follow - up ct should be recommended . if the lesion remains on follow - up ct , the patient should then undergo histological confirmation . for the lesions confirmed as bac or adenocarcinoma , the standard curative surgery such as lobectomy should be considered . several researchers have reported , although in retrospective studies , that limited resection may be an acceptable alternative in patients with t1 n0 m0 disease and insufficient pulmonary reserve ( 17 , 18 ) . recent reports have suggested that patients with no invasive lesions such as bac or pnggo on ct could be possible candidates for limited resection ( 19 - 21 ) . according to five - year follow - up results in a prospective limited resection clinical trial , lung tumors 2 cm or less in diameter with hrct findings of ggo , and without evident pleural indentations or vascular convergence may be safely managed with only limited resection ( 22 ) . however , considering the probable slow - growing nature of ggo lesions , five years of follow - up is not long enough to conclude that the disease is cured . moreover , a limited resection would be acceptable only if a bac without an invasive component is correctly diagnosed by frozen section of the tumor . this issue must be studied in further investigations . a critique of our study is required . first , our investigation is limited to the relatively small number of pathologically confirmed nodular ggos and relatively short period of follow - up . our study could be regarded as a preliminary report of long - term follow - up . long - term follow - up in a larger series with a prospective design will be required to elucidate the natural history of nodular ggo and to determine whether surgical intervention is acceptable or unnecessary , which may contribute to reduction of over diagnosis bias . . a recently developed computerized scheme ( 23 , 24 ) may help detect and characterize nodular ggos . fourth , serial ct scans in several individuals were performed with different reconstruction thickness in the same person . therefore a fine structural change could have been missed . in conclusion , mnggos had growth potential ; most of them were pathologically adenocarcinoma or bac . pnggos were stable for several months to years and most of them were aah , bac or focal interstitial fibrosis . these results suggest that mnggo may be a radiological manifestation of advanced cancer with a short doubling time compared to pnggo , which is supported by results of a previous study ( 3 ) . our study also showed that , regardless of the initial size of the lesion , the rate of malignancy and the rate of adenocarcinoma were high with the mnggos . they reported that , the frequency of malignancy in mnggo was much higher than in solid nodules , and that the distribution by malignancy type was strikingly different in the mnggos as compared with solid nodules . the malignancies in mnggos were typically bacs or adenocarcinomas with bronchioloalveolar features , whereas for the solid nodules the malignancies were typically other subtypes of adenocarcinoma . according to the 1999 who classification , bac is defined as a noninvasive tumor in adenocarcinoma ( 10 ) . aah has been suggested to be a possible precursor to bac , and many studies have been conducted to characterize aah ( 11 - 16 ) . both aah and bac without fibroblast proliferation display a replacement growth pattern . if this is true , hrct images could be used to distinguish precursor lesions and early ( in situ ) carcinomas from invasive carcinomas . among the histologically confirmed mnggo in our study , we could not find any aah lesions ; all lesions were bac or adenocarcinoma except for one aspergillosis . in our study , the separate classification of pnggo and mnggo was helpful in the differentiation between aah and adenocarcinoma . interestingly , the grading of the internal solid portion of the mnggo also did not contribute the differentiation of bac from adenocarcinoma . in the three cases of proven bac in our study , the grade of the internal solid portion on ct was 3 or 4 ; that is , the internal solid portion on ct exceeded 50% of the lesion . the potential for malignancy was very high in most mnggos regardless of size . to avoid resection of focal ggos due to acute inflammation or bleeding , a three - month follow - up ct should be recommended . if the lesion remains on follow - up ct , the patient should then undergo histological confirmation . for the lesions confirmed as bac or adenocarcinoma , the standard curative surgery such as lobectomy should be considered . several researchers have reported , although in retrospective studies , that limited resection may be an acceptable alternative in patients with t1 n0 m0 disease and insufficient pulmonary reserve ( 17 , 18 ) . recent reports have suggested that patients with no invasive lesions such as bac or pnggo on ct could be possible candidates for limited resection ( 19 - 21 ) . according to five - year follow - up results in a prospective limited resection clinical trial , lung tumors 2 cm or less in diameter with hrct findings of ggo , and without evident pleural indentations or vascular convergence may be safely managed with only limited resection ( 22 ) . however , considering the probable slow - growing nature of ggo lesions , five years of follow - up is not long enough to conclude that the disease is cured . moreover , a limited resection would be acceptable only if a bac without an invasive component is correctly diagnosed by frozen section of the tumor . this issue must be studied in further investigations . a critique of our study is required . first , our investigation is limited to the relatively small number of pathologically confirmed nodular ggos and relatively short period of follow - up . our study could be regarded as a preliminary report of long - term follow - up . long - term follow - up in a larger series with a prospective design will be required to elucidate the natural history of nodular ggo and to determine whether surgical intervention is acceptable or unnecessary , which may contribute to reduction of over diagnosis bias . . a recently developed computerized scheme ( 23 , 24 ) may help detect and characterize nodular ggos . fourth , serial ct scans in several individuals were performed with different reconstruction thickness in the same person . pnggos were stable for several months to years and most of them were aah , bac or focal interstitial fibrosis .

objectiveto evaluate the inter - group differences in growth and the pathological results of nodular ground - glass opacities ( ggos ) according to their size and focal solid portions.materials and methodsninety - six nodular ggos in 55 individuals followed by ct for at least one month from an initial chest ct were included . forty nodular ggos in 30 individuals were pathologically confirmed to be : adenocarcinoma ( n = 15 ) , bronchioloalveolar carcinoma ( bac ) ( n = 11 ) , atypical adenomatous hyperplasia ( aah ) ( n = 8) , focal interstitial fibrosis ( n = 5 ) and aspergillosis ( n = 1 ) . lesions were categorized based on high - resolution ct findings : pure nodular ggo ( pnggo ) 10 mm , pnggo > 10 mm , mixed nodular ggo ( mnggo) 10 mm , and mnggo > 10 mm . in each group , the change in size during the follow - up period , the pathological results and the rate of malignancy were evaluated.resultsthree mnggo lesions , and none of the pnggo , grew during the follow - up period . resected pnggos 10 mm were aah ( n = 6 ) , bac ( n = 5 ) , and focal interstitial fibrosis ( n = 1 ) . resected pnggos > 10 mm were focal interstitial fibrosis ( n = 4 ) , aah ( n = 2 ) , bac ( n = 2 ) , and adenocarcinoma ( n = 2 ) . resected mnggos 10 mm were adenocarcinoma ( n = 2 ) , and bac ( n = 1 ) . resected mnggos > 10 mm were adenocarcinoma ( n = 11 ) , bac ( n = 3 ) , and aspergillosis ( n = 1).conclusionmixed nodular ggos ( mnggos ) had the potential for growth ; most were pathologically adenocarcinoma or bac . by contrast , pnggos were stable for several months to years ; most were aah , bac , or focal interstitial fibrosis .

MATERIALS AND METHODS Patients CT Imaging Image Reading and Data Analysis RESULTS DISCUSSION Pure Nodular Grand-Glass Opacities The Change in Follow-up The Potential of Malignancy Treatment Strategy of Pure Nodular Grand-Glass Opacities Mixed Nodular Grand-Glass Opacities The Change in Follow-up and the Potential of Malignancy Treatment Strategy of Mixed Nodular Grand-Glass Opacities

from september 2002 to july 2005 , nodular ggos smaller than 3 cm were found on chest ct in 93 individuals ; follow - up thin - section ct ( 1 - 2.5 mm ) were performed in 64 individuals . the remaining 96 nodular ggos in 55 individuals ( m : f = 27:28 ; age range 44 - 80 ; median age 58 ; mean age 59 ) were included in this study . the lesions were followed by ct for at least one month from the initial chest ct ( 1 - 49 months , mean 7 months ) . forty nodular ggos in 30 individuals were pathologically confirmed to be : adenocarcinoma ( n = 15 ) , bac ( n = 11 ) , aah ( n = 8) , focal interstitial fibrosis ( n = 5 ) and aspergillosis ( n = 1 ) by lobectomy ( n = 30 ) , wedge resection ( n = 9 ) and cutting needle biopsy ( n = 1 ) . the lesions were incidentally found in 53 individuals by regular screening or as part of the evaluation of other diseases , one was found by imaging work - up for hemoptysis , and another by ct imaging for persistent whitish sputum . eight individuals had a history of a previous malignancy in a variety of organs : stomach cancer ( n = 2 ) , bladder cancer ( n = 2 ) , breast cancer ( n = 1 ) , chondrosarcoma ( n = 1 ) , osteosarcoma ( n = 1 ) , and malignant fibrous histiocytoma ( n = 1 ) . in all individuals , a thin - section ct was performed at least one time during the follow - up . in all individuals , initial or follow - up chest ct was performed using a variety of scanners including : sensation-16 , lightspeed - ultra , mx-8000 , somatom plus 4 , or hispeed advantage ( ge medical systems ) . we classified each lesion as to its type - pure nodular ggo ( pnggo ) or mixed nodular ggo ( mnggo)-on the basis of the appearance on the thin - section ct scans . subsequently , we categorized nodular ggos into four groups on the basis of the size on initial ct and type on the thin - section ct : pnggo 10 mm , pnggo > 10 mm , mnggo 10 mm , and mnggo > 10 mm . in each group , we evaluated the change in size during the follow - up period , and the pathological result . the change of size during the follow - up period and the pathological results according to the grade of solid portion was assessed . the rate of malignancy and the rate of adenocarcinoma according to the grade of solid portion were also evaluated . from september 2002 to july 2005 , nodular ggos smaller than 3 cm were found on chest ct in 93 individuals ; follow - up thin - section ct ( 1 - 2.5 mm ) were performed in 64 individuals . the remaining 96 nodular ggos in 55 individuals ( m : f = 27:28 ; age range 44 - 80 ; median age 58 ; mean age 59 ) were included in this study . the lesions were followed by ct for at least one month from the initial chest ct ( 1 - 49 months , mean 7 months ) . forty nodular ggos in 30 individuals were pathologically confirmed to be : adenocarcinoma ( n = 15 ) , bac ( n = 11 ) , aah ( n = 8) , focal interstitial fibrosis ( n = 5 ) and aspergillosis ( n = 1 ) by lobectomy ( n = 30 ) , wedge resection ( n = 9 ) and cutting needle biopsy ( n = 1 ) . the lesions were incidentally found in 53 individuals by regular screening or as part of the evaluation of other diseases , one was found by imaging work - up for hemoptysis , and another by ct imaging for persistent whitish sputum . eight individuals had a history of a previous malignancy in a variety of organs : stomach cancer ( n = 2 ) , bladder cancer ( n = 2 ) , breast cancer ( n = 1 ) , chondrosarcoma ( n = 1 ) , osteosarcoma ( n = 1 ) , and malignant fibrous histiocytoma ( n = 1 ) . in all individuals , a thin - section ct was performed at least one time during the follow - up . in all individuals , initial or follow - up chest ct was performed using a variety of scanners including : sensation-16 , lightspeed - ultra , mx-8000 , somatom plus 4 , or hispeed advantage ( ge medical systems ) . we classified each lesion as to its type - pure nodular ggo ( pnggo ) or mixed nodular ggo ( mnggo)-on the basis of the appearance on the thin - section ct scans . subsequently , we categorized nodular ggos into four groups on the basis of the size on initial ct and type on the thin - section ct : pnggo 10 mm , pnggo > 10 mm , mnggo 10 mm , and mnggo > 10 mm . in each group , we evaluated the change in size during the follow - up period , and the pathological result . the change of size during the follow - up period and the pathological results according to the grade of solid portion was assessed . the rate of malignancy and the rate of adenocarcinoma according to the grade of solid portion were also evaluated . fifty - five lesions in 23 individuals were pnggo 10 mm and 13 lesions in 13 individuals were pnggo > 10 mm . eleven lesions in eight individuals were mnggo 10 mm and 17 lesions in 16 individuals were mnggo > 10 mm . two lesions in the mnggos 10 mm and one lesion in the mnggos > 10 mm grew in size during the follow - up period . twelve lesions from the pnggos 10 mm , ten lesions from the pnggo > 10 mm , three lesions from the mnggo 10 mm and 15 lesions from the mnggo > 10 mm were resected . the resected pnggos 10 mm were aah ( n = 6 ) ( fig . 1 ) , bac ( n = 5 ) , and focal interstitial fibrosis ( n = 1 ) . the resected pnggos > 10 mm were focal aah ( n = 2 ) ( fig . 3 ) ( n = 2 ) and interstitial fibrosis ( n = 4 ) ( fig . the resected mnggos 10 mm were adenocarcinoma ( n = 2 ) and bac ( n = 1 ) . the resected mnggos > 10 mm were adenocarcinoma ( n = 11 ) ( fig . 5 ) , bac ( n = 3 ) and aspergillosis ( n = 1 ) ( fig . three lesions that had grown during the follow - up were histologically confirmed as adenocarcinoma with mixed acinar and bronchioloalveolar patterns . in the resected lesions , the rate of malignancy on a lesion basis was 42% ( pnggos 10 mm ) , 40% ( pnggos > 10 mm ) , 100% ( mnggos 10 mm ) and 93% ( mnggos > 10 mm ) . the rate of adenocarcinoma was 0% ( pnggos 10 mm ) , 20% ( pnggos > 10 mm ) , 67% ( mnggos 10 mm ) and 73% ( mnggos > 10 mm ) . the pathology and change in size during the follow - up period according to the grade of the solid portion in the mnggos are presented in table 3 . one lesion that had grown during the follow - up was grade 2 , another was grade 3 , and a third was grade 4 . according to the grade of the solid portion of the mnggos , the rate of malignancy on a lesion by lesion basis was 83% ( grade 1 ) and 100% ( grades 2 - 4 ) . the rate of adenocarcinoma was 67% ( grade 1 ) , 100% ( grade 2 ) , 60% ( grade 3 ) and 67% ( grade 4 ) . the results of this study can be summarized as the following : a ) none of the pnggos showed considerable change in size during the follow - up period ; b ) most of the pnggos were histologically aah , bac , or focal interstitial fibrosis ; c ) we did not find adenocarcinoma among pnggos 10 mm but , there were several adenocarcinomas among pnggos > 10 mm ; d ) several mnggos grew during the follow - up period and they were histologically described as adenocarcinoma ; e ) in mnggos , the possibility of malignancy and the possibility of adenocarcinoma were high , regardless of the grade of the solid portion . none of pnggo showed a change in size during the follow - up period regardless of size . ( 4 ) reported that all adenocarcinomas with noguchi types a ( localized bac ) or b ( localized bac with foci of structural collapse of alveoli ) tumors with localized ggo ( 5 ) on high - resolution ct had a tumor doubling time of more than one year . ( 6 ) reported that , among 19 pnggos over a two - year follow - up , the size increased by more than 5 mm in five patients , increased up to 5 mm in six patients and showed no change in eight patients . the resected lesions were histologically bac , adenocarcinoma , aah , focal fibrosis and lymphoproliferative disease . might be caused by the difference in the follow - up period , the method of measurement and ct parameters and the difference in the disease entities studied . , the focal solid portion appeared during the follow - up , and growth occurred after the appearance of a focal solid portion . most of the pnggos in this study were histologically aah , bac or focal interstitial fibrosis . among pnggos smaller than 10 mm theoretically , pnggos on high resolution ct ( hrct ) should not possess a nonaerated component suggesting adenocarcinoma in their pathologic specimens , since nonaerated components usually appear as focal solid portions on hrct . however , pnggos due to focal interstitial fibrosis remained stable over a several months in our study . in this study the pnggo smaller than 10 mm remained stable for a considerable period of time . by contrast , when the pnggo is larger than 10 mm or shows an increase in size or density , wedge resection of the lesion should be recommended for the purpose of a diagnostic intervention as choice of management . for the differentiation of aah , bac and adenocarcinoma , an adequate tissue sample including the entire area of the lesion should be obtained . our study also showed that , regardless of the initial size of the lesion , the rate of malignancy and the rate of adenocarcinoma were high with the mnggos . aah has been suggested to be a possible precursor to bac , and many studies have been conducted to characterize aah ( 11 - 16 ) . according to five - year follow - up results in a prospective limited resection clinical trial , lung tumors 2 cm or less in diameter with hrct findings of ggo , and without evident pleural indentations or vascular convergence may be safely managed with only limited resection ( 22 ) . first , our investigation is limited to the relatively small number of pathologically confirmed nodular ggos and relatively short period of follow - up . long - term follow - up in a larger series with a prospective design will be required to elucidate the natural history of nodular ggo and to determine whether surgical intervention is acceptable or unnecessary , which may contribute to reduction of over diagnosis bias . in conclusion , mnggos had growth potential ; most of them were pathologically adenocarcinoma or bac . pnggos were stable for several months to years and most of them were aah , bac or focal interstitial fibrosis . none of pnggo showed a change in size during the follow - up period regardless of size . ( 4 ) reported that all adenocarcinomas with noguchi types a ( localized bac ) or b ( localized bac with foci of structural collapse of alveoli ) tumors with localized ggo ( 5 ) on high - resolution ct had a tumor doubling time of more than one year . ( 6 ) reported that , among 19 pnggos over a two - year follow - up , the size increased by more than 5 mm in five patients , increased up to 5 mm in six patients and showed no change in eight patients . the resected lesions were histologically bac , adenocarcinoma , aah , focal fibrosis and lymphoproliferative disease . might be caused by the difference in the follow - up period , the method of measurement and ct parameters and the difference in the disease entities studied . , the focal solid portion appeared during the follow - up , and growth occurred after the appearance of a focal solid portion . most of the pnggos in this study were histologically aah , bac or focal interstitial fibrosis . among pnggos smaller than 10 mm theoretically , pnggos on high resolution ct ( hrct ) should not possess a nonaerated component suggesting adenocarcinoma in their pathologic specimens , since nonaerated components usually appear as focal solid portions on hrct . to discriminate between adenocarcinoma , bac and aah , usually , a focal ggo due to acute inflammation or bleeding disappears during a three month follow - up period . however , pnggos due to focal interstitial fibrosis remained stable over a several months in our study . by contrast , when the pnggo is larger than 10 mm or shows an increase in size or density , wedge resection of the lesion should be recommended for the purpose of a diagnostic intervention as choice of management . for the differentiation of aah , bac and adenocarcinoma , an adequate tissue sample including the entire area of the lesion should be obtained . none of pnggo showed a change in size during the follow - up period regardless of size . ( 4 ) reported that all adenocarcinomas with noguchi types a ( localized bac ) or b ( localized bac with foci of structural collapse of alveoli ) tumors with localized ggo ( 5 ) on high - resolution ct had a tumor doubling time of more than one year . ( 6 ) reported that , among 19 pnggos over a two - year follow - up , the size increased by more than 5 mm in five patients , increased up to 5 mm in six patients and showed no change in eight patients . the resected lesions were histologically bac , adenocarcinoma , aah , focal fibrosis and lymphoproliferative disease . might be caused by the difference in the follow - up period , the method of measurement and ct parameters and the difference in the disease entities studied . , the focal solid portion appeared during the follow - up , and growth occurred after the appearance of a focal solid portion . most of the pnggos in this study were histologically aah , bac or focal interstitial fibrosis . among pnggos smaller than 10 mm theoretically , pnggos on high resolution ct ( hrct ) should not possess a nonaerated component suggesting adenocarcinoma in their pathologic specimens , since nonaerated components usually appear as focal solid portions on hrct . to discriminate between adenocarcinoma , bac and aah , usually , a focal ggo due to acute inflammation or bleeding disappears during a three month follow - up period . however , pnggos due to focal interstitial fibrosis remained stable over a several months in our study . by contrast , when the pnggo is larger than 10 mm or shows an increase in size or density , wedge resection of the lesion should be recommended for the purpose of a diagnostic intervention as choice of management . for the differentiation of aah , bac and adenocarcinoma , an adequate tissue sample including the entire area of the lesion should be obtained . our study also showed that , regardless of the initial size of the lesion , the rate of malignancy and the rate of adenocarcinoma were high with the mnggos . they reported that , the frequency of malignancy in mnggo was much higher than in solid nodules , and that the distribution by malignancy type was strikingly different in the mnggos as compared with solid nodules . aah has been suggested to be a possible precursor to bac , and many studies have been conducted to characterize aah ( 11 - 16 ) . according to five - year follow - up results in a prospective limited resection clinical trial , lung tumors 2 cm or less in diameter with hrct findings of ggo , and without evident pleural indentations or vascular convergence may be safely managed with only limited resection ( 22 ) . first , our investigation is limited to the relatively small number of pathologically confirmed nodular ggos and relatively short period of follow - up . long - term follow - up in a larger series with a prospective design will be required to elucidate the natural history of nodular ggo and to determine whether surgical intervention is acceptable or unnecessary , which may contribute to reduction of over diagnosis bias . in conclusion , mnggos had growth potential ; most of them were pathologically adenocarcinoma or bac . pnggos were stable for several months to years and most of them were aah , bac or focal interstitial fibrosis . our study also showed that , regardless of the initial size of the lesion , the rate of malignancy and the rate of adenocarcinoma were high with the mnggos . aah has been suggested to be a possible precursor to bac , and many studies have been conducted to characterize aah ( 11 - 16 ) . according to five - year follow - up results in a prospective limited resection clinical trial , lung tumors 2 cm or less in diameter with hrct findings of ggo , and without evident pleural indentations or vascular convergence may be safely managed with only limited resection ( 22 ) . first , our investigation is limited to the relatively small number of pathologically confirmed nodular ggos and relatively short period of follow - up . long - term follow - up in a larger series with a prospective design will be required to elucidate the natural history of nodular ggo and to determine whether surgical intervention is acceptable or unnecessary , which may contribute to reduction of over diagnosis bias . pnggos were stable for several months to years and most of them were aah , bac or focal interstitial fibrosis .

conjugated shape - persistent macrocycles and , in particular , arylene ethynylene macrocycles ( aems ) have received much attention in recent years because advances in synthetic techniques have made them more accessible , and they have potential in many applications . aems are relatively thermally , photolytically , and oxidatively stable ; they often have strong uv absorptions and are often highly fluorescent . additionally , these macrocycles have been of interest because their high polarizability makes them desirable as second - order nonlinear optical materials and as materials for organic semiconductors and devices . their unique two - dimensional structure could potentially allow these materials to circumvent the trade - off between efficiency and transparency observed in linear systems , and c3-symmetric systems can be derivatized to give noncentrosymmetric materials . planar aems have been shown to aggregate in solution , in the liquid crystalline phase , and in the solid phase through weak van der waals interactions . aems can aggregate into columnar mesophases as well as vesicles and have the potential to act as model systems for organic nanotubes . arylene ethynylene macrocycles are synthetically accessible either by statistical cyclization of a single aryl halide ethynyl monomer or by the cyclization of a linear oligomer via a palladium catalyzed cross coupling , in either case at low concentrations . this latter approach requires a linear , stepwise , and often tedious synthesis of the linear oligomer but usually gives a single discrete macrocycle as the sole product . alternatively , the former methodology employs more synthetically accessible precursors but often yields various cyclic and linear oligomers unless specific macrocycle ring sizes are excluded by ring strain or steric interactions on neighboring monomer units . more recently , alkyne metathesis has proven to be an efficient synthetic method to prepare the arylene ethynylene scaffold . metathesis allows the formation of the thermodynamically most favored product(s ) but may not offer the functional group tolerance of the palladium - catalyzed protocols . derivatization of the monomers should allow the formation of electron - rich and/or electron - poor ring systems and thus the tuning of their physical properties . the incorporation of side chains is often crucial to allow the solubility of the highly rigid macrocycles . despite the recent attention given arylene ethynylene macrocycles , the incorporation of biphenyl and terphenyl moieties is rare , and such macrocycles have not been fully explored . the few reported examples of biphenyl or teraryl units include bipyridyl , m - terphenyl , m - terpyridinyl , and 11,12-dihydroindolo[2,3-a]carbazole ( nominal p - terphenylenes ) and tetram - phenylene subunits but no simple biphenyl or p - terphenylene containing macrocycles have been reported . the presence of such biphenyl units disrupts the fully planar geometry of the macrocycle and would therefore be expected to attenuate any aggregation via stacking of the planar rings . the deviation from planarity also would affect the conjugation around the macrocycle , and change their electronic properties in comparison to their fully planar analogs . more significantly , the title c3 macrocycles offer the possibility to synthesize short segments of single - walled carbon nanotubes . ethynylene units in shape - persistent macrocycles are usually used as rigid spacers that prevent diaryl steric interactions . however , alkynes are also reactive moieties , and if the alkynes in the title macrocycles can be incorporated into ortho - substituted aromatic rings via cycloaddition reactions with cyclopentadienone synthons , the vertices of the triangular macrocycles could be folded out of the plane to form the walls of the nanotube segment . the phenylene rings in such a cycloaddition product are geometrically disposed to produce a fully fused nanotube segment upon oxidative cyclodehydrogenation ( figure 2 ) . this synthetic path toward carbon nanotube segments relies on the title macrocycles as relatively strain - free templates that are elaborated with additional phenyl rings in a stepwise increase of strain until the fused tube is achieved . alternatively , macrocycles containing cyclopentadienone moieties could be constructed and undergo cycloadditions with diarylalkynes to give similar cyclooligophenylenes . a similar strain strategy pioneered by bertozzi and jasti converts a more highly curved precursor containing sp centers to the all - sp nanotube segment . jasti , itami , and others have used this route to prepare a variety of [ n]cycloparaphenylenes , and jasti and mullen have recently prepared [ n]cycloparaphenylenes that could in principle give belts of longer length upon oxidative cyclodehydrogenation of pendant phenyl substituents . bodwell has also prepared highly curved nanotube segments by incorporating polycyclic arenes in cyclophanes . scott has proposed a slightly different approach that uses bowl - shaped templates upon which the nanotube can be grown . to further our synthetic proposal , the synthesis of c3-symmetric biphenyl and terphenyl arylene ethynylene macrocycles and their alkyl derivatives , along with their photophysical properties , are described below . as discussed above , the construction of cyclooligomers is usually achieved by one of two means : a statistical coupling of simple monomer units or a cyclization of a linear oligomer of appropriate length . the former method involves a shorter synthetic route and was the first one attempted for the construction of the c3-symmetric macrocycles 1a , 1b , 2a , and 2b . since both synthetic approaches require monomers 3a , 3b , 4a , and 4b , they were prepared first . the diethyltriazene and triisopropylsilyl groups on the termini of 3 and 4 are protecting groups that can be removed under orthogonal reaction conditions to give the aryl iodide or terminal alkyne , respectively . a statistical macrocyclization would require both groups to be deprotected to give the ab monomer , while the synthesis of a linear trimer which could be subsequently cyclized could be accomplished using a split - pool strategy ( scheme 1 ) . triazene 5 was prepared in 97% yield by diazotization of 4-iodoaniline followed by quenching with diethylamine . boronation of 5 with 1.2 equiv of bis(pinacalato)diboron , cl2pd(dppf ) , and dry koac in dmso gave 6 in 74% yield . however , this boronation also produced a significant quantity of biphenyl 7 which could only be removed by recrystallization from 2-propanol . in an attempt to minimize the formation of this undesired homodimer , a 3-fold excess of bis(pinacalato)boron no homodimerization was observed , but the excess diboron proved equally difficult to remove during purification . the most effective purification protocol involves recrystallization from 2-propanol to remove the homodimer 7 followed by column chromatography to remove the residual palladium and excess diboron . alternatively , 6 was prepared by diethylamine addition to the diazotized aminophenylboronic acid pinacol ester in 85% yield . this second protocol not only provided an overall higher yield of the phenylene synthon 6 but also a high enough purity after workup that the crude product could be used in further reactions without further purification , in stark contrast to the first protocol . alkyne coupling partner 8 was prepared in 99% yield according to literature procedures from 1-bromo-2-iodobenzene and triisopropylsilylacetylene . suzuki coupling of the boronate ester 6 and alkyne 8 was performed using cl2pd(dppf ) and k3po4 in dme to give the biphenyl monomer 3a in 95% yield . it should be noted that other palladium catalysts ( notably pd(pph4)3 ) , bases , and solvents did not give comparable yields . terphenyl monomer 4a was prepared in 65% yield from triazene 3a by treatment with iodomethane to give 9 followed by a suzuki coupling with triazene 6 . despite the more reactive iodide and lack of hindering ortho group in 9 compared to 8 , the lower yield for the second suzuki coupling was the result of competitive protiodeiodination to give 2-(triisopropylsilylethynyl)biphenyl . the terphenyl triazene also undergoes photolytic decomposition , especially in the presence of silica gel or florisil ; alumina was used in the chromatography of all terphenyl triazenes described , and little such decomposition was observed on this stationary phase . since the macrocycles constructed from 3a and 4a were anticipated to have low solubility , alkyl - substituted analogues 3b and 4b were also prepared ( scheme 3 ) . octylaniline 10 was iodinated with an ammonium dichloroiodate with high regiospecificity give iodoarene 11 , which was diazotized and quenched with diethylamine gave triazene 12 . the sonogashira coupling of 12 with triisopropylsilylacetylene gave 13 , which was then converted to iodoarene 14 in 86% yield over four steps . as with the unsubstituted analogues , biphenyl monomer 3b was obtained by suzuki miyaura coupling of 6 and 14 , and terphenyl monomer 4b was obtained from 3b by deprotection of the triazene in 3b with methyl iodide to give iodoarene 15 ( in 94% yield ) which was then coupled with another equivalent of 6 . initial attempts to couple 6 and 14 produced very poor yields of 3b along with significant quantities of ( 3-octylphenylethynyl)triisopropylsilane , the protiodeiodination product of 14 . the concentration of reactants was increased 5-fold in an attempt to make the coupling more competitive with protiodeiodination , and the yield of the desired biphenyl 3b was increased to 82% . the careful exclusion of water , a potential source of protons , did not increase the yield of coupled product 3b appreciably . additionally , the yield of the suzuki coupling to produce the terphenyl monomer 4b was 68% , lower than that for 3b , presumably for the same reasons discussed above for 3a . the doubly deprotected monomeric iodoalkynes 16a and 17a were obtained by diethyltriazene removal in methyl iodide followed by fluoride deprotection of the ethynyl protecting groups ( scheme 2 ) in 88% and 71% yields over two steps , respectively . the instability of terphenyl 17a required that this free alkyne be utilized immediately after preparation and explains the lower yield of its preparation in comparison to the biphenyl 16a . biphenyl monomer 16a was subjected to sonogashira reaction conditions using cl2pd(pph3)2 at low concentration ( 18 mm ) for 10 days at room temperature to give a product mixture containing a mixture of linear and cyclic oligomers . poor solubility and similar polarities of the reaction products precluded chromatographic separation or purification , but the presence of 1a was evident by peaks in the h nmr ( figure 3 ) that matched those in pure samples of 1a obtained by the alternate synthesis described below . stephens castro coupling of terphenyl monomer 17a at 182 mm and sonogashira coupling at 155 mm with pd(pph3)2cl2 gave only an insoluble yellow product , which had an h nmr spectrum consistent with a mixture of linear and cyclic oligomers . the sonogashira coupling of 17a was attempted again , using pd(pph3)4 and at lower concentration , 18 mm . after 12 days at room temperature , this reaction yielded a solid that when washed repeatedly with methylene chloride proved to be macrocycle 2a . the cyclic trimer was isolated in 20% crude yield but could not be separated from an impurity of unknown structure . similar protocols were used to convert the alkyl - substituted monomers 3b and 4b to cyclooligomers . double deprotection of 3b and 4b to give 16b and 17b proceeded as with the unsubstituted analogues in 68% and 98% yields over two steps , respectively . compound 16b was subjected to sonogashira coupling conditions at 28 mm ; four fluorescent compounds were identified by tlc , but only two compounds were isolated by five iterations of flash chromatography , the cyclic trimer 1b in 36% yield , and the cyclic tetramer 1c in 24% . in an effort to improve the yield of the cyclooligomers , the concentration was lowered to 1.5 mm in the sonogashira coupling reaction of 17b . a 51% yield of the cyclic trimer 2b was recovered by column chromatography as well as cyclic dimer 2c in 12% yield . the yield of the cyclic trimer may also have been higher in the cyclooligomerization of 17b because of the use of pd(pph3)4 instead of pdcl2(pph3)2 . the isolation of the substituted macrocycles was greatly facilitated by their much higher solubility , attributable to their long alkyl side chains . analytically pure samples of 1a and 2a were obtained by constructing each through a linear , stepwise approach ( schemes 4 and 5 , respectively ) . this split pool approach began with the removal of the diethyltriazene of 3a to give 18a in 98% yield and the removal of the ethynyl protecting group of 3a using tbaf to yield 19a in 90% yield ( scheme 3 ) . sonogashira coupling of fragments 18a and 19a gave the protected dimer 20a in 75% yield . unmasking the aryl iodide by removal of the diethyltriazene was followed by a second coupling with fragment 19a , which led to the linear trimer 22a in 67% yield over the two steps . deprotection of 23a with tbaf gave an iodoarylethyne which was immediately subjected to sonogashira coupling conditions without purification . the cyclization was performed by slowly adding a solution of the linear trimer iodoarylethyne with a syringe pump to a solution of the catalysts in triethylamine . the final and highest concentration of the linear trimer was 3.7 mm , and after the addition was complete , the reaction was stirred for another 12 h before workup . a split - pool approach to the terphenyl macrocycle 2a also occurred in a similar sequence , but the additional p - phenylene unit within the monomer unit contributed to solubility problems ( scheme 4 ) . both deprotections of 3b proceeded in 91% yield to give the iodide 18b and terminal alkyne 19b , which were coupled under sonogashira conditions to give dimer 20b in 76% yield . a significant quantity of unreacted 18b was also recovered , along with a similar amount of a third organic product which is presumed to be the hay coupled dialkyne arising from dimerization of 19b . conversion of the triazene to the iodide 21b and sonogashira coupling with another 1.3 equiv of 19b gave the trimer 22b in 52% combined yield . the lower yield compared to the biphenyl system can be attributed to losses during chromatography of the sparingly soluble synthons , and the larger excess of the terminal alkyne was utilized to minimize yield loss due to hay coupling . removal of the tips group gave the sparingly soluble iodoarylethyne linear trimer ; the loss of the relatively small alkyl groups in the tips group significantly lowered its solubility , and its subsequent cyclization was carried out without further chromatographic purification or complete characterization . as with the biphenyl trimer , the cyclization was carried out under high dilution sonogashira conditions ( 2.1 mm ) achieved using a syringe pump . the product mixture was purified by removing the solvent and centrifugation of the residue slurried with dichloromethane . the insoluble organic products floated on the chlorinated solvent while the inorganic catalysts and byproducts thereof formed a solid pellet . the formation of both linear and cyclic oligomers of various sizes during cyclooligomerization is not unexpected and has been shown to occur in various systems under a wide array of reaction conditions . in some of these reports , the cyclotrimeric and cyclotetrameric products isolated from o - iodoethynylenebenzene precursors were relatively unstrained . other studies have reported the production of strained cyclic dimeric species along with unstrained cyclotrimers and cyclotetramers . on the basis of the macrocycles described above , the cyclic dimer , cyclic trimer , and cyclotetramer of the 1,2-phenylene , 1,4-biphenylene , and 1,4-terphenylene ethynylene macrocycles were computationally modeled . geometry optimization and single - point energies were calculated at various levels of theory using gaussian 03 , and the alkyl chains were omitted for computational ease . the structures shown are from the b3lyp/6 - 31g(d ) geometry optimization , but the structures for the geometries optimized with every method do not differ appreciably . the energies tabulated in table 3 are per repeat unit and normalized to the cyclotrimer for each analogous series . for the o - arylene ethynylene cyclooligomers , it is no surprise that the d2h cyclodimer 24c is much higher in energy than either the d3h cyclotrimer 24a or d2d tetramer 24b . the incomplete treatment of the closed -system in the molecular mechanics force field is most likely the source of the difference in repeat unit strain calculated for the cyclotetramer 24b ; the puckered ring of 24b is predicted to be as nearly strainless as cyclotrimer 24a by semiempirical , ab initio , and density functional methods but not by molecular mechanics . the c14h8 repeat units in cyclodimer c2h1d are 10 kcal / mol higher in energy than those in c31a , and the two para - substituted rings are predicted to be coplanar with one another , which gives c21d the appearance of an extended cyclophane . the aryl aryl dihedral angle ( 57 ) as well as a similar alkyne bond angle to that of the cyclic trimer . these structural similarities , despite the pucker in the cyclotetramer ring , contribute its lack of strain ; the cyclotrimer and cyclotetramer are nearly isoenergetic on the basis of each repeat unit . the constrained cyclic array of the terphenyl dimer c2 symmetric 2d forces the two para - substituted rings to be nearly coplanar , while being orthogonal to the ortho - substituted ring . the c20h12 repeat units are calculated to be 8 kcal / mol more strained in 2d than in 2a , a smaller difference than that calculated in the biphenyl macrocycles . the optimized geometry of the cyclotrimer 2a is c3 symmetric and features a nearly all - planar system in which only the central p - phenylene of the terphenyl unit is twisted out of the plane . as a result , there are three planar diphenylacetylene moieties within 2a , a structural feature that is shared by the optimized geometry of c2 symmetric 2e . the lack of angle strain in the alkykyl moieties in both 2a and 2e is a contributing factor making them nearly isoenergetic . in all cases , the cyclodimers have the highest energy per monomer unit compared to the respective cyclotrimers or cyclotetramers , which in each case are nearly isoenergetic . there is a relationship between the number of p - phenylene units in the macrocycle and the relative strain of the cyclodimer ; presumably , the angle strain of the alkynyl moieties is shared among additional phenylene units and the overall strain of the repeat unit is reduced . the average sp carbon bond angle is 155.4 in 24c , 166.5 in 1d , and 170.0 in 2d , and it is evident that smaller deviations from the ideal bond angle in the alkynyl carbons is accompanied by a reduction in the strain energy . the structural similarities shared between 1a and 1e as well as 2a and 2e shown in table 1 reflect their isoenergetic relationships . h interaction present in the biphenyl and terphenyl macrocycles that is as significant in setting the aryl aryl dihedral as the distance between the 2- and 2-hydrogens ; these distances are only significant in the cyclic trimers and tetramers since the cyclic dimer has a near orthogonal biphenyl dihedral angle ( table 2 ) . the observation of cyclic dimer 2c in the macrocyclization of 3b suggests that the strain calculated per repeat unit is not great enough to prevent the irreversible , kinetic formation of 2c . the larger strain calculated for the biphenyl dimer 1d suggests that strain may be playing a role in favoring the formation of cyclotrimer 1b and cyclotetramer 1c in the macrocyclization of 3a . energies are in kcal mol per repeat unit of cyclooligomer and are referenced to the cyclic trimer repeat unit energy for each set of cyclooligomers . the absorption and emission spectra of the novel macrocycles described above were recorded in a variety of solvents , since solvent polarity has been shown to affect the absorption and emission wavelength as well as the quantum yields ( ) of organic molecules . their poor solubility in some solvents such as pentane limited full comparisons of all of the macrocycles , but in benzene , thf and chcl3 , their max and max were determined . compound 1b exhibited an absorption maximum at 296 nm , with shoulders near 264 and 330 nm . cyclotetramer 2b exhibited two nearly equally intense absorptions at 282 and 301 nm , and these two maxima were observed in other solvents as well . cyclotrimer and cyclodimer 2b and 2c exhibited more similar spectra in pentane , with single dominant absorption maxima at 312 and 306 nm , respectively . it should be noted that accurate molar absorptivities were not obtained for 2b and 2c because of the formation of insoluble precipitate during the measurement of the spectra in pentane . all of the macrocycles were more soluble in benzene , and their absorption spectra are shown in figure 4 . the spectra of linear trimers 23a and 23b were also obtained in benzene . the max of biphenylene linear trimer 23a , 299 nm , does not change appreciably upon cyclization to 1a , which has a max of 298 nm . alternatively , the max of terphenylene linear trimer 23b , 295 nm , is shifted bathochromatically by 8 nm upon cyclization to cyclic trimer 2a ( max = 303 nm ) . this could arise from an increase in conjugation upon moving from the linear to the cyclic system . both 1b and 2b are red - shifted by 56 nm compared to their unsubstituted analogs , 1a and 2a . cyclic tetramer 1c is also red - shifted 6 nm compared to cyclic trimer 1b and has a much broader and less intense uv absorption band . all of the terphenylene macrocycles also showed broad absorptions , but cyclic dimer 2c and cyclic trimer 2b had very similar spectra . the absorption spectra of the macrocycles in thf show similar trends to those seen in the less polar benzene and pentane . all of the terphenylene macrocycles 2a c exhibit a bathochromatic shift in their absorption maxima in chcl3 compared to thf , while no such shift is observed for the biphenylene macrocycles 1a c . just as was observed in benzene , the max of linear trimer 23a , 298 nm , is very similar to that of cyclic trimer 1a , 297 nm , while terphenylene linear trimer 23b has a max of 293 , 10 nm blueshifted compared to the cyclic trimer 2a which has a max of 303 nm . alkyl substitution again redshifts the max of cyclic trimers , from 297 nm for 1a to 302 nm for 1b and from 303 for 2a to 315 nm for 2b , which was the largest max observed in the solvents examined . as observed in previous solvents , biphenyl cyclotetramer 1c exhibits two max at 286 and 308 nm , and cyclic dimer 2c exhibits a broad maximum with a relatively small molar absorptivity ( figure 5 ) . there is little to no solvent dependence on absorption for macrocycles 1a c and 2a c . cyclotrimers 1a , 1b and 2a or for cyclotetramer 1c or cyclodimer 2c . only substituted biphenylene cyclotrimer 1b and terphenylene cyclotrimer 2b exhibited a significant solvatochromic shifts , and while cyclotetramer 1c exhibits two nearly identical max in pentane , thf and chloroform , it has only a single broad max in benzene . the relationship between the structure of the macrocycle and its absorption spectrum should depend on the extent of conjugation around the macrocyclic ring . the three central p - phenylene aromatic rings of the terphenylene moiety in 2b are 47 out of the macrocyclic plane , as are the three p - phenylenes in 1b . it is possible that the conjugation around the macrocyclic ring is not interrupted to a significant extent , thus causing 2b to exhibit a more red - shifted max compared to 1b which has a smaller system . this trend exists in all solvents tested , although the extent of the shift increases as solvent polarity decreased . chcl3 and thf show a shift of 12 nm each , while nonpolar solvents benzene and pentane cause a larger red shift of 9 and 18 nm , respectively . several groups have examined the varying effects of ring strain on absorption properties of conjugated ethynylic systems . in the case of the biphenylene and terphenylene systems , 1e suggest that all biphenylene macrocycles synthesized , 1a , 1b , and 1c , are all essentially strain - free . aryl dihedral angle is also nearly identical in 1a and 1e , and both the planar cyclic trimer and puckered cyclic tetramer differ only in the disposition of the identical biphenylene ethynylene units ; these units are coplanar in the cyclic trimer and are not in the cyclic tetramer . in all solvents examined excepting benzene where the solvent may have partially obscured the spectrum , trimer 1b showed a single , broad max at around 300 nm , and tetramer 1c showed two max , one 15 nm shorter and another 5 nm longer wavelength ( table 3 ) . it should be noted that the two - dimensional -network is disrupted in the cyclotetramer by the ring pucker , which may explain the lower molar absorptivities of the cyclic tetramer in each solvent examined . cyclic dimer 2c and cyclic trimer 2b exhibit very similar spectra in benzene and thf , and slightly shifted spectra in pentane and chloroform . deformation from planarity by the ethynyl - substituted p - phenylene ring in the terphenyl cyclotrimer , 2b is amplified from 6 to near orthongonality , 87 , by removing a single repeat unit to form the terphenyl cyclodimer 2c . despite this interruption in the cyclic -nework , the uv this suggests that either -conjugation is not occurring in both systems and absorptions are a result of individual subunits or that delocalization about the macrocycles is not disrupted . in all cases , 2b exhibits greater molar absorptivity which could be the result of a larger number of absorbing moieties in the cyclic trimer versus the cyclic dimer . solutions of all of the macrocycles 1 and 2 as well as the linear trimers 23a and 23b are highly fluorescent ( figure 6 ) . the emission properties of these compounds were analyzed in benzene and their fluorescence quantum yields were determined using pyrene as a standard . all macrocycles displayed large stokes shifts which are indicative of large conjugated arylene ethynylene macrocycles ( see table 4 ) . the absorbance spectrum of biphenylene linear trimer 23a and biphenylene cyclic trimer 1a are very similar , but the single emission maximum exhibited by linear trimer 23a at 381 nm shifts to 418 nm upon ring closure to form 1a . no such large shift is observed upon ring closure of terphenylene linear trimer 23b with an emission maxima of 381 and 399 nm to terphenylene cyclic trimer 2a with emission maxima of 390 and 406 nm . comparison of the fluorescence spectra of 1a to 1b and 2a to 2b indicates that n - octyl substitution causes only small shifts in the emission maxima . cyclic tetramer 1c displays two em at 382 and 401 nm , at much shorter wavelengths than 1a and 1b ; the puckering of the macrocyclic ring not only affects the absorbance spectrum of 1c , but its emission spectrum . all terphenylene macrocycles emit two em at similar wavelengths to 1c , at 390 and 405 nm . cyclic dimer 2c shows similar emission properties to that of the cyclic trimer even though the p - phenylenes are orthogonal to the o - phenylene . the dihedral angles of the terphenyl moiety do not appear to perturb the optical properties of these systems . while 1a and 1b exhibit stokes shifts of 120 nm , all of the other macrocycles exhibit stokes shifts of 80 and 100 nm . the larger stokes shifts for the biphenylene cyclic trimers could indicate a more complete planarization of the macrocycle in 1a and 1b than the other macrocycles ; extending the conjugation throughout the biphenylene cyclotrimers requires only a single close h h and a single close c h contact , while the biphenylene cyclotetramer can not achieve planarity and greater conjugation without involving a significant amount of angle strain and the terphenylenes would require four close h one possible cause for the stokes shifts observed could be aggregation of the macrocycles in solution , which has been observed for similar structures . to test this hypothesis , we obtained the nmr spectra of solutions of 1b in benzene at concentrations higher than those used to obtain the fluorescence spectrum of 1b ( figure 7 ) . if 1b had been aggregating in the 0.066 mm solution used in the fluorescence experiment , it should be doing so at higher concentrations as well . since the nmr chemical shifts of all of the aromatic protons in 1b show no concentration dependence above that concentration , it seems likely that no aggregation would have taken place at the lower concentration . concentration - dependent nmr chemical shifts of 1b in c6d6 ; fluorescence spectra were observed at 0.066 mm . all macrocycles exhibit fairly low quantum yields ( ) in benzene compared to larger arylene ethynylene macrocycles ( table 4 ) . however , these systems do not contain long linear conjugated pathways which has been correlated to high quantum yields . quantum yields for the biphenyl macrocycles range from 0.02 for 1b to 0.04 for 1c while the quantum yields for terphenyl macrocycles are slightly more efficient ranging 0.04 for both 2a and 23b to 0.06 for 2b . in an effort to synthesize precursors of short carbon single - walled nanotubes , cyclic trimers containing biphenylene and p - terphenylene ethynylene units were constructed via linear , split - pool approaches to give unsubstituted macrocycles 1a and 2a . more soluble alkyl - substituted analogues 1b and 2b were also synthesized , utilizing statistical macrocyclizations of monomers made possible by the increased solubility . these statistical macrocyclizations also yielded a cyclotetramer 1c in the biphenylene system and a cyclodimer 2c in the p - terphenylene system ; these alternative cyclooligomers were separable from the cyclotrimers by exhaustive column chromatography . computational geometry optimizations suggest that the cyclic dimer is not energetically accessible in the statistical macrocyclization of 16b , while a lesser degree of angle strain in the terphenylene monomer 17b allows formation of cyclodimer 2c . all of the macrocycles obtained absorb around 300 nm , but the cyclotrimers 1a and 1b exhibit larger stokes shifts in their fluorescence emission spectra than the other macrocycles observed . further studies are currently being conducted to determine if multiple cycloadditions can be carried out on the alkynes present in these macrocycles to convert them into arylene cyclooligomers that can be oxidized to make carbon nanobelts . 4-iodoaniline ( 10.004 g , 45.67 mmol , 1.00 equiv ) was dissolved in 380 ml of acetonitrile , 160 ml of water , and 16.0 ml of concentrated hydrochloric acid and cooled to 0 c . a solution of 1.1 equiv of nano2 ( 3.321 g , 48.14 mmol , 1.05 equiv ) in 20 ml water was added slowly via syringe and the mixture stirred 45 min at 0 c . the mixture was transferred to a flask containing k2co3 ( 21.001 g , 151.9 mmol , 3.32 equiv ) and diethylamine ( 9.5 ml , 91.81 mmol , 2.00 equiv ) in 250 ml of h2o at 0 c . the reaction was allowed to slowly warm to room temperature and stirred for 2 h before being extracted with diethyl ether . the combined organic layers washed with brine , dried over magnesium sulfate , filtered , and concentrated in vacuo . the crude product was then purified by flash chromatography using 5% diethyl ether in hexanes to afford 13.43 g of the desired product as an orange oil ( 97% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.61 ( d , j = 8.8 hz , 2h ) , 7.16 ( d , j = 8.8 hz , 2h ) , 3.74 ( q , j = 7.3 , 4h ) , 1.25 ( br t , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 150.9 , 137.6 , 122.4 , 88.9 ; ir ( cm ) 2974 , 2933 , 2871 , 1475 , 1420 , 1391 , 1341 , 1238 , 1198 , 1108 , 1093 , 1001 , 828 ; ms ( ci - isobutane ) [ mh ] 304.6 m / z . a 25 ml round - bottomed flask was charged with 1-bromo-2-iodobenzene ( 2.697 g , 9.53 mmol , 1.00 equiv ) , cl2pd(pph3)2 ( 0.198 g , 0.282 mmol , 0.03 equiv ) , cui ( 0.051 g , 0.267 mmol , 0.03 equiv ) , triisopropylsilylethynylene ( 2.3 ml , 10.25 mmol , 1.07 equiv ) , and 20 ml of 1:1 thf / et3n . the solution was stirred at room temperature for 24 h before being concentrated in vacuo . the crude residue was dissolved in diethyl ether and washed with saturated nh4cl ( aq ) . the organic layers were dried over mgso4 , filtered , and concentrated in vacuo . purification of the crude material by flash column chromatography yielded 3.18 g of the product as a yellow oil ( 99% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.56 ( d , j = 8.2 hz , 1h ) , 7.50 ( d j = 7.74 hz , 1h ) , 7.22 ( t , j = 7.74 hz , 1h ) , 7.13 ( t , j = 7.95 hz , 1h ) , 1.13 ( br s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 134.1 , 132.6 , 129.6 , 127.0 , 126.0 , 125.9 , 105.0 , 96.4 , 18.9 , 11.6 ; ir ( cm ) 2943 , 2865 , 2161 , 1464 , 1220 , 1047 , 908 , 883 , 834 , 753 , 678 ; ms ( ci - isobutane ) [ mh ] 295.5 , 296.4 m / z method a : 1,1-diethyl-3-(4-iodophenyl)triazene ( 1.931 g , 6.370 mmol , 1.00 equiv ) was combined with bis(pinacolato)diboron ( 1.947 g , 7.667 mmol , 1.20 equiv ) , cl2pd(dppf ) ( 0.143 g , 0.195 mmol , 0.03 equiv ) , and koac that had been dried under vacuum ( 1.875 g , 19.11 mmol , 3.00 ) . deoxygenated dmso ( 52 ml ) was added , and the reaction was heated to 80 c and monitored by tlc ( 5% diethyl ether in hexanes ) . upon consumption of triazene starting material , the organic layers were combined , washed with satd nh4cl ( aq ) , dried over mgso4 , and concentrated in vacuo . crude material was purified by flash chromatography using 5% diethyl ether in hexanes as the eluent to afford 1.931 g ( 74% yield ) of the desired product as a white solid , mp 119120 c . the product can also be purified by filtration through a silica plug ( 10% ethyl acetate in hexanes ) followed by recrystallization from 2-propanol : h nmr ( 500 mhz , cdcl3 ) 7.77 ( d , j = 8.3 hz , 2h ) , 7.40 ( d , j = 8.3 hz , 2h ) , 3.77 ( q , j = 7.3 , 4h ) , 1.35 ( s , 12h ) , 1.27 ( br t , j = 6.8 hz , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 153.6 , 135.5 , 119.7 , 83.5 , 24.8 ; ir ( cm ) 2979 , 1602 , 1391 , 1351 , 1320 , 1139 , 1087 , 857 , 655 ; hrms ( esi ) m / z calcd for c16h26bn3o2h ( [ m + h ] ) 304.2196 , found 304.2194 . method b : 6 n hcl ( 27.10 ml , 162.51 mmol , 8.9 equiv ) was added dropwise to a solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline ( 4.035 g , 18.42 mmol , 1 equiv ) in 62.40 ml of diethyl ether , 48.00 ml of tetrahydrofuran , and 9.60 ml of acetonitrile chilled to 5 c in an ice salt bath . a solution of nano2 ( 4.3255 g , 62.69 mmol , 3.5 equiv ) in 21.60 ml of water and 9.69 ml of acetonitrile was added dropwise , and the reaction was stirred at 5 c for 30 min before being slowly transferred via cannula to a flask containing diethylamine ( 43.45 ml , 419.98 mmol , 23 equiv ) and k2co3 ( 12.640 g , 91.46 mmol , 5 equiv ) in 79.20 ml of water and 174.00 ml of acetonitrile at 0 c . the reaction was stirred for 45 min while being warmed to room temperature before being diluted with satd nacl and extracted with et2o . the organics were washed with h2o , dried over mgso4 , filtered , and concentrated in vacuo to give a brown crystal . crude material was purified by extraction with hexanes and concentration in vacuo to afford 4.7567 g ( 85% yield ) of orange crystals : h nmr ( 500 mhz , cdcl3 ) 7.77 ( d , j = 8.3 hz , 2h ) , 7.40 ( d , j = 8.3 hz , 2h ) , 3.77 ( q , j = 7.3 , 4h ) , 1.35 ( s , 12h ) , 1.27 ( br t , j = 6.8 hz , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 153.6 , 135.5 , 119.7 , 83.5 , 24.8 ; ir ( cm ) 2979 , 1602 , 1391 , 1351 , 1320 , 1139 , 1087 , 857 , 655 ; hrms ( esi ) m / z calcd for c16h26bn3o2h ( [ m + h ] ) 304.2196 , found 304.2194 . ( 2-bromophenylethynyl)triisopropylsilane ( 2.067 g , 6.13 mmol , 1.00 equiv ) was added to a flask fitted with a sealed reflux condenser and charged with 3,3-diethyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)triaz-1-ene ( 2.787 g , 9.19 mmol , 1.5 equiv ) , powdered potassium phosphate ( tribasic ) ( 6.51 g , 30.65 mmol , 5 equiv ) , and pdcl2(dppf ) ( 0.150 g , 0.184 mmol , 0.03 equiv ) . the flask was purged with nitrogen , and 50 ml of dexoygenated 1,2-dimethoxyethane was added via syringe . the reaction was stirred at reflux for 624 h , until tlc indicated completion ( some side products run at the same rf as the bromide , making exact assignment of completion difficult ) . the reaction mixture was then cooled , and the dme was removed in vacuo . the combined organic layers were washed with brine , dried over magnesium sulfate , filtered , and concentrated in vacuo . purification was effected by flash column chromatography over neutral alumina with a mobile phase of 5% diethyl ether in hexanes to obtain 2.66 g of the biphenyl product as an orange oil ( 95% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.59 ( d , j = 8.3 hz , 3h ) , 7.43 ( d , j = 8.3 hz , 2h ) , 7.38 ( d , j = 7.8 hz , 1h ) , 7.33 ( t , j = 7.3 hz , 1h ) , 7.23 ( t , j = 7.8 hz , 1h ) , 3.76 ( q , j = 7.3 hz , 4h ) , 1.27 ( t , j = 7.3 hz , 6h ) , 1.03 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 150.48 , 144.0 , 137.1 , 133.9 , 129.7 , 129.3 , 128.4 , 126.5 , 121.7 , 119.9 , 106.6 , 93.9 , 18.6 , 11.3 ; ir ( cm ) 2940 , 2864 , 2151 , 1464 , 1397 , 1330 , 1235 , 1096 , 883 , 835 , 761 , 677 ; hrms ( esi ) m / z calcd for c27h39n3sih ( [ m + h ] ) 434.2992 , found 434.2991 . compound 3a ( 1.855 g , 4.277 mmol , 1.00 equiv ) was dissolved in 10 ml of methyl iodide in a sealable reaction flask , degassed , backfilled with nitrogen , sealed , and heated to 125 c for 44 h. after cooling , the methyl iodide was removed by evaporation and the product purified by flash chromatography over silica with 5% diethyl ether in hexanes to isolate 1.950 g of a yellow oil ( 99% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.70 ( d , j = 8.3 hz , 2h ) , 7.59 ( d , j = 7.8 hz , 1h ) , 7.277.37 ( m , 5h ) , 1.01 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 143.1 , 140.1 , 137.0 , 133.7 , 131.2 , 129.0 , 128.5 , 127.2 , 121.9 , 105.9 , 94.6 , 93.1 , 18.5 , 11.3 ; ir ( cm ) 2940 , 2863 , 2152 , 1469 , 1386 , 1000 , 883 , 820 , 758 , 677 ; hrms ( ei ) m / z calcd for c23h29isi ( [ m ] ) 460.1083 , found 460.1091 . tbaf ( 1 m ) in thf ( 2.0 ml , 2.00 mmol , 4.35 equiv ) was added to a solution of 9 ( 0.212 g , 0.460 mmol , 1.00 equiv ) in 3 ml of thf . the reaction was stirred at room temperature and monitored by tlc . upon disappearance of starting material , the reaction was concentrated to one - third of its original volume , diluted with 25 ml of h2o , and extracted with diethyl ether ( 3 50 ml ) . the crude material was purified via flash chromatography using 5% diethyl ether in hexane to afford 0.134 g ( 96% yield ) of a reddish oil : h nmr ( 500 mhz , cdcl3 ) 7.78 ( d , j = 7.0 hz , 2h ) , 7.64 ( d , j = 7.5 hz , 1h ) , 7.42 ( t , j = 6.5 hz , 1h ) , 7.377.34 ( m , 4h ) , 3.08 ( s , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 143.1 , 139.7 , 137.1 , 133.9 , 131.1 , 129.2 , 129.0 , 127.3 , 120.3 , 93.6 , 80.6 ; ir ( neat , cm ) 3274 , 3061 , 1583,1472 ; hrms ( esi ) m / z calcd for c14h10i ( [ m + h ] ) 16a ( 0.060 g , 0.197 mmol , 1.00 equiv ) , cl2pd(pph3)2 ( 0.007 g , 0.010 mmol , 0.05 equiv ) , and cui ( 0.004 g , 0.020 mmol , 0.11 equiv ) were combined and dissolved in 10.6 ml of thf and 0.2 ml of et3n . the reaction was stirred for 10 days at room temperature before being diluted with h2o and extracted with et2o ( 3 20 ml ) . the organics were washed over brine , dried over mgso4 , filtered , and concentrated in vacuo . the crude material was run through a flash column to afford a white solid mixture of cyclic and linear oligomers that resisted further purification . compound 3a ( 0.655 g , 1.510 mmol , 1 equiv ) was dissolved in 5 ml of methanol . a solution of 1 m tbaf in thf ( 7.6 ml , 7.50 mmol , 4.97 equiv ) was added ; the reaction was then stirred at room temperature and monitored by tlc . upon disappearance of starting material , the reaction was concentrated to one - third its original volume , diluted with 50 ml of h2o , and extracted with diethyl ether ( 3 50 ml ) . the organic layers were combined , dried over mgso4 , and concentrated in vacuo . the crude material was purified via flash chromatography using 5% diethyl ether in hexane to afford 0.376 g ( 90% yield ) of a yellow oil : h nmr ( 500 mhz , cdcl3 ) 7.62 ( d , j = 8.0 hz , 1h ) , 7.59 ( d , j = 8.2 hz , 2h ) , 7.50 ( d , j = 8.2 hz , 2h ) , 7.417.40 hz ( m , 2h ) , 7.307.27 ( m , 1h ) , 3.80 ( q , j = 7.0 hz , 4h ) , 3.06 ( s , 1h ) , 1.30 ( t , j = 7.0 hz , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 150.6 , 144.4 , 136.9 , 133.8 , 129.7 , 129.5 , 128.9 , 126.6 , 120.3 , 119.9 , 83.3 , 80.0 . ir ( cm ) 3284 , 1330 , 1229 , 837 ; hrms ( photospray ionization ) m / z calcd for c18h20n3 ( [ m + h ] ) 278.1657 , found 278.1664 . a 50 ml round - bottomed flask was charged with 9 ( 0.223 g , 0.484 mmol , 1.00 equiv ) , 19a ( 0.180 g , 0.649 mmol , 1.34 equiv ) , and cl2pd(pph3)2 ( 0.020 g , 0.0285 mmol , 0.06 equiv ) and flushed with n2 . to this 20 ml of deoxygenated thf / et3n ( 1:1 v / v ) was added , and the reaction flask was sparged with n2 for 5 min . cui was added and the reaction stirred at 40 c for 18 h. upon completion , the reaction was diluted with 25 ml of h2o and extracted with diethyl ether ( 3 25 ml ) . the organics were combined , washed with satd nh4cl , dried over mgso4 , and concentrated in vacuo . the crude material was purified via flash chromatography using 5% diethyl ether in hexanes to give 0.226 g ( 77% ) of the desired product as a yellow oil : h nmr ( 500 mhz , cdcl3 ) 7.72 ( d , j = 8.5 hz , 2h ) , 7.70 ( d , j = 7.9 hz , 1h ) , 7.62 ( d , j = 7.8 hz , 1h ) , 7.577.54 ( m , 4h ) , 7.48 ( d , j = 8.0 hz , 1h ) , 7.43 ( d , j = 7.9 hz , 2h ) , 7.40 ( d , j = 8.3 hz , 1h ) , 7.387.27 ( m , 4h ) , 3.82 ( q , 7.2 hz , 4h ) , 1.31 ( t , j = 7.1 hz , 6h ) , 1.05 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 150.6 , 143.7 , 143.5 , 140.2 , 137.2 , 133.8 , 133.0 130.9 , 129.8 , 129.5 , 129.2 , 129.1 , 128.5 , 127.1 , 126.7 , 122.5 , 121.8 , 121.4 , 119.9 , 106.0 , 94.4 , 92.3 , 89.8 , 18.55 , 11.3 ; ir ( cm ) 2942 , 2860 , 2356 , 1460 , 1334 , 1229 , 830 ; hrms ( maldi ) m / z calcd for c41h48n3si ( [ m + h ] ) 610.3618 , found 610.3616 . compound 20a ( 0.206 g , 0.338 mmol , 1.00 equiv ) and 4 ml of mei were placed in a sealed tube flask under an atmosphere of n2 . the reaction was heated to 125 c for 18 h before being allowed to cool to room temperature . the residue was dissolved in 10 ml of ch2cl2 , washed with h2o , dried over mgso4 , and concentrated in vacuo . the crude product was purified via flash chromatography using 5% etoac in hexanes as the eluent and afforded 0.202 g ( 94% yield ) of the desired product as a yellow oil : h nmr ( 500 mhz , cdcl3 ) 7.81 ( d , j = 8.5 hz , 2h ) , 7.69 ( d , j = 7.7 hz , 1h ) , 7.62 ( d , j = 7.5 hz , 1h ) , 7.59 ( d , j = 8.0 hz , 2h ) , 7.46 ( d , j = 8.0 hz , 2h ) , 7.417.38 ( m , 2h ) , 7.377.35 ( m , 5h ) , 7.327.29 ( m , 1h ) 1.05 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 143.3 , 142.5 , 140.5 , 140.0 , 137.0 , 133.8 , 133.0 , 131.3 , 130.9 , 129.3 , 129.2 , 128.6 , 128.5 , 127.4 , 127.2 , 122.1 , 121.8 , 121.5 , 106.0 , 94.5 , 93.4 , 92.8 , 89.1 , 18.6 , 11.3 ; ir ( cm ) 2940 , 2865 , 2148 , 1467 , 1000 , 756 ; hrms ( esi ) m / z calcd for c37h38isi ( [ m + h ] ) 637.1787 , found 637.1777 . compound 21a ( 0.091 g , 0.142 mmol , 1.00 equiv ) , 3,3-diethyl-1-(2-ethynylbiphenyl-4-yl)triaz-1-ene ( 0.035 g , 0.153 mmol , 1.08 equiv ) , cl2pd(pph3)2 ( 0.005 g , 0.007 mmol , 0.05 equiv ) , and cui ( 0.002 g , 0.010 mmol , 0.07 equiv ) were dissolved in 5 ml of deoxygenated thf and 5 ml of deoxygenated triethylamine . the solution was heated to 40 c overnight before being diluted with satd nh4cl and extracted with etoac . the organics were washed with satd nh4cl , dried over mgso4 , filtered , and concentrated in vacuo . the crude material was purified by flash chromatography using 10% etoac in hexanes to afford 0.075 g of the desired product as an off - white waxy solid ( 67% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.727.66 ( m , 5h ) , 7.62 ( d , j = 7.5 hz , 1h ) , 7.587.52 ( m , 6h ) , 7.507.28 ( m , 8h ) , 3.77 ( q , j = 7.2 hz , 4h ) , 1.28 ( t , j = 7.2 hz ) , 1.02 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 143.7 , 143.3 , 143.1 , 140.4 , 140.2 , 137.2 , 133.8 , 133.0 , 132.9 , 131.0 , 130.9 , 129.85 , 129.4 , 129.3 , 129.27 , 128.18 , 128.5 , 127.2 , 127.0 , 126.6 , 122.7 , 122.2 , 121.8 , 121.5 , 121.4 , 119.0 , 106.0 , 94.4 , 92.6 , 92.2 , 90.2 , 89.3 , 18.5 , 11.2 ; ir ( neat , cm ) : 2932 , 2860 , 2154 , 1473 , 1096 , 836 , 744 ; hrms ( esi ) m / z calcd for c55h56n3si ( [ m + h ] ) 786.4244 , found 786.4257 . compound 22a ( 0.039 g , 0.0478 mmol , 1.0 equiv ) was sealed in a flask with 1 ml of mei and heated to 120 c for 48 h. excess mei was allowed to evaporate after the reaction mixture cooled . the crude residue was purified by flash chromatography using 10% etoac in hexanes as eluent to afford 0.039 g of the desired product as a light yellow waxy solid ( 98% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.81 ( d , j = 8.0 hz , 2h ) , 7.70 7.67 ( m , 4h ) , 7.61 ( d , j = 8.0 hz , 1h ) , 7.57 ( d , j = 8.5 hz , 2h ) , 7.477.35 ( m , 14h ) , 7.327.27 ( m , 1h ) 1.05 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 143.3 , 142.9 , 140.6 , 140.5 , 140.0 , 137.0 , 133.8 , 133.1 , 133.0 , 131.2 , 130.9 , 129.4 , 129.4 , 129.3 , 129.2 , 128.7 , 128.5 , 128.5 , 127.4 , 127.3 , 127.1 , 122.3 , 122.2 , 121.9 , 121.6 , 121.4 , 106.04 , 94.4 , 93.4 , 92.7 , 92.6 , 89.5 , 89.3 , 18.5 , 11.2 ; ir ( neat , cm ) 2802 , 2847 , 2356 , 1726 , 1457 , 1273 , 761 ; hrms ( esi ) m / z calcd for c51h46isi ( [ m + h ] ) 813.2413 , found 813.2406 . compound 23a ( 0.039 g , 0.0463 mmol , 1.00 equiv ) was dissolved in 0.25 ml of thf before 0.100 ml of 1 m tbaf in thf ( 0.100 ml , 0.100 mmol , 2.16 equiv ) was added . after 5 min , the reaction showed no signs of starting material by tlc and was diluted with et2o . the organics were washed with satd nh4cl ( aq ) , dried over mgso4 , filtered , and rotovapped . the crude material was purified by flash chromatography using 5% etoac in hexanes to afford the desired compound which was used in the following reaction without further characterization . compound 24a ( assuming 100% conversion in previous reaction : 0.032 g , 0.0463 mmol , 1.00 equiv ) was dissolved in 1 ml of et3n and charged in a gastight syringe in a syringe pump . pd(dba)2 ( 0.039 g 0.068 mmol , 1.42 equiv ) , cui ( 0.015 g , 0.079 mmol , 1.71 equiv ) , and pph3 ( 0.076 g , 0.290 mmol , 6.26 equiv ) were dissolved in 11.6 ml of et3n . the iodoalkyne trimer was added at a rate of 0.1 ml per hour , and the reaction was allowed to stir for an additional 12 h after the addition was complete . the reaction was diluted with sat nh4cl ( aq ) and extracted with etoac . the organic phases were dried over mgso4 , filtered , and concentrated in vacuo . the crude material was purified using 15% ch2cl2 in hexanes to afford 11 mg ( 45% yield ) of the desired macrocycle as a white solid : mp 282291 c ; h nmr ( 500 mhz , cdcl3 ) 7.75 ( d , j = 8.4 hz , 6h ) , 7.67 ( d , j = 7.4 hz , 3h ) , 7.507.48 ( m , 9h ) , 7.45 ( t , j = 7.8 hz , 3h ) , 7.36 ( t , j = 7.4 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 143.7 , 140.9 , 133.4 , 131.5 , 130.0 , 129.9 , 129.5 , 128.1 , 123.1 , 122.2 , 93.4 , 90.3 ; hrms ( maldi ) m / z calcd for c42h24 ( [ m ] ) 528.1872 , found 528.1864 . compound 9 ( 1.923 g , 4.18 mmol , 1.00 equiv ) was added to a flask with 6 ( 1.90 g , 6.27 mmol , 1.5 equiv ) , boronate , k3po4 ( 4.52 g , 21.3 mmol , 5.1 equiv ) , and pdcl2(dppf ) ( 0.10 g , 0.125 mmol , 0.03 equiv ) . the reaction was refluxed for 20 h and cooled to room temperature , the solvent was removed in vacuo , and the organic products were extracted from water with diethyl ether . the combined organic layers were washed with brine , dried over magnesium sulfate , filtered , and concentrated in vacuo . purification over neutral alumina with 25% methylene chloride in hexanes afforded 1.38 g of the product as a pale yellow solid : mp 110111 c ( 65% yield);. h nmr ( 500 mhz , cdcl3 ) 7.587.65 ( m , 7h ) , 7.51 ( d , j = 8.3 hz , 2h ) , 7.364.42 ( m , 2h ) , 7.29 ( t , j = 7.3 hz , 1h ) , 3.79 ( q , j = 7.3 hz , 4h ) , 1.29 ( t , j = 7.3 hz , 6h ) , 1.01 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 150.6 , 144.0 , 140.0 , 139.1 , 137.7 , 133.7 , 129.7 , 129.2 , 128.4 , 127.5 , 126.8 , 126.4 , 122.0 , 120.8 , 106.4 , 94.1 , 18.6 , 11.3 ; ir ( cm ) 2940 , 2864 , 2361 , 2151 , 1463 , 1329 , 1233 , 1092 , 995 , 882 , 823 , 764 , 674 , 639 ; hrms ( esi ) m / z calcd for c33h43n3sih ( [ m + h ] ) 510.3305 , found 510.3309 . compound 4a ( 0.096 g , 0.188 mmol , 1 equiv ) was dissolved in 2.5 ml of methyl iodide in a sealable reaction flask , degassed , backfilled with nitrogen , sealed , and heated to 125 c for 40 h. after cooling , the methyl iodide was removed by evaporation and the product purified by flash chromatography over neutral alumina with 50% methylene chloride in hexanes to afford 0.092 mg of the product as a yellow solid : mp 8183 c ( 91% yield ) ; h nmr ( 500 mhz , cdcl3 ) 7.77 ( d , j = 8.3 hz , 2h ) , 7.64 ( d , j = 8.3 hz , 2h ) , 7.61 ( d , j = 7.8 hz , 1h ) , 7.54 ( d , j = 8.3 hz , 2h ) , 7.277.38 ( m , 5h ) , 1.00 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 143.7 , 140.5 , 140.1 , 138.9 , 137.9 , 137.8 , 129.9 , 128.91 , 128.87 , 128.5 , 127.0 , 126.4 , 122.0 , 106.2 , 95.2 , 93.0 , 18.6 , 11.3 . ir ( cm ) 2939 , 2862 , 2152 , 1473 , 1384 , 1064 , 1000 , 882 , 837 , 812 , 578 , 662 ; hrms ( photospray ionization ) m / z calcd for c29h33sii ( [ m ] ) 536.1396 , found 536.1383 . compound 18b ( 0.229 g , 0.427 mmol , 1.00 equiv ) was stirred with tbaf ( 1 m in thf ) ( 5.55 ml , 5.55 mmol , 13 equiv ) with 4 ml of thf and 1 ml of methanol for 3 days at room temperature . the solvent was removed in vacuo , and the organic products were extracted from water with methylene chloride . the combined organic layers were dried over magnesium sulfate , filtered , and concentrated to produce 0.127 g of an orange solid ( 78% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.78 ( d , j = 8.6 hz , 2h ) , 7.68 ( d , j = 8.1 hz , 2h ) , 7.617.65 ( m , 3h ) , 7.387.43 ( m , 4h ) , 7.307.34 ( m , 1h ) , 3.08 ( s , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 143.7 , 140.2 , 139.6 , 139.1 , 137.8 , 134.0 , 129.7 , 129.5 , 129.0 , 128.9 , 127.1 , 126.4 , 120.3 , 93.1 , 83.0 , 80.4 ; hrms ( photospray ionization ) m / z calcd for c20h14i ( [ m + h ] ) 381.0140 , found 381.0151 compound 4a ( 0.240 g , 0.471 mmol , 1.00 equiv ) was stirred with tbaf ( 1 m in thf ) ( 1.41 ml , 1.41 mmol , 3.0 equiv ) with 2.0 ml of thf and 6 drops of methanol for 40 h at room temperature . the organic products were extracted from water with methylene chloride , dried over magnesium sulfate , filtered , and concentrated in vacuo . purification over neutral alumina with 50% methylene chloride in hexanes yielded 0.152 g of 1,1-diethyl-3-(4-(4-(2-ethynyl)phenyl)phenyl)phenyl)triaz-1-ene as an orange solid : mp 117119 c ( 91% yield ) ; h nmr ( 500 mhz , cdcl3 ) 7.567.68 ( m , 7h ) , 7.51 ( d , j = 8.3 hz , 2h ) , 7.40 ( m , 2h ) , 7.29 ( m , 1h ) , 3.77 ( q , j = 7.3 hz , 4h ) , 3.07 ( s , 1h ) , 1.26 ( br t , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 150.6 , 144.0 , 140.1 , 138.7 , 137.2 , 133.9 , 129.6 , 129.5 , 129.0 , 127.4 , 126.9 , 126.8 , 126.3 , 120.8 , 120.3 , 83.2 , 80.3 ; ir ( cm ) 3280 , 1471 , 1427 , 1324 , 1233 , 1098 , 1003 , 828 , 761 , 659 , 634 ; hrms ( apci ) m / z calcd for c24h24n3 ( [ m ] ) 354.1970 , found 354.1965 . compounds 18b ( 0.100 g , 0.186 mmol , 1.00 equiv ) and 19b ( 0.069 g , 0.196 mmol , 1.05 equiv ) were dissolved with cui ( 0.001 g , 0.0056 mmol , 0.03 equiv ) and pdcl2(ph3p)2 ( 0.006 g , 0.009 mmol , 0.05 equiv ) in 0.5 ml of deoxygenated triethylamine and 2.0 ml of deoxygenated thf . the reaction was stirred at room temperature , periodically reloading solvent and palladium catalyst as needed over the course of several days . when tlc failed to show any further conversion , the solvents were removed in vacuo , and the products were extracted from saturated ammonium chloride solution with methylene chloride . the combined organic layers were dried over sodium sulfate , filtered , and concentrated . flash chromatography over neutral alumina provided 0.108 g ( 76% yield ) of 20b as a yellow oil as well as 13% recovery of unreacted iodide 18b : h nmr ( 500 mhz , cdcl3 ) 7.77 ( d , j = 8.3 hz , 2h ) , 7.73 ( d , j = 8.3 hz , 2h ) , 7.517.69 ( m , 12h ) , 7.48 ( d , j = 7.8 hz , 1h ) , 7.337.45 ( m , 6h ) , 7.28 ( t , j = 7.3 hz , 1h ) , 3.78 ( q , j = 6.8 hz , 4h ) , 1.27 ( br t , 6h ) , 1.00 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 150.7 , 143.7 , 143.5 , 140.1 , 139.9 , 139.2 , 139.1 , 137.3 , 133.7 , 133.0 , 131.8 , 129.8 , 129.7 , 129.4 , 129.2 , 128.8 , 128.65 , 128.55 , 128.4 , 127.5 , 127.0 , 126.9 , 126.5 , 126.3 , 122.3 , 120.8 , 106.2 , 94.2 , 92.3 , 90.2 , 18.5 , 11.3 ; ir ( cm ) 3280 , 2978 , 2932 , 2360 , 1471 , 1427 , 1390 , 1322 , 1233 , 1098 , 828 , 761 , 698 , 659 ; hrms ( ei ) m / z calcd for c53h55n3si ( [ m ] ) 761.4165 , found 761.4160 . compound 20b ( 0.096 g , 0.126 mmol , 1.00 equiv ) was dissolved in 2.5 ml of methyl iodide in a sealable reaction flask , degassed , backfilled with nitrogen , sealed , and heated to 125 c for 40 h. after cooling , the methyl iodide was removed by evaporation and the product purified by flash chromatography over silica with 25% methylene chloride in hexanes to afford 0.065 mg of a white , crystalline solid : mp 109112 c ( 86% yield ) ; h nmr ( 500 mhz , cdcl3 ) 7.78 ( m , 4h ) , 7.517.69 ( m , 10h ) , 7.26746 ( m , 10h ) , 1.01 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 143.7 , 143.1 , 140.7 , 140.3 , 140.0 , 139.1 , 139.0 , 137.9 , 137.8 , 133.7 , 133.0 , 131.8 , 129.9 , 129.8 , 129.4 , 129.2 , 128.93 , 128.86 , 128.5 , 127.2 , 127.0 , 126.9 , 126.5 , 126.3 , 122.2 , 122.0 , 121.6 , 106.3 , 94.2 , 93.1 , 92.4 , 90.1 , 18.6 , 11.3 ; ir ( cm ) 2939 , 2861 , 2151 , 1473 , 1384 , 1064 , 1000 , 882 , 813 , 758 , 664 ; hrms m / z calcd for c37h38isi ( [ mh ] ) 637.1782 , found 637.1777 . compound 21b ( 0.086 g , 0.109 mmol , 1.00 equiv ) was dissolved in 0.5 ml of deoxygenated thf and 0.2 ml of deoxygenated triethylamine with 19b ( 0.042 g , 0.120 mmol , 1.10 equiv ) , pdcl2(ph3p)2 ( 0.006 g , 0.0087 mmol , 0.08 equiv ) , and cui ( 0.0006 g , 0.0033 mmol , 0.03 equiv ) . the reaction was stirred at room temperature for 24 h , the solvent was removed in vacuo , and the organic products were extracted from saturated ammonium chloride solution with methylene chloride . the combined organic layers were dried over sodium sulfate , filtered , and concentrated . four rounds of purification by flash column chromatography over alumina with 40% methylene chloride in hexanes yielded 0.067 g of trimer 22b as a white , crystalline solid : mp 167171 c ( 61% yield ) ; h nmr ( 500 mhz , cdcl3 ) 7.227.79 ( m , 36h ) , 3.75 ( q , j = 7.3 hz , 4h ) , 1.25 ( br t , 6h ) , 1.00 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 150.6 , 143.6 , 143.4 , 143.1 , 140.8 , 140.6 , 140.3 , 140.2 , 140.1 , 139.8 , 139.3 , 139.1 , 139.0 , 137.3 , 133.6 , 133.1 , 133.0 , 132.9 , 131.8 , 131.7 , 129.8 , 129.7 , 129.4 , 129.1 , 128.7 , 128.5 , 128.4 , 127.5 , 127.4 , 127.1 , 127.0 , 126.8 , 126.4 , 126.2 , 122.4 , 122.3 , 122.2 , 121.9 , 121.5 , 120.8 , 106.2 , 94.1 , 92.3 , 92.3 , 90.3 , 90.2 , 90.0 , 18.5 , 11.2 ; ir ( cm ) 2929 , 2862 , 1474 , 1440 , 1343 , 1233 , 1091 , 1003 , 882 , 821 , 760 , 665 , 553 ; hrms ( esi ) m / z calcd for c69h57n2si ( [ m ( c4h10n ) ] ) 941.4291 , found 941.4255 . compound 22b ( 0.067 g , 0.066 mmol , 1.00 equiv ) was dissolved in 2.5 ml of methyl iodide in a sealable reaction flask , degassed , backfilled with nitrogen , sealed , and heated to 125 c for 40 h. after cooling , the methyl iodide was removed by evaporation . the residue was filtered through a silica plug with methylene chloride to provide 0.068 g of the product iodide ( 99% yield ) as a colorless waxy solid , which was reacted without further purification since poor solubility precluded further chromatography : h nmr ( 500 mhz , cdcl3 ) 7.78 ( d , j = 8.8 hz , 6h ) , 7.527.70 ( m , 15h ) , 7.357.48 ( m , 14h ) , 7.29 ( m , 1h ) , 0.99 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 143.7 , 143.1 , 140.7 , 140.5 , 140.3 , 139.9 , 139.2 , 139.1 , 139.0 , 137.9 , 133.7 , 133.0 , 131.8 , 131.7 , 129.9 , 129.9 , 129.8 , 129.4 , 128.9 , 128.6 , 127.2 , 126.9 , 126.8 , 126.4 , 126.4 , 126.3 , 122.3 , 122.2 , 122.0 , 121.5 , 106.2 , 94.1 , 93.1 , 92.3 , 90.1 , 18.6 , 11.3 ; ir ( cm ) 2921 , 2860 , 2360 , 2342 , 2155 , 1473 , 1441 , 1384 , 1000 , 822 , 814 , 761 , 667 . compound 23b ( 0.070 g , 0.067 mmol , 1.00 equiv ) was stirred with 4.0 ml of thf , 6 drops of methanol , and tbaf ( 1 m in thf ) ( 0.20 ml , 0.20 mmol , 3.0 equiv ) for 48 h , during which time a precipitate was observed in the reaction . extraction from water with methylene chloride , drying of the organic layers with magnesium sulfate , filtration , and concentration in vacuo yielded 0.53 g of crude trimer ( 90% yield ) , which was used in the next step without further purification or characterization . the crude linear iodoarylethyne trimer was dissolved in 10 ml of deoxygenated thf and placed in a syringe pump . 1.5 equiv of pd(dba)2 ( 0.046 g , 0.080 mmol , 1.5 equiv ) , 1.3 equiv of cui ( 0.013 g , 0.068 mmol , 1.3 equiv ) , and 5.9 equiv of triphenylphosphine ( 0.082 g , 0.313 mmol , 5.9 equiv ) were dissolved in 5.0 ml of deoxygenated triethylamine and 10 ml of deoxygenated thf . the catalyst solution was heated to reflux under nitrogen , and the trimer solution was added at 0.6 ml / h , followed by continued refluxing overnight . the solvents were then removed in vacuo and the organic products extracted from saturated ammonium chloride solution with methylene chloride , washed with water and brine , and concentrated . the solids were then filtered with whatman quantitative filter paper ( 1 m pore size ) and washed with methylene chloride ; centrifugation in methylene chloride yielded 0.008 g ( 21% yield ) of a colorless solid floating on top of the supernatant . no melting transition was observed below 300 c : h nmr ( 500 mhz , cdcl3 ) 7.81 ( d , j = 8.3 hz , 2h ) , 7.70 ( d , j = 7.8 hz , 2h ) , 7.69 ( d , j = 6.3 hz , 1h)7.64 ( d , j = 8.3 hz , 2h ) , 7.52 ( d , j = 7.8 hz , 1h ) , 7.49 ( d , j = 7.8 hz , 2h ) , 7.44 ( t , j = 6.8 hz , 1h ) , 7.37 ( t , j = 7.3 hz , 1h ) ; h nmr ( 500 mhz , c6d4cl2 ) 7.82 ( d , j = 8.0 hz , 6h ) , 7.70 ( d , j = 7.5 hz , 3h ) , 7.67 ( d , j = 8.0 hz , 6h ) , 7.58 ( d , j = 8.5 hz , 6h ) , 7.51 ( d , j = 8.5 hz , 6h ) , 7.47 ( d , j = 7.5 hz , 3h ) , 7.36 ( t , j = 7.3 hz , 3h ) , 7.29 ( t , j = 7.3 hz , 3h ) ; cosy ( 500 mhz , o - c6d4cl2 ) 7.82 7.67 , 7.70 7.29 , 7.58 7.51 , 7.47 7.36 , 7.36 7.29 ; hmqc ( 500 mhz , o - c6d4cl2 ) 7.82 130.3 , 7.70 133.0 , 7.68 126.6 , 7.59 127.1 , 7.52 132.0 , 7.47 129.5 , 7.36 128.9 , 7.29 127.5 ; hmbc ( o-500 mhz , c6d4cl2 , selected peaks ) 7.82 143.3 , 7.82 139.5 , 7.70 143.3 , 7.70 90.7 , 7.68 140.4 , 7.68 139.8 , 7.59 139.5 , 7.59 122.6 , 7.52 140.4 , 7.52 92.7 , 7.47 139.8 , 7.47 121.6 , 7.36 143.3 , 7.29 121.6 ; ir ( cm ) 3056 , 3031 , 2217 , 1922 , 1498 , 1473 , 1442 , 1395 , 1104 , 1004 , 819 , 758 , 733 , 703 ; hrms ( maldi ) m / z calcd for c60h36 ( [ m ] ) 756.2817 , found 756.2822 . compound 17a ( 0.053 g , 0.139 mmol , 1.00 equiv ) was dissolved in 0.8 ml of thf and 0.1 ml of triethylamine ( both solvents distilled and deoxygenated ) with cui ( 0.3 mg , 0.0016 mmol , 0.01 equiv ) and pdcl2(ph3p)2 ( 0.005 g , 0.007 mmol , 0.05 equiv ) and stirred at room temperature . the reaction was monitored by tlc and periodically reloaded with catalysts and solvent until tlc failed to indicate a significant change over a 24 h period . the solid precipitate formed was filtered from solution and washed with methylene chloride to yield a solid : h nmr ( 500 mhz , c6d4cl2 ) 7.80 ( d ) , 7.267.7.2 ( m ) , 3.07 ( s ) . compound 17a ( 0.053 g , 0.139 mmol , 1.00 equiv ) was dissolved in 7.5 ml of thf and 0.1 ml of triethylamine ( both solvents were distilled and deoxygenated ) with cui ( 0.3 mg , 0.0016 mmol , 0.01 equiv ) and pd(ph3p)4 ( 0.005 g , 0.007 mmol , 0.05 equiv ) and stirred at room temperature . the reaction was monitored by tlc and periodically reloaded with catalysts and solvent until tlc failed to indicate a significant change over a 24 h period . the solid precipitate formed was filtered from solution and washed with methylene chloride to yield 20% yield of macrocycle 2a with slight contamination evidenced by : h nmr ( 500 mhz , cdcl3 ) 7.78 ( d , j = 7.8 hz ) , 7.62 ( d , j = 7.8 hz ) . [ bnn(ch3)3]icl2 ( 3.535 g , 10.15 mmol , 1.05 equiv ) and caco3 ( 3.141 g , 31.38 mmol , 3.21 equiv ) were added to a stirred solution of 4-n - octylaniline ( 1.995 g , 9.72 mmol , 1 equiv ) in 80 ml of dry ch2cl2 and 35 ml of anhydrous meoh . the reaction stirred at room temperature for 2 h prior to being vacuum filtered through a pad of celite . the filtrate was washed with satd na2so4 ( 40 ml ) followed by satd nh4cl ( 40 ml ) . the organics were dried over mgso4 , filtered , and concentrated in vacuo to give a brown oil that was purified by chromatography ( 10% ethyl acetate in hexane on silica ) to give 3.08 g of a brown oil ( 96% yield ) . this product was used in subsequent reactions without further purification : h nmr ( 500 mhz , cdcl3 ) 7.43 ( d , j = 1.9 hz , 1h ) , 6.93 ( dd , j = 1.9 , 8.3 hz , 1h ) , 6.65 ( d , j = 8.3 hz , 1h ) , 3.93 ( br s , 2h ) , 2.44 ( t , j = 7.3 hz , 2h ) , 1.52 ( m , 2h ) , 1.231.31 ( m , 10h ) , 0.88 ( t , j = 7.3 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 144.4 , 138.4 , 134.8 , 129.4 , 114.6 , 84.4 , 34.5 , 31.8 , 31.6 , 29.5 , 29.4 , 29.2 , 22.6 , 14.0 ; ir ( cm ) 3407 , 3317 , 2954 , 2916 , 2850 , 1617 , 1496 , 1467 , 1405 , 1306 , 1152 , 1028 , 819 , 665 ; hrms ( maldi ) m / z calcd for c14h22ni ( [ m + h ] ) 332.0870 , found 332.0870 . hcl ( 1.1 ml , 13.2 mmol , 7.8 equiv ) was added dropwise to a solution of 11 ( 0.558 g , 1.68 mmol , 1 equiv ) in 10.0 ml of tetrahydrofuran and 0.6 ml of acetonitrile chilled to 5 c in an ice salt bath . a solution of nano2 ( 0.415 g , 6.02 mmol , 3.58 equiv ) in 1.8 ml of water and 0.8 ml acetonitrile was added dropwise , and the reaction was stirred at 5 c for 30 min before being slowly transferred via cannula to a flask containing diethylamine ( 3.5 ml , 42.5 mmol , 25 equiv ) and k2co3 ( 2.32 g , 16.8 mmol , 10 equiv ) in 7.0 ml of water at 0 c . the reaction was stirred for 45 min while warming to room temperature before being diluted with satd nacl and extracted with et2o . the organics were washed with h2o , dried over mgso4 , filtered , and concentrated in vacuo . crude material was purified by flash chromatography using hexanes to afford 0.686 g of an orange oil ( 98% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.66 ( d , j = 1.9 hz , 1h ) , 7.25 ( d , j = 8.6 hz , 1h ) , 7.08 ( dd , j = 1.9 , 8.2 hz , 1h ) , 3.78 ( q , j = 7.2 hz , 4h ) , 2.52 ( t , j = 7.5 hz , 2h ) , 1.57 ( m , 2h ) , 1.231.33 ( m , 12h ) , 0.88 ( t , j = 6.4 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 148.3 , 141.5 , 138.6 , 128.8 , 117.1 , 96.5 , 34.9 , 31.8 , 31.4 , 29.4 , 29.22 , 29.16 , 22.6 , 14.1 ; ir ( cm ) 2923 , 2853 , 1463 , 1432 , 1389 , 1331 , 1266 , 1234 , 1202 , 1105 ; hrms ( maldi ) m / z calcd for c18h30n3i ( [ m + h ] ) 416.1563 , found 416.1558 . compound 12 ( 0.250 g , 0.602 mmol , 1.00 equiv ) , ( triisopropylsilyl)acetylene ( 0.21 ml , 0.934 mmol , 1.55 equiv ) , cl2pd(pph3)2 ( 0.0227 g , 0.0323 mmol , 0.054 equiv ) , and cui ( 0.0039 g , 0.0204 mmol , 0.034 equiv ) were combined in a 50 ml round - bottomed flask and purged with n2 . ten milliliters of deoxygenated thf and 10 ml of deoxygenated et3n were added by syringe , and the reaction was stirred at 40 c for 18 h. the reaction mixture was then diluted with h2o ( 50 ml ) and extracted with etoac ( 3 50 ml ) . the combined organic phases were washed with satd nh4cl ( 2 50 ml ) , dried over mgso4 , filtered , and concentrated in vacuo . the crude material was purified by flash chromatography using 5% et2o in hexanes to give 0.256 g of a brown / orange oil ( 92% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.34 ( d , j = 8.3 hz , 1h ) , 7.28 ( d , j = 1.9 hz , 1h ) , 3.77 ( q , j = 7.3 hz , 4h ) , 2.53 ( t , j = 7.3 hz , 2h ) , 1.551.60 ( m , 2h ) , 1.221.32 ( m , 18h ) , 1.13 ( s , 21h ) , 0.88 ( t , j = 6.3 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 150.5 , 139.2 , 133.5 , 129.2 , 118.2 , 116.5 , 105.9 , 35.2 , 31.9 , 31.5 , 29.5 , 29.2 , 22.7 , 18.8 , 14.1 , 11.4 ; ir ( cm ) 2925 , 2862 , 2147 , 1463 , 1398 , 1330 , 1242 , 1201 , 1092 , 883 ; hrms ( maldi ) m / z calcd for c29h51n3si ( [ m + h ] ) 470.3930 , found 470.3917 . compound 13 ( 0.485 g , 1.032 mmol , 1 equiv ) was dissolved in 5 ml of mei in a sealed tube . the reaction flask was evacuated and backfilled with n2 before being sealed and heated to 125 c for 20 h. the reaction was cooled to room temperature before excess mei was evaporated by n2 bubbling . the crude residue was purified by flash chromatography using hexanes as eluent to afford 0.51 g of a yellow oil ( 99% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.70 ( d , j = 8.2 hz , 1h ) , 7.29 ( d , j = 2.1 hz , 1h ) , 6.81 ( dd , j = 2.1 , 8.0 hz , 1h ) , 2.51 ( t , j = 7.7 hz , 2h ) , 1.231.31 ( m , 10h ) , 1.16 ( s , 21h ) , 0.88 ( t , j = 7.1 , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 142.8 , 138.4 , 133.2 , 129.9 , 129.8 , 108.2 , 97.1 , 94.6 , 31.9 , 31.2 , 29.4 , 29.2 , 22.6 , 18.7 , 14.1 , 11.4 ; ir ( cm ) 2923 , 2862 , 2366 , 2150 , 1459 , 1394 , 1017 , 883 , 765 , 735 , 665 ; hrms ( maldi ) m / z calcd for c25h41isi ( [ m + h ] ) 497.2100 , found 497.2098 . boronic ester 6 ( 0.620 g , 2.04 mmol , 2.00 equiv ) and 14 ( 0.508 g , 1.02 mmol , 1.00 equiv ) were dissolved in deoxygenated dme and further deoxygenated for 10 min by n2 bubbling . to this solution were added cl2pd(dppf ) ( 0.061 g , 0.0834 mmol , 0.082 equiv ) and dry k3po4 ( 1.045 g , 4.922 mmol , 4.83 equiv ) , and the reaction was deoxygenated by n2 bubbling for an additional 10 min . the reaction was heated to 90 c for 20 h before being cooled to room temperature and diluted with h2o . the reaction was extracted with et2o ( 3 30 ml ) , and organics were washed with satd nacl before being dried over mgso4 , filtered , and concentrated in vacuo . purification by flash chromatography over neutral alumina with 5% diethyl ether in hexanes gave 0.56 g of a yellow oil ( 82% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.58 ( d , j = 8.3 hz , 2h ) , 7.41 ( d , j = 8.8 hz , 3h ) , 7.29 ( d , j = 7.8 hz , 1h ) , 7.16 ( d , j = 7.8 hz , 1h ) , 3.77 ( q , j = 7.0 hz , 4h ) , 2.60 ( t , j = 7.8 hz , 2h ) , 1.64 ( m , 2h ) , 1.251.38 ( m , 16 h ) , 1.03 ( s , 21h ) , 0.88 ( t , j = 6.8 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 150.3 , 141.5 , 141.3 , 137.2 , 133.7 , 129.8 , 129.2 , 128.8 , 121.5 , 119.9 , 106.9 , 93.2 , 35.3 , 31.9 , 31.4 , 29.5 , 29.4 , 29.3 , 22.7 , 18.7 , 14.1 , 11.3 ; ir ( cm ) 2926 , 2862 , 2363 , 2341 , 2146 , 1463 , 1380 , 1234 , 1095 , 908 , 883 , 827 , 734 , 666 ; hrms ( esi ) m / z calcd for c35h55n3sih ( [ m + h ] ) 546.4244 , found 546.4235 . compound 3b ( 0.349 g , 0.639 mmol , 1.00 equiv ) was dissolved in 5 ml of mei in a sealed tube . the flask was evacuated and purged with n2 before being sealed and heated to 125 c for 44 h. the reaction was cooled to room temperature before excess mei was removed by n2 bubbling . the crude residue was purified by flash chromatography using 5% et2o in hexanes to afford 0.336 g of the desired compound as a colorless oil ( 92% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.69 ( d , j = 8.3 hz , 2h ) , 7.39 ( s , 1h ) , 7.30 ( d , j = 8.8 hz , 2h ) , 7.21 ( d , j = 7.8 hz , 1h ) , 7.17 ( dd , j = 7.8 , 1.5 hz , 1h ) , 2.60 ( t , j = 7.8 hz , 2h ) , 1.63 ( m , 2h ) , 1.251.37 ( m , 10h ) , 1.01 ( s , 21h ) , 0.88 ( t , j = 7.3 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 142.3 , 140.5 , 140.1 , 136.9 , 133.5 , 131.3 , 128.9 , 128.8 , 106.2 , 93.9 , 92.8 , 35.4 , 31.9 , 31.4 , 29.4 , 29.3 , 29.2 , 22.7 , 18.6 , 14.1 , 11.3 ; ir ( cm ) 2924 , 2861 , 2366 , 2333 , 2145 , 1464 , 1385 , 1000 , 883 , 816 , 667 ; hrms ( esi ) m / z calcd for c31h45sii ( [ m ] ) 572.2335 , found 572.2344 . compound 15 ( 0.299 g , 0.522 mmol , 1.00 equiv ) was dissolved in 2 ml of thf before 0.63 ml of 1 m tbaf ( in thf containing 5% h2o ) was added . the reaction was then concentrated , and the residue was dissolved in et2o and washed with satd nh4cl . the organic phase was dried over mgso4 , filtered , and concentrated in vacuo . the crude material was purified by flash chromatography using hexanes to afford 0.22 g of a colorless oil ( 74% yield ) : h nmr : ( cdcl3 , 500 mhz ) 7.90 ( d , j = 7.0 hz , 2h ) , 7.49 ( s , 1h ) , 7.37 ( d , j = 7.0 hz , 2h ) , 7.287.24 ( m , 2h ) , 3.07 ( s , 1h ) , 2.64 ( t , j = 7.8 hz , 2h ) , 1.68 ( m , 2h ) , 1.251.37 ( m , 10h ) , 0.88 ( t , j = 7.3 hz , 3h ) ; c nmr ( cdcl3 , 125 mhz ) 142.3 , 140.6 , 139.7 , 137.1 , 133.9 , 131.1 , 129.4 , 129.2 , 120.0 , 93.4 , 83.2 , 80.2 , 77.1 , 35.3 , 31.9 , 31.2 , 29.5 , 29.3 , 29.2 , 22.7 , 14.2 ; ir ( neat , cm ) 3293 , 1479.7 , 1384 , 1007 , 814 ; hrms ( esi ) m / z calcd for c22h25ih ( [ m + h ] ) 417.1071 , found 417.1090 . compound 16b ( 0.031 g , 0.074 mmol , 1.00 equiv ) was dissolved in 2.6 ml of 1:1 phch3/et3n . cl2pd(pph3)2 ( 0.002 g , 0.0028 mmol , 0.038 equiv ) and cui ( 0.001 g , 0.005 mmol , 0.07 equiv ) were added neat . the reaction was stirred at room temperature for 2 h and monitored by tlc ( 10% etoac in hexanes ) . it was then diluted with h2o and extracted with et2o ( 3 20 ml ) , and the combined organic phases were washed with satd nacl before being dried over mgso4 , filtered , and concentrated in vacuo . purification required 5 silica gel flash columns using 5% ch2cl2 in hexanes to afford 2.5 mg of the cyclotrimer 1b and 1.2 mg of the cyclotetramer 1c , both as white waxy solids ( 12% and 6% yield , respectively ) . cyclic trimer 1b : h nmr ( cdcl3 , 500 mhz ) 7.73 ( d , j = 6.5 hz , 6h ) , 7.48 ( s , 3h ) , 7.47 ( d , j = 8.0 hz , 6h ) , 7.39 ( d , j = 8.5 hz , 3h ) , 7.23 ( d , j = 8.0 hz , 3h ) , 2.65 ( t , j = 7.7 hz , 6h ) , 1.67 ( quintet , j = 7.5 hz , 6h ) , 1.401.26 ( m , 30h ) , 0.89 ( t , j = 6.5 hz , 9h ) ; c nmr ( 125 mhz , cdcl3 ) 140.55 , 140.24 , 137.32 , 132.75 , 130.87 , 129.42 , 129.35 , 129.20 , 122.35 , 121.33 , 92.48 , 90.02 , 35.56 , 32.06 , 31.46 , 29.64 , 29.49 , 29.42 , 22.84 , 14.27 ; hrms ( maldi ) m / z calcd for c66h72 ( [ m ] ) 864.5634 , found 864.5624 . cyclic tetramer 1c : h nmr ( cdcl3 , 500 mhz ) 7.65 ( d , j = 8.5 hz , 6h ) , 7.49 ( d , j = 1.5 hz , 3h ) , 7.45 ( d , j = 8.5 hz , 6h ) , 7.31 ( d , j = 8.5 hz , 3h ) , 7.20 ( dd , j = 8.0 hz , 1.5 hz , 3h ) , 2.64 ( t , j = 7.5 hz , 3h ) , 1.67 ( quintet , j = 7.0 hz , 6h ) 1.401.26 ( m , 30h ) , 0.88 ( t , j = 7.0 hz , 9h ) ; c nmr ( 125 mhz , cdcl3 ) 140.56 , 137.14 , 133.21 , 131.40 , 129.68 , 129.47 , 129.25 , 122.39 , 121.35 , 92.20 , 89.88 , 35.56 , 32.05 , 31.44 , 29.63 , 29.52 , 29.41 , 22.83 , 14.27 ; hrms ( maldi ) m / z calcd for c88h96 ( [ m ] ) 1152.75065 , found 1152.7470 . compound 15 ( 0.276 g , 0.482 mmol , 1.00 equiv ) was added to a round - bottomed flask fitted with a sealed reflux condenser , as were 6 ( 0.248 g , 0.819 mmol , 1.7 equiv ) , tribasic potassium phosphate ( 0.563 g , 2.65 mmol , 5.5 equiv ) , and pdcl2(dppf ) ( 0.018 g , 0.022 mmol , 0.045 equiv ) . the flask was purged with nitrogen and 4.0 ml deoxygenated 1,2-dimethoxyethane added . the reaction was heated to reflux for 5 h and cooled to room temperature and the solvent removed under vacuum . the product was extracted from water with ether , and the combined organic layers washed with brine , dried over magnesium sulfate , filtered , and concentrated . purification by flash column chromatography over neutral alumina with 25% methylene chloride in hexanes afforded 0.204 g of a dark yellow oil ( 68% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.63 ( d , j = 8.4 hz , 2h ) , 7.577.61 ( m , 4h ) , 7.50 ( d , j = 8.8 hz , 2h ) , 7.42 ( d , j = 1.5 hz , 1h ) , 7.31 ( d , j = 8.0 hz , 1h ) , 7.19 ( dd , j = 1.7 , 7.7 hz , 1h ) , 3.78 ( q , j = 7.1 hz , 4h ) , 2.61 ( t , j = 7.7 hz , 2h ) , 1.65 ( m , 2h ) , 1.251.38 ( m , 16h ) , 1.01 ( s , 21h ) , 0.89 ( t , j = 6.9 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 150.5 , 141.7 , 141.4 , 139.8 , 139.1 , 137.7 , 133.5 , 129.6 , 129.1 , 128.7 , 127.3 , 126.3 , 121.7 , 120.7 , 106.7 , 93.4 , 35.4 , 31.9 , 31.4 , 29.5 , 29.4 , 29.3 , 22.6 , 18.6 , 14.1 , 11.3 ; ir ( cm ) 2926 , 2864 , 2146 , 1462 , 1396 , 1352 , 1235 , 1100 , 882 , 818 , 770 , 663 ; hrms ( esi ) m / z calcd for c37h49n2si ( [ m ( c4h10n ) ] ) 549.3665 , found 549.3659 . compound 4b ( 0.0631 g , 0.101 mmol , 1.00 equiv ) was added to 2.5 ml of iodomethane in a flask fitted with a sealable valve and the flask degassed and backfilled with nitrogen . the flask was sealed and the reaction heated to 125 c for 44 h. after cooling , the methyl iodide was evaporated and the product filtered through a silica plug with 25% dichloromethane in hexanes to afford 0.065 g ( quantitative yield ) of the iodide as a reddish oil : h nmr ( 500 mhz , cdcl3 ) 7.77 ( d , j = 8.3 hz , 2h ) , 7.64 ( d , j = 8.3 hz , 2h ) , 7.53 ( d , j = 8.3 hz , 2h ) , 7.42 ( d , j = 1.4 hz , 1h ) , 7.33 ( d , j = 8.3 hz , 2h ) , 7.29 ( d , j = 7.8 hz , 1h ) , 7.19 ( dd , j = 1.4 , 7.8 hz , 1h ) , 2.60 ( t , j = 7.8 hz , 2h ) , 1.65 ( m , 2h ) , 1.251.39 ( m , 10h ) , 1.00 ( s , 21h ) , 0.89 ( t , j = 6.9 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 141.9 , 141.1 , 140.6 , 140.1 , 138.6 , 137.8 , 133.5 , 129.8 , 129.1 , 128.9 , 128.8 , 126.3 , 121.7 , 106.6 , 93.5 , 92.8 , 35.3 , 31.8 , 31.3 , 29.4 , 29.3 , 29.2 , 22.6 , 18.5 , 14.0 , 11.3 ; ir ( cm ) 2923 , 2861 , 2363 , 2148 , 1476 , 1382 , 1064 , 1000 , 882 , 810 , 771 , 734 , 666 ; hrms ( ei ) m / z calcd for c37h50isi ( [ m + h ] ) 649.2726 , found 649.2714 . 2-(2-triisopropylsilyl)ethynyl-4-octyl-4-iodoterphenyl ( 0.0519 g , 0.080 mmol , 1.00 equiv ) was dissolved in 2 ml of thf with 5 drops of methanol , to which tbaf ( 1 m in thf ) ( 1.05 ml , 1.05 mmol , 13 equiv ) was added . the reaction was stirred overnight at room temperature , and the solvents were evaporated . the crude product was purified by column chromatography over silica with 100% hexanes followed by 25% dichloromethane in hexanes to yield 0.039 g of white crystals : mp 6971 c ( 98% yield ) ; h nmr ( 500 mhz , cdcl3 ) 7.76 ( d , j = 8.6 hz , 2h ) , 7.66 ( d , j = 8.6 hz , 2h ) , 7.60 ( d , j = 8.4 hz , 2h ) , 7.46 ( d , j = 1.7 hz , 1h ) , 7.38 ( d , j = 8.4 hz , 2h ) , 7.31 ( d , j = 8.0 hz , 1h ) , 7.23 ( dd , j = 1.9 , 8.0 hz , 1h ) , 3.04 ( s , 1h ) , 2.62 ( t , j = 7.7 hz , 2h ) , 1.65 ( m , 2h ) , 1.251.37 ( m , 10h ) , 0.89 ( t , j = 7.1 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 142.0 , 141.0 , 139.6 , 138.8 , 137.8 , 133.8 , 129.7 , 129.3 , 128.9 , 126.4 , 120.0 , 93.0 , 83.4 , 79.8 , 35.3 , 31.8 , 31.2 , 29.4 , 29.2 , 29.2 , 22.6 , 14.0 ; ir ( cm ) 3288 , 2922 , 2854 , 2360 , 2340 , 1478 , 1386 , 1000 , 895 , 808 , 788 , 731 , 657 , 611 ; hrms ( ei ) m / z calcd for c28h29i ( [ m ] ) 492.1314 , found 492.1312 . compound 17b ( 0.0364 g , 0.074 mmol , 1.00 equiv ) was dissolved with cui ( 0.0009 g , 0.0047 mmol , 0.06 equiv ) and pd(ph3p)4 ( 0.0243 g , 0.021 mmol , 0.28 equiv ) in 40 ml of dry , deoxygenated thf and 10 ml of triethylamine . the reaction was stirred at room temperature under nitrogen for 2 weeks , with periodic reloading of catalyst ( only 3 mol % of pd at a time ) . when tlc indicated the reaction had gone to completion , the solvents were removed in vacuo , and the reaction was purified over a silica column with 15% dichloromethane in hexanes . two products were recovered , 3.2 mg of cyclodimer 2c ( 12% yield ) and 13.8 mg of cyclotrimer 2b ( 51% yield ) , both as colorless waxy solids . 2c : h nmr ( 500 mhz , cdcl3 ) 7.75 ( d , j = 8.3 hz , 4h ) , 7.67 ( d , j = 8.3 hz , 4h ) , 7.60 ( d , j = 8.3 hz , 4h ) , 7.51 ( d , j = 1.5 hz , 2h ) , 7.41 ( d , j = 8.3 hz , 4h ) , 7.37 ( d , j = 7.8 hz , 2h ) , 7.22 ( dd , j = 7.8 , 1.5 hz , 2h ) , 2.66 ( t , j = 7.8 hz , 4h ) , 1.68 ( quintet , j = 6.8 hz , 4h ) , 1.271.41 ( m , 20 h ) , 0.89 ( t , j = 6.8 hz , 6h ) ; cosy ( 500 mhz , cdcl3 ) 7.75 7.67 , 7.60 7.41 , 7.37 7.22 , 2.66 1.68 , 1.68 1.38 , 1.30 0.89 ; hmqc ( cdcl3 ) 7.75 130.0 , 7.67 126.3 , 7.60 126.9 , 7.51 132.6 , 7.41 131.8 , 7.37 129.3 , 7.22 129.0 , 2.66 35.5 , 1.68 31.3 , 1.36 29.4 , 1.31 22.6 , 1.26 31.8 , 1.22 26.7 , 1.21 33.5 , 0.89 14.1 ; maldi - tof ( dhb matrix ) [ m ] m / z calcd for c56h56 728.4832 , found 728.6150 . 2b : h nmr ( 500 mhz , cdcl3 ) 7.80 ( d , j = 8.3 hz , 6h ) , 7.68 ( d , j = 8.3 hz , 6h ) , 7.64 ( d , j = 7.8 hz , 6h ) , 7.52 ( s , 3h ) , 7.49 ( d , j = 7.8 hz , 6h ) , 7.42 ( d , j = 7.8 hz , 3h ) , 7.26 ( m , 3h ) , 2.67 ( t , j = 7.8 hz , 6h ) , 1.69 ( quintet , j = 8.3 hz , 6h ) , 1.291.42 ( m , 30h ) , 0.90 ( quintet , j = 8.3 hz , 9h ) ; c nmr ( 125 mhz , cdcl3 ) 142.1 , 140.7 , 140.6 , 139.8 , 139.3 , 132.6 , 131.7 , 130.0 , 129.2 , 127.1 , 126.4 , 122.5 , 121.2 , 91.8 , 90.5 , 35.4 , 31.9 , 31.3 , 29.7 , 29.5 , 29.3 , 22.7 , 14.1 ; hmqc ( cdcl3 ) 7.80 130.1 , 7.68 126.4 , 7.64 127.1 , 7.49 131.7 , 7.52 132.6 , 7.42 129.2 , 7.26 129.9 , 2.67 35.4 , 1.69 31.3 , 1.36 29.5 , 1.29 22.7 , 0.89 14.1 ; hmbc ( cdcl3 , selected peaks ) 7.80 140.7 , 7.80 139.3 , 7.68 140.6 , 7.68 139.8 , 7.64 139.3 , 7.64 122.5 , 7.52 90.5 , 7.52 35.4 , 7.49 140.7 , 7.49 91.8 , 7.42 142.1 , 7.42 139.8 , 7.42 121.2 , 7.26 140.6 , 2.67 129.2 , 2.67 132.6 , 2.67 142.1 , 2.67 31.3 , 2.67 29.3 , 1.69 29.3 , 1.36 29.7 , 0.90 31.9 , 0.90 22.7 ; maldi - tof ( dhb matrix ) m / z calcd for c84h84 ( [ m ] ) 1092.7 , found 1092.7 . hrms ( maldi ) m / z calcd for c84h84 ( [ m ] ) 1092.6573 , found 1092.6612 .

a series of single - walled carbon nanotube precursors , c3h - symmetric cyclotri(ethynylene)(biphenyl-2,4-diyl ) and cyclotri(ethynylene)(p - terphenyl-2,4-diyl ) , have been prepared by a linear stepwise oligomerization cyclization route and by statistical intermolecular cyclooligomerization . in addition to producing these members of a novel class of arylene ethynylene macrocycles , 1 and 2 , the latter statistical process produces the smaller cyclic dimer , cyclodi(ethynylene)(p - terphenyl-2,4-diyl ) and the larger cyclic tetramer cyclotetra(ethynylene)(biphenyl-2,4-diyl ) . these macrocycles display large stokes shifts in their fluorescence spectra . their biphenyl or terphenyl connectivity prevents these macrocycles from achieving full planarity in the ground state , and the ethynylene moieties could provide synthetic access to cyclic arylene oligomers and discrete carbon nanotube segments .

Introduction Results and Discussion Conclusions Experimental Section

arylene ethynylene macrocycles are synthetically accessible either by statistical cyclization of a single aryl halide ethynyl monomer or by the cyclization of a linear oligomer via a palladium catalyzed cross coupling , in either case at low concentrations . despite the recent attention given arylene ethynylene macrocycles , the incorporation of biphenyl and terphenyl moieties is rare , and such macrocycles have not been fully explored . the few reported examples of biphenyl or teraryl units include bipyridyl , m - terphenyl , m - terpyridinyl , and 11,12-dihydroindolo[2,3-a]carbazole ( nominal p - terphenylenes ) and tetram - phenylene subunits but no simple biphenyl or p - terphenylene containing macrocycles have been reported . more significantly , the title c3 macrocycles offer the possibility to synthesize short segments of single - walled carbon nanotubes . compound 16b was subjected to sonogashira coupling conditions at 28 mm ; four fluorescent compounds were identified by tlc , but only two compounds were isolated by five iterations of flash chromatography , the cyclic trimer 1b in 36% yield , and the cyclic tetramer 1c in 24% . on the basis of the macrocycles described above , the cyclic dimer , cyclic trimer , and cyclotetramer of the 1,2-phenylene , 1,4-biphenylene , and 1,4-terphenylene ethynylene macrocycles were computationally modeled . all macrocycles displayed large stokes shifts which are indicative of large conjugated arylene ethynylene macrocycles ( see table 4 ) . all of the macrocycles obtained absorb around 300 nm , but the cyclotrimers 1a and 1b exhibit larger stokes shifts in their fluorescence emission spectra than the other macrocycles observed . compound 3a ( 1.855 g , 4.277 mmol , 1.00 equiv ) was dissolved in 10 ml of methyl iodide in a sealable reaction flask , degassed , backfilled with nitrogen , sealed , and heated to 125 c for 44 h. after cooling , the methyl iodide was removed by evaporation and the product purified by flash chromatography over silica with 5% diethyl ether in hexanes to isolate 1.950 g of a yellow oil ( 99% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.70 ( d , j = 8.3 hz , 2h ) , 7.59 ( d , j = 7.8 hz , 1h ) , 7.277.37 ( m , 5h ) , 1.01 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 143.1 , 140.1 , 137.0 , 133.7 , 131.2 , 129.0 , 128.5 , 127.2 , 121.9 , 105.9 , 94.6 , 93.1 , 18.5 , 11.3 ; ir ( cm ) 2940 , 2863 , 2152 , 1469 , 1386 , 1000 , 883 , 820 , 758 , 677 ; hrms ( ei ) m / z calcd for c23h29isi ( [ m ] ) 460.1083 , found 460.1091 . the crude material was purified via flash chromatography using 5% diethyl ether in hexane to afford 0.134 g ( 96% yield ) of a reddish oil : h nmr ( 500 mhz , cdcl3 ) 7.78 ( d , j = 7.0 hz , 2h ) , 7.64 ( d , j = 7.5 hz , 1h ) , 7.42 ( t , j = 6.5 hz , 1h ) , 7.377.34 ( m , 4h ) , 3.08 ( s , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 143.1 , 139.7 , 137.1 , 133.9 , 131.1 , 129.2 , 129.0 , 127.3 , 120.3 , 93.6 , 80.6 ; ir ( neat , cm ) 3274 , 3061 , 1583,1472 ; hrms ( esi ) m / z calcd for c14h10i ( [ m + h ] ) 16a ( 0.060 g , 0.197 mmol , 1.00 equiv ) , cl2pd(pph3)2 ( 0.007 g , 0.010 mmol , 0.05 equiv ) , and cui ( 0.004 g , 0.020 mmol , 0.11 equiv ) were combined and dissolved in 10.6 ml of thf and 0.2 ml of et3n . a 50 ml round - bottomed flask was charged with 9 ( 0.223 g , 0.484 mmol , 1.00 equiv ) , 19a ( 0.180 g , 0.649 mmol , 1.34 equiv ) , and cl2pd(pph3)2 ( 0.020 g , 0.0285 mmol , 0.06 equiv ) and flushed with n2 . compound 4a ( 0.096 g , 0.188 mmol , 1 equiv ) was dissolved in 2.5 ml of methyl iodide in a sealable reaction flask , degassed , backfilled with nitrogen , sealed , and heated to 125 c for 40 h. after cooling , the methyl iodide was removed by evaporation and the product purified by flash chromatography over neutral alumina with 50% methylene chloride in hexanes to afford 0.092 mg of the product as a yellow solid : mp 8183 c ( 91% yield ) ; h nmr ( 500 mhz , cdcl3 ) 7.77 ( d , j = 8.3 hz , 2h ) , 7.64 ( d , j = 8.3 hz , 2h ) , 7.61 ( d , j = 7.8 hz , 1h ) , 7.54 ( d , j = 8.3 hz , 2h ) , 7.277.38 ( m , 5h ) , 1.00 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 143.7 , 140.5 , 140.1 , 138.9 , 137.9 , 137.8 , 129.9 , 128.91 , 128.87 , 128.5 , 127.0 , 126.4 , 122.0 , 106.2 , 95.2 , 93.0 , 18.6 , 11.3 . the combined organic layers were dried over magnesium sulfate , filtered , and concentrated to produce 0.127 g of an orange solid ( 78% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.78 ( d , j = 8.6 hz , 2h ) , 7.68 ( d , j = 8.1 hz , 2h ) , 7.617.65 ( m , 3h ) , 7.387.43 ( m , 4h ) , 7.307.34 ( m , 1h ) , 3.08 ( s , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 143.7 , 140.2 , 139.6 , 139.1 , 137.8 , 134.0 , 129.7 , 129.5 , 129.0 , 128.9 , 127.1 , 126.4 , 120.3 , 93.1 , 83.0 , 80.4 ; hrms ( photospray ionization ) m / z calcd for c20h14i ( [ m + h ] ) 381.0140 , found 381.0151 compound 4a ( 0.240 g , 0.471 mmol , 1.00 equiv ) was stirred with tbaf ( 1 m in thf ) ( 1.41 ml , 1.41 mmol , 3.0 equiv ) with 2.0 ml of thf and 6 drops of methanol for 40 h at room temperature . compound 20b ( 0.096 g , 0.126 mmol , 1.00 equiv ) was dissolved in 2.5 ml of methyl iodide in a sealable reaction flask , degassed , backfilled with nitrogen , sealed , and heated to 125 c for 40 h. after cooling , the methyl iodide was removed by evaporation and the product purified by flash chromatography over silica with 25% methylene chloride in hexanes to afford 0.065 mg of a white , crystalline solid : mp 109112 c ( 86% yield ) ; h nmr ( 500 mhz , cdcl3 ) 7.78 ( m , 4h ) , 7.517.69 ( m , 10h ) , 7.26746 ( m , 10h ) , 1.01 ( s , 21h ) ; c nmr ( 125 mhz , cdcl3 ) 143.7 , 143.1 , 140.7 , 140.3 , 140.0 , 139.1 , 139.0 , 137.9 , 137.8 , 133.7 , 133.0 , 131.8 , 129.9 , 129.8 , 129.4 , 129.2 , 128.93 , 128.86 , 128.5 , 127.2 , 127.0 , 126.9 , 126.5 , 126.3 , 122.2 , 122.0 , 121.6 , 106.3 , 94.2 , 93.1 , 92.4 , 90.1 , 18.6 , 11.3 ; ir ( cm ) 2939 , 2861 , 2151 , 1473 , 1384 , 1064 , 1000 , 882 , 813 , 758 , 664 ; hrms m / z calcd for c37h38isi ( [ mh ] ) 637.1782 , found 637.1777 . the crude material was purified by flash chromatography using 5% et2o in hexanes to give 0.256 g of a brown / orange oil ( 92% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.34 ( d , j = 8.3 hz , 1h ) , 7.28 ( d , j = 1.9 hz , 1h ) , 3.77 ( q , j = 7.3 hz , 4h ) , 2.53 ( t , j = 7.3 hz , 2h ) , 1.551.60 ( m , 2h ) , 1.221.32 ( m , 18h ) , 1.13 ( s , 21h ) , 0.88 ( t , j = 6.3 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 150.5 , 139.2 , 133.5 , 129.2 , 118.2 , 116.5 , 105.9 , 35.2 , 31.9 , 31.5 , 29.5 , 29.2 , 22.7 , 18.8 , 14.1 , 11.4 ; ir ( cm ) 2925 , 2862 , 2147 , 1463 , 1398 , 1330 , 1242 , 1201 , 1092 , 883 ; hrms ( maldi ) m / z calcd for c29h51n3si ( [ m + h ] ) 470.3930 , found 470.3917 . the crude residue was purified by flash chromatography using hexanes as eluent to afford 0.51 g of a yellow oil ( 99% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.70 ( d , j = 8.2 hz , 1h ) , 7.29 ( d , j = 2.1 hz , 1h ) , 6.81 ( dd , j = 2.1 , 8.0 hz , 1h ) , 2.51 ( t , j = 7.7 hz , 2h ) , 1.231.31 ( m , 10h ) , 1.16 ( s , 21h ) , 0.88 ( t , j = 7.1 , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 142.8 , 138.4 , 133.2 , 129.9 , 129.8 , 108.2 , 97.1 , 94.6 , 31.9 , 31.2 , 29.4 , 29.2 , 22.6 , 18.7 , 14.1 , 11.4 ; ir ( cm ) 2923 , 2862 , 2366 , 2150 , 1459 , 1394 , 1017 , 883 , 765 , 735 , 665 ; hrms ( maldi ) m / z calcd for c25h41isi ( [ m + h ] ) 497.2100 , found 497.2098 . to this solution were added cl2pd(dppf ) ( 0.061 g , 0.0834 mmol , 0.082 equiv ) and dry k3po4 ( 1.045 g , 4.922 mmol , 4.83 equiv ) , and the reaction was deoxygenated by n2 bubbling for an additional 10 min . purification by flash chromatography over neutral alumina with 5% diethyl ether in hexanes gave 0.56 g of a yellow oil ( 82% yield ) : h nmr ( 500 mhz , cdcl3 ) 7.58 ( d , j = 8.3 hz , 2h ) , 7.41 ( d , j = 8.8 hz , 3h ) , 7.29 ( d , j = 7.8 hz , 1h ) , 7.16 ( d , j = 7.8 hz , 1h ) , 3.77 ( q , j = 7.0 hz , 4h ) , 2.60 ( t , j = 7.8 hz , 2h ) , 1.64 ( m , 2h ) , 1.251.38 ( m , 16 h ) , 1.03 ( s , 21h ) , 0.88 ( t , j = 6.8 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 150.3 , 141.5 , 141.3 , 137.2 , 133.7 , 129.8 , 129.2 , 128.8 , 121.5 , 119.9 , 106.9 , 93.2 , 35.3 , 31.9 , 31.4 , 29.5 , 29.4 , 29.3 , 22.7 , 18.7 , 14.1 , 11.3 ; ir ( cm ) 2926 , 2862 , 2363 , 2341 , 2146 , 1463 , 1380 , 1234 , 1095 , 908 , 883 , 827 , 734 , 666 ; hrms ( esi ) m / z calcd for c35h55n3sih ( [ m + h ] ) 546.4244 , found 546.4235 . the crude material was purified by flash chromatography using hexanes to afford 0.22 g of a colorless oil ( 74% yield ) : h nmr : ( cdcl3 , 500 mhz ) 7.90 ( d , j = 7.0 hz , 2h ) , 7.49 ( s , 1h ) , 7.37 ( d , j = 7.0 hz , 2h ) , 7.287.24 ( m , 2h ) , 3.07 ( s , 1h ) , 2.64 ( t , j = 7.8 hz , 2h ) , 1.68 ( m , 2h ) , 1.251.37 ( m , 10h ) , 0.88 ( t , j = 7.3 hz , 3h ) ; c nmr ( cdcl3 , 125 mhz ) 142.3 , 140.6 , 139.7 , 137.1 , 133.9 , 131.1 , 129.4 , 129.2 , 120.0 , 93.4 , 83.2 , 80.2 , 77.1 , 35.3 , 31.9 , 31.2 , 29.5 , 29.3 , 29.2 , 22.7 , 14.2 ; ir ( neat , cm ) 3293 , 1479.7 , 1384 , 1007 , 814 ; hrms ( esi ) m / z calcd for c22h25ih ( [ m + h ] ) 417.1071 , found 417.1090 . the flask was sealed and the reaction heated to 125 c for 44 h. after cooling , the methyl iodide was evaporated and the product filtered through a silica plug with 25% dichloromethane in hexanes to afford 0.065 g ( quantitative yield ) of the iodide as a reddish oil : h nmr ( 500 mhz , cdcl3 ) 7.77 ( d , j = 8.3 hz , 2h ) , 7.64 ( d , j = 8.3 hz , 2h ) , 7.53 ( d , j = 8.3 hz , 2h ) , 7.42 ( d , j = 1.4 hz , 1h ) , 7.33 ( d , j = 8.3 hz , 2h ) , 7.29 ( d , j = 7.8 hz , 1h ) , 7.19 ( dd , j = 1.4 , 7.8 hz , 1h ) , 2.60 ( t , j = 7.8 hz , 2h ) , 1.65 ( m , 2h ) , 1.251.39 ( m , 10h ) , 1.00 ( s , 21h ) , 0.89 ( t , j = 6.9 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 141.9 , 141.1 , 140.6 , 140.1 , 138.6 , 137.8 , 133.5 , 129.8 , 129.1 , 128.9 , 128.8 , 126.3 , 121.7 , 106.6 , 93.5 , 92.8 , 35.3 , 31.8 , 31.3 , 29.4 , 29.3 , 29.2 , 22.6 , 18.5 , 14.0 , 11.3 ; ir ( cm ) 2923 , 2861 , 2363 , 2148 , 1476 , 1382 , 1064 , 1000 , 882 , 810 , 771 , 734 , 666 ; hrms ( ei ) m / z calcd for c37h50isi ( [ m + h ] ) 649.2726 , found 649.2714 .

invasive meningococcal disease ( imd ) may result in death within 24 h , even with antibiotic treatment . patients who survive imd are also at high risk of suffering at least 1 permanent sequela , which may include limb loss , cognitive deficits , hearing loss , or seizure disorders . each year approximately 1.2 million cases of imd with 135 000 deaths are estimated worldwide . meningitidis has at least 13 serologically distinct groups , classified according to the antigenic structure of the polysaccharide capsule . six serogroups of n. meningitidis ( a , b , c , y , w , and more recently x ) are responsible for the majority of imd cases worldwide . the risk of invasive disease is higher for serogroup c and b compared with other serogroups , and is higher for serogroup c than for serogroup b. disease caused by n. meningitidis serogroups ( a , c , y , and w ) is preventable using conjugate vaccines targeting the respective serogroup - specific polysaccharide capsules . currently , most meningococcal disease in developed countries is caused by menb . in the united states , nearly one - third of all cases of meningococcal disease are caused by capsular group b strains . in many european countries , group b strains cause a disproportionate number of imd cases in infants < 1 y of age ( table 1 ) . rates of imd infection generally decline with age , although disease prevalence rises slightly during the teenage years , presumably due to the high carriage rate in this group attributable to increased peer contact and social behavior . , the european commission granted a marketing authorization for the first meningococcal serogroup b ( menb ) vaccine , 4cmenb , a vaccine containing three recombinant proteins , and outer membrane vesicles ( omv ) derived from menb . in august 2013 , this article discusses the different aspects that should be considered to enable a successful implementation of this vaccine in national immunization programs . most of the menb disease is endemic with incidences varying by country over the years ; the highest incidence of imd caused by serogroup b meningococci is in the infant age group ( < 1 y ) ( table 1 ) . in europe , the overall imd incidence ranges from 0.3 to 1.1 per 100 000 persons ( 20092012 ) . in the united states , the incidence of menb disease is historically low , i.e. , 0.05 per 100 000 ( 2011 ) . the epidemiology data of the us from 19982007 shows that the incidence of imd caused by serogroup b peaked among infants aged 03 mo , whereas menc disease peaked among 45 mo . in canada ( data from 19912011 ) the menb disease incidence ranged from 0.10.9 per 100 000 per year ; a peak in imd incidence was observed in infants at 45 mo . in australia ( 2011 ) , the incidence rate was 0.8 per 100 000 . infants < 1 y accounted for 18% of the cases , children 14 y for 17% , and persons aged 1519 y for 20% of the cases . in new zealand ( 2012 ) , the incidence rate was 1.2 per 100 000 . menb can also cause severe epidemics dominated by one particular strain , which may persist for 10 y or longer , as seen in the past in cuba , brazil , norway , and new zealand . vaccines against menb disease have proved difficult to produce , because the capsular polysaccharide on the serogroup b bacterium is poorly immunogenic as it exhibits structural similarity to human neural ( adhesion ) molecules and is therefore not a useful target . consequently , vaccine developers focused on other outer membrane structures and initially meningococcal outer membrane vesicles ( omv ) were used as basis for the development of several menb vaccines . omv produced from a representative outbreak strain has been shown to be successful in controlling various epidemics of menb disease , such as menzb that was used to control an epidemic in new zealand ( 20046 ) . the bactericidal activity induced by these omv vaccines is largely directed at the pora outer membrane protein ( omp ) . however , pora is a highly variable , and therefore monovalent strain - specific omv vaccines are not generally useful for prevention of endemic imd caused by diverse strains . in order to obtain broader protection multivalent pora omv vaccines have been developed , such as bivalent , hexavalent , and nonavalent omv vaccine combinations . in addition , omv vaccine formulations based on neisseria lactamica have been designed to provide broad coverage . more recently , native omv vaccines , without the use of detergents , based on genetically detoxified lps have been developed . other approaches that have been applied are vaccines containing multiple recombinant proteins , such as 4cmenb or rlp2086 . several of these vaccines are at stages of clinical development , and in january 2013 , 4cmenb was the first menb vaccine that has been approved by the european medicines agency ( ema ) for use on the european market . 4cmenb is based on novel antigens identified by reverse vaccinology and is composed of factor h binding protein ( fhbp ) , involved in regulation of complement activation , nada , involved in cell adhesion , invasion and induction of cytokines , and nhba , heparin - binding protein . nhba and fhbp were fused to gna1030 ( unknown function ) and gna2091 ( unknown function ) , respectively , to enhance protein stability and immunogenicity . in addition to these proteins , omv from the new zealand epidemic strain ( nz98/254 ; p1.7 - 2,4 , st41/44 ) were added to the formulation as major ( pora ) antigen and for additional potential adjuvant activity besides the alum adjuvant . most of the menb disease is endemic with incidences varying by country over the years ; the highest incidence of imd caused by serogroup b meningococci is in the infant age group ( < 1 y ) ( table 1 ) . in europe , the overall imd incidence ranges from 0.3 to 1.1 per 100 000 persons ( 20092012 ) . in the united states , the incidence of menb disease is historically low , i.e. , 0.05 per 100 000 ( 2011 ) . the epidemiology data of the us from 19982007 shows that the incidence of imd caused by serogroup b peaked among infants aged 03 mo , whereas menc disease peaked among 45 mo . in canada ( data from 19912011 ) the menb disease incidence ranged from 0.10.9 per 100 000 per year ; a peak in imd incidence was observed in infants at 45 mo . in australia ( 2011 ) , the incidence rate was 0.8 per 100 000 . infants < 1 y accounted for 18% of the cases , children 14 y for 17% , and persons aged 1519 y for 20% of the cases . in new zealand ( 2012 ) , the incidence rate was 1.2 per 100 000 . menb can also cause severe epidemics dominated by one particular strain , which may persist for 10 y or longer , as seen in the past in cuba , brazil , norway , and new zealand . vaccines against menb disease have proved difficult to produce , because the capsular polysaccharide on the serogroup b bacterium is poorly immunogenic as it exhibits structural similarity to human neural ( adhesion ) molecules and is therefore not a useful target . consequently , vaccine developers focused on other outer membrane structures and initially meningococcal outer membrane vesicles ( omv ) were used as basis for the development of several menb vaccines . omv produced from a representative outbreak strain has been shown to be successful in controlling various epidemics of menb disease , such as menzb that was used to control an epidemic in new zealand ( 20046 ) . the bactericidal activity induced by these omv vaccines is largely directed at the pora outer membrane protein ( omp ) . however , pora is a highly variable , and therefore monovalent strain - specific omv vaccines are not generally useful for prevention of endemic imd caused by diverse strains . in order to obtain broader protection multivalent pora omv vaccines have been developed , such as bivalent , hexavalent , and nonavalent omv vaccine combinations . in addition , omv vaccine formulations based on neisseria lactamica have been designed to provide broad coverage . more recently , native omv vaccines , without the use of detergents , based on genetically detoxified lps have been developed . other approaches that have been applied are vaccines containing multiple recombinant proteins , such as 4cmenb or rlp2086 . several of these vaccines are at stages of clinical development , and in january 2013 , 4cmenb was the first menb vaccine that has been approved by the european medicines agency ( ema ) for use on the european market . 4cmenb is based on novel antigens identified by reverse vaccinology and is composed of factor h binding protein ( fhbp ) , involved in regulation of complement activation , nada , involved in cell adhesion , invasion and induction of cytokines , and nhba , heparin - binding protein . nhba and fhbp were fused to gna1030 ( unknown function ) and gna2091 ( unknown function ) , respectively , to enhance protein stability and immunogenicity . in addition to these proteins , omv from the new zealand epidemic strain ( nz98/254 ; p1.7 - 2,4 , st41/44 ) were added to the formulation as major ( pora ) antigen and for additional potential adjuvant activity besides the alum adjuvant . today , meningococci are classified into serogroups ( by type of capsular polysaccharide ) usually performed by bacterial agglutination test or pcr and fine types determined by sequencing epitope encoding regions of pora ( vr1 , vr2 ) and feta . multilocus sequence typing ( mlst ) is a molecular technique that has been developed and has been increasingly used for menb typing . the loci of each house - keeping gene define the allelic profile or so - called sequence type ( st ) . related sts with identical alleles at four or more loci the combination of antigen sequence typing of pora and feta together with mlst seems to provide a robust framework , which can be complemented by sequence typing of other antigens and measurement of their expression . after implementation of a menb vaccine in a national immunization program , it is important to have a surveillance system that provides complete and accurate data of the circulating menb strains . this is essential to be able to identify antigenic changes that may lead to vaccine failures . n. meningitidis has the capability adapt surface structures to changing environments by a variety of genetic mechanisms . horizontal gene transfer is a common occurrence in the neisseria genus and is responsible for large numbers of genetically heterogeneous menb strains , especially at the omp level whereby a variety of combinations are present . in addition , menb strains could also escape immune responses against vaccine antigens by changing the expression levels of the target antigens . for example , only 50% of invasive meningococcal isolates are known to produce nada , present in 4cmenb , in detectable quantities ( nada expression is phase variable ) with a significant proportion of the nada negative isolates not having the nada gene at all . nhba on the other hand , appears to be present in all isolates tested so far , but protein sequence variability is high . the fhbp shows significant variability resulting in limited cross - protective antibody responses and immune selection under vaccine pressure may occur . therefore , in addition to traditional typing of the serogroup , fine and genetic typing , data are required on the fhbp , nhba and nada genotypes ( dna sequencing ) , and phenotypes of invasive circulating strains for the post - implementation surveillance of the 4cmenb vaccine . the meningococcal antigen typing system ( mats ) was developed to predict 4cmenb strain coverage , using serum bactericidal antibody assay with human complement ( hsba ) taking antigen expression levels and cross protection into account . however , the mats assay is a complicated assay that can not be performed in any reference laboratory and it has been suggested that selected reference laboratories should carry out mats . moreover , the vaccine producer itself developed the assay and is the only producer so far . also , if post - implementation surveillance methods that take into account antigen expression levels will be used , vaccine failures should be carefully defined . as aforementioned vaccine pressure since no other menb vaccine has completed clinical development , this article focuses on clinical data obtained with 4cmenb ( table 2 ) . three large phase 23 randomized controlled clinical studies have been performed to study the immunogenicity and safety of the investigational vaccine , 4cmenb , in adolescents , and infants ( 212 mo ) . the immunogenicity of 4cmenb was determined by measuring serum bactericidal antibody ( sba ) titers against menb reference strains that primarily expressed just one particular vaccine antigen , i.e. , strain 44/76-sl ( matched with the vaccine for fhbp ) , strain 5/99 ( matched with the vaccine for nada ) , and strain nz98/254 , the vaccine strain for the omv component . summarizing , 4cmenb was shown to be safe in adolescents and infants ( primary series and booster dose ) . however , high fever ( 3838.5 c ) rates , up to 80% , were reported in the infant groups , especially when 4cmenb was given concomitantly with routine vaccines . evidence suggests that the rise in body temperature induced by the vaccine can be tempered by prophylactic use of paracetamol without influencing the immunogenicity . in general , good sba titers were found against the selected menb reference strains and no influence of clinical relevance was observed regarding the immune response against routine infant vaccines . the potential of 4cmenb to protect against wild - type circulation strains should be proven after implementation of the vaccine in routine schemes . * routine vaccination : with 7-valent pneumococcal and combined diphtheria , tetanus , acellular pertussis , inactivated polio , hepatitis b , hemophilus influenzae type b dtap - ipv - hepb - hib vaccine . menc , serogroup c conjugate vaccine ; mmrv , measles - mumps - rubella - varicella vaccine . in a recent study , waning of sba titers was observed at 40 mo of age after primary immunization with 4cmenb at 6 , 8 , and 12 mo of age . thus , a robust surveillance program post - implementation is recommended , allowing early recognition of any decline in vaccine effectiveness and the need for a booster vaccination later in childhood or adolescence . asymptomatic nasopharyngeal carriage of n. meningitidis is common , with an average carriage rate of approximately 10% . commensal association of particular strains with a host is a long - term relationship , often lasting several months . in contrast to imd , which is most common in infants and declines through childhood , the prevalence of n. meningitidis carriage is highest among teenagers and low in young children . the carriage rate was shown to be < 3% in children younger than 4 y and increased up to 2437% in the age - group 1524 y , but may differ per country and over the time . apart from age , other risk factors for higher carriage are active and passive smoking , gender ( slightly more male ) , recent respiratory infections , and regular visits to public venues , such as youth clubs and discotheques . asymptomatic infection with or carriage of pathogenic and non - pathogenic strains may help to protect against meningococcal disease . this may explain the higher risk of disease in infants that may have never been a carrier ( naive ) . cellular immunity and cytokine production in relation to meningococcal disease and carriage are poorly understood and deserve more attention . there is evidence that the loss of capsule enhances the capability of meningococci to colonize the human nasopharynx and to avoid human defense systems . capsule production in meningococcal strains can be switched on and off at a high frequency . moreover , meningococci can change from serogroup by capsular switching . in a population - based study , a substantial proportion of invasive serogroup b , c , and y isolates demonstrated capsular switching , indicating that this is a common natural phenomenon . the implementation of menb vaccination in national immunization programs might have an effect on population - level meningococcal carriage state . the highest incidence of imd caused by serogroup b is in the infant age group ( < 1 y ) . therefore , implementation of a new menb vaccine in existing routine infant immunization schedules seems the most logical strategy . however , it is noteworthy that a substantial disease burden occurs in very young infants ( i.e. , those younger than 35 mo of age ) , and these cases will probably not be vaccine - preventable using a 2- , 4- , and 6-mo schedule . results from clinical trials suggest that a 2- , 3- , or 4-mo infant schedule may be acceptable , although further information is needed given the lower immunogenicity that was observed using this schedule . in some countries , this accelerated schedule fits well in the national immunization program , while in other countries the routine infant vaccinations are given at a 2- , 4- , and 6-mo schedule . official recommendations for infants is , according to the product information , three primary doses starting at the age of 2 mo and a booster vaccination between the age of 12 and 23 mo . 4cmenb can be given concomitantly with vaccines against diphtheria , tetanus , acellular pertussis , hemophilus influenza type b , inactivated poliomyelitis , hepatitis b , heptavalent pneumonoccal conjugate , measles , mumps , rubella or varicella . another strategy may be maternal vaccination , however , because young children remain vulnerable for imd in the first years of their life , the infants will not be protected when maternal antibody levels have diminished . probably multiple vaccinations are then still necessary to prevent menb imd during young childhood . after the implementation of the menb vaccine , it is recommended to investigate the influence on meningococcal carriage . so far , three studies have examined the effect of menb omv vaccines on carriage ; in these studies high vaccine coverage had no effect on rates of meningococcal carriage . recently , results were presented of a large study in the uk of nearly 3000 young adults immunized with 4cmenb and/or quadravalent meningococcal a , c , w , y conjugate vaccines examining the effect on meningococcal carriage rates . in this study , prior to vaccination 33% of the samples ( n = 930 ) yielded neisseria cultures , mostly n. meningitides ( 98% ) , mainly of serogroups b and y. primary analysis at one month after the vaccination series did not reveal significant impact of the 4cmenb vaccine , but at later time points 4cmenb was associated with a decrease in carriage of menbcwy strains ( 24.2% ) . these results raise the possibility of an impact on individual carriage , which may lead to greater herd protection in settings where the vaccines are implemented broadly . if immunization with a menb vaccine were to influence nasopharyngeal carriage , a mass immunization campaign of adolescents and young adults , the age of peak nasopharyngeal carriage , may reduce circulation of strains covered by the vaccine leading to reduced rates of disease ( i.e. , herd immunity ) . this strategy alone , however , is probably not sufficient to protect young children . based on modeling data that have been published regarding the cost - effectiveness of a new menb vaccine it is not expected that the vaccine is cost - effective at present , considering the commonly accepted threshold of 50 000 per qaly . only when the menb incidence will increase considerably or . another important influence on cost - effectiveness will be whether the vaccine results in herd effects , by reduction of carriage rate , which is not yet known . the duration of protection and the need for booster doses in childhood will be other key cost considerations . apart from cost - effectiveness , the success of a vaccine program is dependent on public acceptability and feasibility . meningococcal disease is highly feared by the public , which may encourage the uptake of the menb vaccine . on the other hand , parental acceptability may be influenced by vaccine concerns , which include safety , undefined effectiveness , or more practical concerns regarding many vaccines and multiple injections at single visits . with respect to vaccine safety , parents can fear the risk of fever associated with 4cmenb vaccine , when administered to infants . an adequate system of post - implementation surveillance to detect and evaluate ( potentially rare and serious ) adverse effects and will be an essential component of maintaining public confidence . in addition , post - implementation surveillance data should be sufficient to monitor the vaccine effectiveness and to be able to detect possible changes in the clonal , antigenic , and phenotypic profiles of circulating strains under vaccine selection pressure . today , meningococci are classified into serogroups ( by type of capsular polysaccharide ) usually performed by bacterial agglutination test or pcr and fine types determined by sequencing epitope encoding regions of pora ( vr1 , vr2 ) and feta . multilocus sequence typing ( mlst ) is a molecular technique that has been developed and has been increasingly used for menb typing . the loci of each house - keeping gene define the allelic profile or so - called sequence type ( st ) . related sts with identical alleles at four or more loci the combination of antigen sequence typing of pora and feta together with mlst seems to provide a robust framework , which can be complemented by sequence typing of other antigens and measurement of their expression . after implementation of a menb vaccine in a national immunization program , it is important to have a surveillance system that provides complete and accurate data of the circulating menb strains . this is essential to be able to identify antigenic changes that may lead to vaccine failures . n. meningitidis has the capability adapt surface structures to changing environments by a variety of genetic mechanisms . horizontal gene transfer is a common occurrence in the neisseria genus and is responsible for large numbers of genetically heterogeneous menb strains , especially at the omp level whereby a variety of combinations are present . in addition , menb strains could also escape immune responses against vaccine antigens by changing the expression levels of the target antigens . for example , only 50% of invasive meningococcal isolates are known to produce nada , present in 4cmenb , in detectable quantities ( nada expression is phase variable ) with a significant proportion of the nada negative isolates not having the nada gene at all . nhba on the other hand , appears to be present in all isolates tested so far , but protein sequence variability is high . the fhbp shows significant variability resulting in limited cross - protective antibody responses and immune selection under vaccine pressure may occur . therefore , in addition to traditional typing of the serogroup , fine and genetic typing , data are required on the fhbp , nhba and nada genotypes ( dna sequencing ) , and phenotypes of invasive circulating strains for the post - implementation surveillance of the 4cmenb vaccine . the meningococcal antigen typing system ( mats ) was developed to predict 4cmenb strain coverage , using serum bactericidal antibody assay with human complement ( hsba ) taking antigen expression levels and cross protection into account . however , the mats assay is a complicated assay that can not be performed in any reference laboratory and it has been suggested that selected reference laboratories should carry out mats . moreover , the vaccine producer itself developed the assay and is the only producer so far . also , if post - implementation surveillance methods that take into account antigen expression levels will be used , vaccine failures should be carefully defined . as aforementioned vaccine pressure since no other menb vaccine has completed clinical development , this article focuses on clinical data obtained with 4cmenb ( table 2 ) . three large phase 23 randomized controlled clinical studies have been performed to study the immunogenicity and safety of the investigational vaccine , 4cmenb , in adolescents , and infants ( 212 mo ) . the immunogenicity of 4cmenb was determined by measuring serum bactericidal antibody ( sba ) titers against menb reference strains that primarily expressed just one particular vaccine antigen , i.e. , strain 44/76-sl ( matched with the vaccine for fhbp ) , strain 5/99 ( matched with the vaccine for nada ) , and strain nz98/254 , the vaccine strain for the omv component . summarizing , 4cmenb was shown to be safe in adolescents and infants ( primary series and booster dose ) . however , high fever ( 3838.5 c ) rates , up to 80% , were reported in the infant groups , especially when 4cmenb was given concomitantly with routine vaccines . evidence suggests that the rise in body temperature induced by the vaccine can be tempered by prophylactic use of paracetamol without influencing the immunogenicity . in general , good sba titers were found against the selected menb reference strains and no influence of clinical relevance was observed regarding the immune response against routine infant vaccines . the potential of 4cmenb to protect against wild - type circulation strains should be proven after implementation of the vaccine in routine schemes . * routine vaccination : with 7-valent pneumococcal and combined diphtheria , tetanus , acellular pertussis , inactivated polio , hepatitis b , hemophilus influenzae type b dtap - ipv - hepb - hib vaccine . menc , serogroup c conjugate vaccine ; mmrv , measles - mumps - rubella - varicella vaccine . in a recent study , waning of sba titers was observed at 40 mo of age after primary immunization with 4cmenb at 6 , 8 , and 12 mo of age . thus , a robust surveillance program post - implementation is recommended , allowing early recognition of any decline in vaccine effectiveness and the need for a booster vaccination later in childhood or adolescence . asymptomatic nasopharyngeal carriage of n. meningitidis is common , with an average carriage rate of approximately 10% . commensal association of particular strains with a host is a long - term relationship , often lasting several months . in contrast to imd , which is most common in infants and declines through childhood , the prevalence of n. meningitidis carriage is highest among teenagers and low in young children . the carriage rate was shown to be < 3% in children younger than 4 y and increased up to 2437% in the age - group 1524 y , but may differ per country and over the time . apart from age , other risk factors for higher carriage are active and passive smoking , gender ( slightly more male ) , recent respiratory infections , and regular visits to public venues , such as youth clubs and discotheques . asymptomatic infection with or carriage of pathogenic and non - pathogenic strains may help to protect against meningococcal disease . this may explain the higher risk of disease in infants that may have never been a carrier ( naive ) . cellular immunity and cytokine production in relation to meningococcal disease and carriage are poorly understood and deserve more attention . there is evidence that the loss of capsule enhances the capability of meningococci to colonize the human nasopharynx and to avoid human defense systems . capsule production in meningococcal strains can be switched on and off at a high frequency . moreover , meningococci can change from serogroup by capsular switching . in a population - based study , a substantial proportion of invasive serogroup b , c , and y isolates demonstrated capsular switching , indicating that this is a common natural phenomenon . the implementation of menb vaccination in national immunization programs might have an effect on population - level meningococcal carriage state . the highest incidence of imd caused by serogroup b is in the infant age group ( < 1 y ) . therefore , implementation of a new menb vaccine in existing routine infant immunization schedules seems the most logical strategy . however , it is noteworthy that a substantial disease burden occurs in very young infants ( i.e. , those younger than 35 mo of age ) , and these cases will probably not be vaccine - preventable using a 2- , 4- , and 6-mo schedule . results from clinical trials suggest that a 2- , 3- , or 4-mo infant schedule may be acceptable , although further information is needed given the lower immunogenicity that was observed using this schedule . in some countries , this accelerated schedule fits well in the national immunization program , while in other countries the routine infant vaccinations are given at a 2- , 4- , and 6-mo schedule . official recommendations for infants is , according to the product information , three primary doses starting at the age of 2 mo and a booster vaccination between the age of 12 and 23 mo . 4cmenb can be given concomitantly with vaccines against diphtheria , tetanus , acellular pertussis , hemophilus influenza type b , inactivated poliomyelitis , hepatitis b , heptavalent pneumonoccal conjugate , measles , mumps , rubella or varicella . another strategy may be maternal vaccination , however , because young children remain vulnerable for imd in the first years of their life , the infants will not be protected when maternal antibody levels have diminished . probably multiple vaccinations are then still necessary to prevent menb imd during young childhood . after the implementation of the menb vaccine , it is recommended to investigate the influence on meningococcal carriage . so far , three studies have examined the effect of menb omv vaccines on carriage ; in these studies high vaccine coverage had no effect on rates of meningococcal carriage . recently , results were presented of a large study in the uk of nearly 3000 young adults immunized with 4cmenb and/or quadravalent meningococcal a , c , w , y conjugate vaccines examining the effect on meningococcal carriage rates . in this study , prior to vaccination 33% of the samples ( n = 930 ) yielded neisseria cultures , mostly n. meningitides ( 98% ) , mainly of serogroups b and y. primary analysis at one month after the vaccination series did not reveal significant impact of the 4cmenb vaccine , but at later time points 4cmenb was associated with a decrease in carriage of menbcwy strains ( 24.2% ) . these results raise the possibility of an impact on individual carriage , which may lead to greater herd protection in settings where the vaccines are implemented broadly . if immunization with a menb vaccine were to influence nasopharyngeal carriage , a mass immunization campaign of adolescents and young adults , the age of peak nasopharyngeal carriage , may reduce circulation of strains covered by the vaccine leading to reduced rates of disease ( i.e. , herd immunity ) . based on modeling data that have been published regarding the cost - effectiveness of a new menb vaccine it is not expected that the vaccine is cost - effective at present , considering the commonly accepted threshold of 50 000 per qaly . only when the menb incidence will increase considerably or the vaccine price will become very competitive it may become cost - effective . another important influence on cost - effectiveness will be whether the vaccine results in herd effects , by reduction of carriage rate , which is not yet known . the duration of protection and the need for booster doses in childhood will be other key cost considerations . apart from cost - effectiveness , the success of a vaccine program is dependent on public acceptability and feasibility . meningococcal disease is highly feared by the public , which may encourage the uptake of the menb vaccine . on the other hand , parental acceptability may be influenced by vaccine concerns , which include safety , undefined effectiveness , or more practical concerns regarding many vaccines and multiple injections at single visits . with respect to vaccine safety , parents can fear the risk of fever associated with 4cmenb vaccine , when administered to infants . an adequate system of post - implementation surveillance to detect and evaluate ( potentially rare and serious ) adverse effects and will be an essential component of maintaining public confidence . in addition , post - implementation surveillance data should be sufficient to monitor the vaccine effectiveness and to be able to detect possible changes in the clonal , antigenic , and phenotypic profiles of circulating strains under vaccine selection pressure .

effective polysaccharide(conjugate ) vaccines against neisseria meningitidis serogroups a , c , w , and y have been widely used , but serogroup b meningococci remain a major cause of severe invasive meningococcal disease ( imd ) worldwide , especially in infants . recently , a vaccine , 4cmenb ( bexsero ) , containing three recombinant proteins , and outer membrane vesicles ( omv ) derived from a serogroup b meningococcal strain ( menb ) has been licensed in europe and australia and is indicated for persons aged 2 mo or older . this article discusses what should be considered to enable a successful implementation of a broad coverage menb vaccine in national immunization programs . epidemiology data , vaccine characteristics including vaccine coverage , immunogenicity , post - implementation surveillance and costs are relevant aspects that should be taken into account when selecting an appropriate immunization strategy . the potential impact on strain variation and carriage , as well as monitoring vaccine effectiveness , and rare but potentially serious adverse events are points that need to be included in a post - implementation surveillance plan .

Introduction Epidemiology Vaccine development Discussion None Clinical data Carriage Implementation in national immunization programs

invasive meningococcal disease ( imd ) may result in death within 24 h , even with antibiotic treatment . six serogroups of n. meningitidis ( a , b , c , y , w , and more recently x ) are responsible for the majority of imd cases worldwide . the risk of invasive disease is higher for serogroup c and b compared with other serogroups , and is higher for serogroup c than for serogroup b. disease caused by n. meningitidis serogroups ( a , c , y , and w ) is preventable using conjugate vaccines targeting the respective serogroup - specific polysaccharide capsules . , the european commission granted a marketing authorization for the first meningococcal serogroup b ( menb ) vaccine , 4cmenb , a vaccine containing three recombinant proteins , and outer membrane vesicles ( omv ) derived from menb . in august 2013 , this article discusses the different aspects that should be considered to enable a successful implementation of this vaccine in national immunization programs . most of the menb disease is endemic with incidences varying by country over the years ; the highest incidence of imd caused by serogroup b meningococci is in the infant age group ( < 1 y ) ( table 1 ) . the epidemiology data of the us from 19982007 shows that the incidence of imd caused by serogroup b peaked among infants aged 03 mo , whereas menc disease peaked among 45 mo . infants < 1 y accounted for 18% of the cases , children 14 y for 17% , and persons aged 1519 y for 20% of the cases . menb can also cause severe epidemics dominated by one particular strain , which may persist for 10 y or longer , as seen in the past in cuba , brazil , norway , and new zealand . vaccines against menb disease have proved difficult to produce , because the capsular polysaccharide on the serogroup b bacterium is poorly immunogenic as it exhibits structural similarity to human neural ( adhesion ) molecules and is therefore not a useful target . consequently , vaccine developers focused on other outer membrane structures and initially meningococcal outer membrane vesicles ( omv ) were used as basis for the development of several menb vaccines . omv produced from a representative outbreak strain has been shown to be successful in controlling various epidemics of menb disease , such as menzb that was used to control an epidemic in new zealand ( 20046 ) . however , pora is a highly variable , and therefore monovalent strain - specific omv vaccines are not generally useful for prevention of endemic imd caused by diverse strains . in order to obtain broader protection multivalent pora omv vaccines have been developed , such as bivalent , hexavalent , and nonavalent omv vaccine combinations . in addition , omv vaccine formulations based on neisseria lactamica have been designed to provide broad coverage . more recently , native omv vaccines , without the use of detergents , based on genetically detoxified lps have been developed . other approaches that have been applied are vaccines containing multiple recombinant proteins , such as 4cmenb or rlp2086 . several of these vaccines are at stages of clinical development , and in january 2013 , 4cmenb was the first menb vaccine that has been approved by the european medicines agency ( ema ) for use on the european market . 4cmenb is based on novel antigens identified by reverse vaccinology and is composed of factor h binding protein ( fhbp ) , involved in regulation of complement activation , nada , involved in cell adhesion , invasion and induction of cytokines , and nhba , heparin - binding protein . in addition to these proteins , omv from the new zealand epidemic strain ( nz98/254 ; p1.7 - 2,4 , st41/44 ) were added to the formulation as major ( pora ) antigen and for additional potential adjuvant activity besides the alum adjuvant . most of the menb disease is endemic with incidences varying by country over the years ; the highest incidence of imd caused by serogroup b meningococci is in the infant age group ( < 1 y ) ( table 1 ) . the epidemiology data of the us from 19982007 shows that the incidence of imd caused by serogroup b peaked among infants aged 03 mo , whereas menc disease peaked among 45 mo . infants < 1 y accounted for 18% of the cases , children 14 y for 17% , and persons aged 1519 y for 20% of the cases . menb can also cause severe epidemics dominated by one particular strain , which may persist for 10 y or longer , as seen in the past in cuba , brazil , norway , and new zealand . vaccines against menb disease have proved difficult to produce , because the capsular polysaccharide on the serogroup b bacterium is poorly immunogenic as it exhibits structural similarity to human neural ( adhesion ) molecules and is therefore not a useful target . consequently , vaccine developers focused on other outer membrane structures and initially meningococcal outer membrane vesicles ( omv ) were used as basis for the development of several menb vaccines . omv produced from a representative outbreak strain has been shown to be successful in controlling various epidemics of menb disease , such as menzb that was used to control an epidemic in new zealand ( 20046 ) . however , pora is a highly variable , and therefore monovalent strain - specific omv vaccines are not generally useful for prevention of endemic imd caused by diverse strains . in order to obtain broader protection multivalent pora omv vaccines have been developed , such as bivalent , hexavalent , and nonavalent omv vaccine combinations . in addition , omv vaccine formulations based on neisseria lactamica have been designed to provide broad coverage . more recently , native omv vaccines , without the use of detergents , based on genetically detoxified lps have been developed . other approaches that have been applied are vaccines containing multiple recombinant proteins , such as 4cmenb or rlp2086 . several of these vaccines are at stages of clinical development , and in january 2013 , 4cmenb was the first menb vaccine that has been approved by the european medicines agency ( ema ) for use on the european market . 4cmenb is based on novel antigens identified by reverse vaccinology and is composed of factor h binding protein ( fhbp ) , involved in regulation of complement activation , nada , involved in cell adhesion , invasion and induction of cytokines , and nhba , heparin - binding protein . nhba and fhbp were fused to gna1030 ( unknown function ) and gna2091 ( unknown function ) , respectively , to enhance protein stability and immunogenicity . in addition to these proteins , omv from the new zealand epidemic strain ( nz98/254 ; p1.7 - 2,4 , st41/44 ) were added to the formulation as major ( pora ) antigen and for additional potential adjuvant activity besides the alum adjuvant . after implementation of a menb vaccine in a national immunization program , it is important to have a surveillance system that provides complete and accurate data of the circulating menb strains . horizontal gene transfer is a common occurrence in the neisseria genus and is responsible for large numbers of genetically heterogeneous menb strains , especially at the omp level whereby a variety of combinations are present . nhba on the other hand , appears to be present in all isolates tested so far , but protein sequence variability is high . therefore , in addition to traditional typing of the serogroup , fine and genetic typing , data are required on the fhbp , nhba and nada genotypes ( dna sequencing ) , and phenotypes of invasive circulating strains for the post - implementation surveillance of the 4cmenb vaccine . the meningococcal antigen typing system ( mats ) was developed to predict 4cmenb strain coverage , using serum bactericidal antibody assay with human complement ( hsba ) taking antigen expression levels and cross protection into account . also , if post - implementation surveillance methods that take into account antigen expression levels will be used , vaccine failures should be carefully defined . as aforementioned vaccine pressure since no other menb vaccine has completed clinical development , this article focuses on clinical data obtained with 4cmenb ( table 2 ) . three large phase 23 randomized controlled clinical studies have been performed to study the immunogenicity and safety of the investigational vaccine , 4cmenb , in adolescents , and infants ( 212 mo ) . , strain 44/76-sl ( matched with the vaccine for fhbp ) , strain 5/99 ( matched with the vaccine for nada ) , and strain nz98/254 , the vaccine strain for the omv component . summarizing , 4cmenb was shown to be safe in adolescents and infants ( primary series and booster dose ) . the potential of 4cmenb to protect against wild - type circulation strains should be proven after implementation of the vaccine in routine schemes . in a recent study , waning of sba titers was observed at 40 mo of age after primary immunization with 4cmenb at 6 , 8 , and 12 mo of age . thus , a robust surveillance program post - implementation is recommended , allowing early recognition of any decline in vaccine effectiveness and the need for a booster vaccination later in childhood or adolescence . the carriage rate was shown to be < 3% in children younger than 4 y and increased up to 2437% in the age - group 1524 y , but may differ per country and over the time . apart from age , other risk factors for higher carriage are active and passive smoking , gender ( slightly more male ) , recent respiratory infections , and regular visits to public venues , such as youth clubs and discotheques . asymptomatic infection with or carriage of pathogenic and non - pathogenic strains may help to protect against meningococcal disease . this may explain the higher risk of disease in infants that may have never been a carrier ( naive ) . cellular immunity and cytokine production in relation to meningococcal disease and carriage are poorly understood and deserve more attention . in a population - based study , a substantial proportion of invasive serogroup b , c , and y isolates demonstrated capsular switching , indicating that this is a common natural phenomenon . the implementation of menb vaccination in national immunization programs might have an effect on population - level meningococcal carriage state . therefore , implementation of a new menb vaccine in existing routine infant immunization schedules seems the most logical strategy . , those younger than 35 mo of age ) , and these cases will probably not be vaccine - preventable using a 2- , 4- , and 6-mo schedule . in some countries , this accelerated schedule fits well in the national immunization program , while in other countries the routine infant vaccinations are given at a 2- , 4- , and 6-mo schedule . after the implementation of the menb vaccine , it is recommended to investigate the influence on meningococcal carriage . recently , results were presented of a large study in the uk of nearly 3000 young adults immunized with 4cmenb and/or quadravalent meningococcal a , c , w , y conjugate vaccines examining the effect on meningococcal carriage rates . in this study , prior to vaccination 33% of the samples ( n = 930 ) yielded neisseria cultures , mostly n. meningitides ( 98% ) , mainly of serogroups b and y. primary analysis at one month after the vaccination series did not reveal significant impact of the 4cmenb vaccine , but at later time points 4cmenb was associated with a decrease in carriage of menbcwy strains ( 24.2% ) . these results raise the possibility of an impact on individual carriage , which may lead to greater herd protection in settings where the vaccines are implemented broadly . if immunization with a menb vaccine were to influence nasopharyngeal carriage , a mass immunization campaign of adolescents and young adults , the age of peak nasopharyngeal carriage , may reduce circulation of strains covered by the vaccine leading to reduced rates of disease ( i.e. based on modeling data that have been published regarding the cost - effectiveness of a new menb vaccine it is not expected that the vaccine is cost - effective at present , considering the commonly accepted threshold of 50 000 per qaly . apart from cost - effectiveness , the success of a vaccine program is dependent on public acceptability and feasibility . meningococcal disease is highly feared by the public , which may encourage the uptake of the menb vaccine . an adequate system of post - implementation surveillance to detect and evaluate ( potentially rare and serious ) adverse effects and will be an essential component of maintaining public confidence . in addition , post - implementation surveillance data should be sufficient to monitor the vaccine effectiveness and to be able to detect possible changes in the clonal , antigenic , and phenotypic profiles of circulating strains under vaccine selection pressure . multilocus sequence typing ( mlst ) is a molecular technique that has been developed and has been increasingly used for menb typing . after implementation of a menb vaccine in a national immunization program , it is important to have a surveillance system that provides complete and accurate data of the circulating menb strains . horizontal gene transfer is a common occurrence in the neisseria genus and is responsible for large numbers of genetically heterogeneous menb strains , especially at the omp level whereby a variety of combinations are present . for example , only 50% of invasive meningococcal isolates are known to produce nada , present in 4cmenb , in detectable quantities ( nada expression is phase variable ) with a significant proportion of the nada negative isolates not having the nada gene at all . nhba on the other hand , appears to be present in all isolates tested so far , but protein sequence variability is high . therefore , in addition to traditional typing of the serogroup , fine and genetic typing , data are required on the fhbp , nhba and nada genotypes ( dna sequencing ) , and phenotypes of invasive circulating strains for the post - implementation surveillance of the 4cmenb vaccine . the meningococcal antigen typing system ( mats ) was developed to predict 4cmenb strain coverage , using serum bactericidal antibody assay with human complement ( hsba ) taking antigen expression levels and cross protection into account . also , if post - implementation surveillance methods that take into account antigen expression levels will be used , vaccine failures should be carefully defined . as aforementioned vaccine pressure since no other menb vaccine has completed clinical development , this article focuses on clinical data obtained with 4cmenb ( table 2 ) . three large phase 23 randomized controlled clinical studies have been performed to study the immunogenicity and safety of the investigational vaccine , 4cmenb , in adolescents , and infants ( 212 mo ) . , strain 44/76-sl ( matched with the vaccine for fhbp ) , strain 5/99 ( matched with the vaccine for nada ) , and strain nz98/254 , the vaccine strain for the omv component . summarizing , 4cmenb was shown to be safe in adolescents and infants ( primary series and booster dose ) . the potential of 4cmenb to protect against wild - type circulation strains should be proven after implementation of the vaccine in routine schemes . in a recent study , waning of sba titers was observed at 40 mo of age after primary immunization with 4cmenb at 6 , 8 , and 12 mo of age . thus , a robust surveillance program post - implementation is recommended , allowing early recognition of any decline in vaccine effectiveness and the need for a booster vaccination later in childhood or adolescence . the carriage rate was shown to be < 3% in children younger than 4 y and increased up to 2437% in the age - group 1524 y , but may differ per country and over the time . apart from age , other risk factors for higher carriage are active and passive smoking , gender ( slightly more male ) , recent respiratory infections , and regular visits to public venues , such as youth clubs and discotheques . this may explain the higher risk of disease in infants that may have never been a carrier ( naive ) . cellular immunity and cytokine production in relation to meningococcal disease and carriage are poorly understood and deserve more attention . in a population - based study , a substantial proportion of invasive serogroup b , c , and y isolates demonstrated capsular switching , indicating that this is a common natural phenomenon . the implementation of menb vaccination in national immunization programs might have an effect on population - level meningococcal carriage state . therefore , implementation of a new menb vaccine in existing routine infant immunization schedules seems the most logical strategy . , those younger than 35 mo of age ) , and these cases will probably not be vaccine - preventable using a 2- , 4- , and 6-mo schedule . in some countries , this accelerated schedule fits well in the national immunization program , while in other countries the routine infant vaccinations are given at a 2- , 4- , and 6-mo schedule . after the implementation of the menb vaccine , it is recommended to investigate the influence on meningococcal carriage . so far , three studies have examined the effect of menb omv vaccines on carriage ; in these studies high vaccine coverage had no effect on rates of meningococcal carriage . recently , results were presented of a large study in the uk of nearly 3000 young adults immunized with 4cmenb and/or quadravalent meningococcal a , c , w , y conjugate vaccines examining the effect on meningococcal carriage rates . in this study , prior to vaccination 33% of the samples ( n = 930 ) yielded neisseria cultures , mostly n. meningitides ( 98% ) , mainly of serogroups b and y. primary analysis at one month after the vaccination series did not reveal significant impact of the 4cmenb vaccine , but at later time points 4cmenb was associated with a decrease in carriage of menbcwy strains ( 24.2% ) . these results raise the possibility of an impact on individual carriage , which may lead to greater herd protection in settings where the vaccines are implemented broadly . if immunization with a menb vaccine were to influence nasopharyngeal carriage , a mass immunization campaign of adolescents and young adults , the age of peak nasopharyngeal carriage , may reduce circulation of strains covered by the vaccine leading to reduced rates of disease ( i.e. based on modeling data that have been published regarding the cost - effectiveness of a new menb vaccine it is not expected that the vaccine is cost - effective at present , considering the commonly accepted threshold of 50 000 per qaly . apart from cost - effectiveness , the success of a vaccine program is dependent on public acceptability and feasibility . meningococcal disease is highly feared by the public , which may encourage the uptake of the menb vaccine . an adequate system of post - implementation surveillance to detect and evaluate ( potentially rare and serious ) adverse effects and will be an essential component of maintaining public confidence . in addition , post - implementation surveillance data should be sufficient to monitor the vaccine effectiveness and to be able to detect possible changes in the clonal , antigenic , and phenotypic profiles of circulating strains under vaccine selection pressure .

poor sleep is ubiquitous , compromising alertness and psychological health . the international prevalence of poor sleep is estimated to range from 17% to 26%13 and is associated with depression using different study designs and populations.1,37 however one example of a subjective sleep instrument is the pittsburgh sleep quality index ( psqi).8 rather than self - reports , objective sleep parameters rely on monitoring of sleep wake activity using electronic instrumentation . one example of an objective sleep instrument is wrist actigraphy , which estimates sleep wake activity based on motor activity.9 a number of studies have shown that results from subjective and objective sleep measures differ . these studies have included healthy men and women,10,11 the depressed,1214 pregnant women,15,16 women with severe premenstrual syndrome,17 the elderly,18 and caregivers.19 understanding the differences between subjective and objective sleep measures among caregivers is important because these populations are often at high risk for poor sleep and other comorbidities , which can have deleterious consequences on both the caregiver and care recipient.2024 caregivers report poorer sleep outcomes , greater anxiety , and greater depression when compared to noncaregivers,19,2528 and caregiver focus groups report that poor sleep has had negative impacts on health.20,29 caregivers of children with chronic health conditions show greater depressive symptoms compared to caregivers of children without chronic health conditions.26 in addition , a graded relationship between care - giving burden and depression was observed when comparing caregivers of children with both neurodevelopmental and behavioral disorders to caregivers of children with either one or none of these disorders.30 therefore , we determined whether self - reported ( subjective ) sleep and wrist actigraphy ( objective ) sleep traits were each associated with depression and investigated the extent to which these associations differed among a sample of female caregivers of children with disabilities . the chile pediatric and adult sleep study ( cpass ) is a cross - sectional study conducted at the centro de rehabilitacin club de leones cruz del sur in punta arenas , chile that was designed to investigate the relationship between the sleep quality of children with disabilities and that of their primary caregivers with cardiometabolic and psychological health . this study was conducted among female primary caregivers of children with disabilities enrolled between september 2012 and july 2013 in two waves . the first wave ( cpass i ) was conducted between september 2012 and december 2012 among caregivers of children aged 612 years receiving routine clinical care for disabilities at the center . the second wave ( cpass ii ) was conducted between april 2013 and july 2013 among caregivers of children with disabilities aged 1021 years at the center . both cpass - i and cpass - ii study protocols have been described in further detail elsewhere.31,32 the primary caregiver was defined as an adult for whom the child s well - being was her principal responsibility . primary caregivers included mothers , grandmothers , and relatives who were not diagnosed with developmental or intellectual disabilities . research personnel invited 239 women , and from this group , 175 agreed to participate ( 73.2% ) , each in the care of one child . after obtaining written informed consent , a research psychologist administered structured questionnaires to women in order to collect information on sociodemographic and lifestyle factors and sleep characteristics . following the structured interview , research staff instructed women to wear actisleep monitors ( actilife ; actigraph r&d , pensacola , fl , usa ) on nondominant wrists for seven consecutive nights . although monitors were waterproof , women were instructed to remove them before showering and swimming . subjective sleep quality was assessed using psqi , which measures sleep quality retrospectively over a 1-month period . the psqi is a validated instrument for sleep research8,33 and consists of 24 questions that generate seven component scores , each with a scale of 03 ; higher scores indicate poorer sleep outcomes . component scores were then totaled for a global psqi score ranging from 0 to 21 , with higher scores indicating worse overall sleep quality . a global psqi score > 5 has been shown to yield a sensitivity of 89.6% and a specificity of 86.5% in distinguishing good ( score 5 ) and poor sleepers ( score>5).8 our analysis explored the following psqi component scores : total time in bed ( hours ) , sleep latency ( minutes ) , sleep duration ( hours ) , and sleep efficiency ( % ) . objective sleep quality and disturbance were assessed with wrist actigraphy , an activity - based monitor to assess sleep wake patterns , which has been shown to be valid in healthy and sleep - disordered populations.3437 actigraphy data were collected using actisleep wrist - monitors that utilized actilife 6 data analysis software . actigraphy - measured sleep data were collected in 1-minute epochs using the zero - crossing modes and the cole - kripke algorithm.9 caregivers wore actisleep monitors for seven consecutive nights on non - dominant wrists . reported actigraphy - measured parameters include total time in bed ( hours ) , sleep latency ( minutes ) , sleep duration ( hours ) , sleep efficiency ( % ) , wake after sleep onset ( waso ; minutes ) ; average awakening length ( minutes ) , and number of awakenings . actigraphy wrist - monitors were inspected and cleaned prior to distribution to ensure that devices were in working condition , and visual inspection of data was performed regularly . depression was assessed using the eight - item patient health questionnaire ( phq-8 ) , which uses a self - administered module for unipolar depression that asks the participant over the last 2 weeks , how often have you been bothered by the following problems ? we did not include the ninth question from the nine - item patient health questionnaire , which assesses for suicidal thoughts , because of the impracticality of timely clinical follow - up of positive responses . also , in other studies , the modification of the nine - item patient health questionnaire to the phq-8 depression questionnaire has been demonstrated to minimally influence overall scale performance , mean scores , or diagnostic cut points.38,39 the eight items of the phq-8 include feigned interest , hopelessness , trouble sleeping , lack of energy , changes in appetite , self - deprecation , trouble concentrating , and changes in physical behavior . the participant s endorsement of each item was ranked on a scale from 0 to 3 : not at all ( 0 ) , several days ( 1 ) , more than half of the days ( 2 ) , and nearly every day ( 3 ) . response to each item was summed for a total phq-8 score ranging from 0 to 24 . validated cutoffs were used to categorize patients into either depressed ( phq-8 score 10 ) or non - depressed ( phq-8 score < 10 ) categories for comparison.38 using the diagnostic statistical manual , fourth edition , a score 10 has been shown to be associated with 88% sensitivity and 88% specificity in diagnosing major depressive disorder38 and validated for use in spanish - speaking populations.40,41 structured interviewer - administered questionnaires collected data on caregivers age , body mass index ( bmi ) , ethnicity , marital status , current smoking status , alcohol consumption , employment status , and education level . bmi was calculated from the caregivers weight measured to the nearest 0.1 kg , and height was measured with a telescopic height instrument to the nearest 0.1 cm . according to the international classification of diseases , tenth revision,42 children s disabilities were grouped as follows : 1 ) mental and behavioral disorders ( eg , autism ) ; 2 ) diseases of the musculoskeletal system and connective tissue , skin , and subcutaneous system ( eg , scoliosis ) ; 3 ) diseases of the nervous system ( eg , cerebral palsy ) ; and 4 ) congenital malformations and chromosomal abnormalities ( eg , down syndrome ) . the fisher s exact and chi - square tests were used to evaluate differences in sociodemographic and lifestyle characteristics by caregiver depression status ( phq-8 score of 10 vs phq-8 score < 10 ) . differences in sleep between depressed women and nondepressed women were analyzed for both subjective and objective sleep parameters using either student s t - test or kruskal wallis test . among depressed and non - depressed groups , overlapping objective and subjective sleep parameters multivariable models were adjusted for bmi ( continuous ) , marital status ( married or living with a partner vs divorced / separated / widowed / never married ) , current smoking status ( yes or no ) , education level ( < high school , high school , or > high school ) , and children s disability group ( mental and behavioral disorders , diseases of musculoskeletal system and connective / skin tissue , diseases of the nervous system , or congenital malformations and chromosomal abnormalities ) . a sensitivity analysis excluding the trouble sleeping item from the phq-8 was performed for the association between subjective sleep and depression . statistical significance levels were set at a p - value < 0.05 for two - sided analysis , and where appropriate , sds , regression coefficients , and standard errors ( ses ) were reported . chan school of public health and the centro de rehabilitacin club de leones cruz del sur in punta arenas , chile . the chile pediatric and adult sleep study ( cpass ) is a cross - sectional study conducted at the centro de rehabilitacin club de leones cruz del sur in punta arenas , chile that was designed to investigate the relationship between the sleep quality of children with disabilities and that of their primary caregivers with cardiometabolic and psychological health . this study was conducted among female primary caregivers of children with disabilities enrolled between september 2012 and july 2013 in two waves . the first wave ( cpass i ) was conducted between september 2012 and december 2012 among caregivers of children aged 612 years receiving routine clinical care for disabilities at the center . the second wave ( cpass ii ) was conducted between april 2013 and july 2013 among caregivers of children with disabilities aged 1021 years at the center . both cpass - i and cpass - ii study protocols have been described in further detail elsewhere.31,32 the primary caregiver was defined as an adult for whom the child s well - being was her principal responsibility . primary caregivers included mothers , grandmothers , and relatives who were not diagnosed with developmental or intellectual disabilities . research personnel invited 239 women , and from this group , 175 agreed to participate ( 73.2% ) , each in the care of one child . after obtaining written informed consent , a research psychologist administered structured questionnaires to women in order to collect information on sociodemographic and lifestyle factors and sleep characteristics . following the structured interview , research staff instructed women to wear actisleep monitors ( actilife ; actigraph r&d , pensacola , fl , usa ) on nondominant wrists for seven consecutive nights . although monitors were waterproof , women were instructed to remove them before showering and swimming . subjective sleep quality was assessed using psqi , which measures sleep quality retrospectively over a 1-month period . the psqi is a validated instrument for sleep research8,33 and consists of 24 questions that generate seven component scores , each with a scale of 03 ; higher scores indicate poorer sleep outcomes . component scores were then totaled for a global psqi score ranging from 0 to 21 , with higher scores indicating worse overall sleep quality . a global psqi score > 5 has been shown to yield a sensitivity of 89.6% and a specificity of 86.5% in distinguishing good ( score 5 ) and poor sleepers ( score>5).8 our analysis explored the following psqi component scores : total time in bed ( hours ) , sleep latency ( minutes ) , sleep duration ( hours ) , and sleep efficiency ( % ) . objective sleep quality and disturbance were assessed with wrist actigraphy , an activity - based monitor to assess sleep wake patterns , which has been shown to be valid in healthy and sleep - disordered populations.3437 actigraphy data were collected using actisleep wrist - monitors that utilized actilife 6 data analysis software . actigraphy - measured sleep data were collected in 1-minute epochs using the zero - crossing modes and the cole - kripke algorithm.9 caregivers wore actisleep monitors for seven consecutive nights on non - dominant wrists . reported actigraphy - measured parameters include total time in bed ( hours ) , sleep latency ( minutes ) , sleep duration ( hours ) , sleep efficiency ( % ) , wake after sleep onset ( waso ; minutes ) ; average awakening length ( minutes ) , and number of awakenings . actigraphy wrist - monitors were inspected and cleaned prior to distribution to ensure that devices were in working condition , and visual inspection of data was performed regularly . depression was assessed using the eight - item patient health questionnaire ( phq-8 ) , which uses a self - administered module for unipolar depression that asks the participant over the last 2 weeks , how often have you been bothered by the following problems ? we did not include the ninth question from the nine - item patient health questionnaire , which assesses for suicidal thoughts , because of the impracticality of timely clinical follow - up of positive responses . also , in other studies , the modification of the nine - item patient health questionnaire to the phq-8 depression questionnaire has been demonstrated to minimally influence overall scale performance , mean scores , or diagnostic cut points.38,39 the eight items of the phq-8 include feigned interest , hopelessness , trouble sleeping , lack of energy , changes in appetite , self - deprecation , trouble concentrating , and changes in physical behavior . the participant s endorsement of each item was ranked on a scale from 0 to 3 : not at all ( 0 ) , several days ( 1 ) , more than half of the days ( 2 ) , and nearly every day ( 3 ) . response to each item was summed for a total phq-8 score ranging from 0 to 24 . validated cutoffs were used to categorize patients into either depressed ( phq-8 score 10 ) or non - depressed ( phq-8 score < 10 ) categories for comparison.38 using the diagnostic statistical manual , fourth edition , a score 10 has been shown to be associated with 88% sensitivity and 88% specificity in diagnosing major depressive disorder38 and validated for use in spanish - speaking populations.40,41 structured interviewer - administered questionnaires collected data on caregivers age , body mass index ( bmi ) , ethnicity , marital status , current smoking status , alcohol consumption , employment status , and education level . bmi was calculated from the caregivers weight measured to the nearest 0.1 kg , and height was measured with a telescopic height instrument to the nearest 0.1 cm . according to the international classification of diseases , tenth revision,42 children s disabilities were grouped as follows : 1 ) mental and behavioral disorders ( eg , autism ) ; 2 ) diseases of the musculoskeletal system and connective tissue , skin , and subcutaneous system ( eg , scoliosis ) ; 3 ) diseases of the nervous system ( eg , cerebral palsy ) ; and 4 ) congenital malformations and chromosomal abnormalities ( eg , down syndrome ) . subjective sleep quality was assessed using psqi , which measures sleep quality retrospectively over a 1-month period . the psqi is a validated instrument for sleep research8,33 and consists of 24 questions that generate seven component scores , each with a scale of 03 ; higher scores indicate poorer sleep outcomes . component scores were then totaled for a global psqi score ranging from 0 to 21 , with higher scores indicating worse overall sleep quality . a global psqi score > 5 has been shown to yield a sensitivity of 89.6% and a specificity of 86.5% in distinguishing good ( score 5 ) and poor sleepers ( score>5).8 our analysis explored the following psqi component scores : total time in bed ( hours ) , sleep latency ( minutes ) , sleep duration ( hours ) , and sleep efficiency ( % ) . objective sleep quality and disturbance were assessed with wrist actigraphy , an activity - based monitor to assess sleep wake patterns , which has been shown to be valid in healthy and sleep - disordered populations.3437 actigraphy data were collected using actisleep wrist - monitors that utilized actilife 6 data analysis software . actigraphy - measured sleep data were collected in 1-minute epochs using the zero - crossing modes and the cole - kripke algorithm.9 caregivers wore actisleep monitors for seven consecutive nights on non - dominant wrists . reported actigraphy - measured parameters include total time in bed ( hours ) , sleep latency ( minutes ) , sleep duration ( hours ) , sleep efficiency ( % ) , wake after sleep onset ( waso ; minutes ) ; average awakening length ( minutes ) , and number of awakenings . actigraphy wrist - monitors were inspected and cleaned prior to distribution to ensure that devices were in working condition , and visual inspection of data was performed regularly . depression was assessed using the eight - item patient health questionnaire ( phq-8 ) , which uses a self - administered module for unipolar depression that asks the participant over the last 2 weeks , how often have you been bothered by the following problems ? we did not include the ninth question from the nine - item patient health questionnaire , which assesses for suicidal thoughts , because of the impracticality of timely clinical follow - up of positive responses . also , in other studies , the modification of the nine - item patient health questionnaire to the phq-8 depression questionnaire has been demonstrated to minimally influence overall scale performance , mean scores , or diagnostic cut points.38,39 the eight items of the phq-8 include feigned interest , hopelessness , trouble sleeping , lack of energy , changes in appetite , self - deprecation , trouble concentrating , and changes in physical behavior . the participant s endorsement of each item was ranked on a scale from 0 to 3 : not at all ( 0 ) , several days ( 1 ) , more than half of the days ( 2 ) , and nearly every day ( 3 ) . response to each item was summed for a total phq-8 score ranging from 0 to 24 . validated cutoffs were used to categorize patients into either depressed ( phq-8 score 10 ) or non - depressed ( phq-8 score < 10 ) categories for comparison.38 using the diagnostic statistical manual , fourth edition , a score 10 has been shown to be associated with 88% sensitivity and 88% specificity in diagnosing major depressive disorder38 and validated for use in spanish - speaking populations.40,41 structured interviewer - administered questionnaires collected data on caregivers age , body mass index ( bmi ) , ethnicity , marital status , current smoking status , alcohol consumption , employment status , and education level . bmi was calculated from the caregivers weight measured to the nearest 0.1 kg , and height was measured with a telescopic height instrument to the nearest 0.1 cm . according to the international classification of diseases , tenth revision,42 children s disabilities were grouped as follows : 1 ) mental and behavioral disorders ( eg , autism ) ; 2 ) diseases of the musculoskeletal system and connective tissue , skin , and subcutaneous system ( eg , scoliosis ) ; 3 ) diseases of the nervous system ( eg , cerebral palsy ) ; and 4 ) congenital malformations and chromosomal abnormalities ( eg , down syndrome ) . the fisher s exact and chi - square tests were used to evaluate differences in sociodemographic and lifestyle characteristics by caregiver depression status ( phq-8 score of 10 vs phq-8 score < 10 ) . differences in sleep between depressed women and nondepressed women were analyzed for both subjective and objective sleep parameters using either student s t - test or kruskal wallis test . among depressed and non - depressed groups , overlapping objective and subjective sleep parameters were compared using either paired t - tests or signed tests for matched pairs . multivariable models were adjusted for bmi ( continuous ) , marital status ( married or living with a partner vs divorced / separated / widowed / never married ) , current smoking status ( yes or no ) , education level ( < high school , high school , or > high school ) , and children s disability group ( mental and behavioral disorders , diseases of musculoskeletal system and connective / skin tissue , diseases of the nervous system , or congenital malformations and chromosomal abnormalities ) . a sensitivity analysis excluding the trouble sleeping item from the phq-8 was performed for the association between subjective sleep and depression . statistical significance levels were set at a p - value < 0.05 for two - sided analysis , and where appropriate , sds , regression coefficients , and standard errors ( ses ) were reported . all statistical analyses were performed using sas version 9.3 ( sas institute inc . , cary , nc , usa ) . chan school of public health and the centro de rehabilitacin club de leones cruz del sur in punta arenas , chile . our sample consisted of 175 female primary caregivers with a mean age of 40.6 years ( sd = 8.7 ) . sociodemographic characteristics of study participants are summarized in table 1 . using a phq-8 score 10 , 26.3% of participants presented with depression . when compared with nondepressed women , depressed women had higher mean bmi ( 31.0 kg / m vs 29.0 kg / m ; p=0.037 ) , were less likely to report being married or living with a partner ( 50.0% vs 72.1% ; p=0.007 ) , were more likely to report being a current smoker ( 63.0% vs 41.1% ; p=0.010 ) , and were less likely to report a completed education beyond high school ( 17.4% vs 37.2% ; p=0.013 ) . the majority of women cared for children with mental and behavioral disorders ( 42.9% ) , followed by children with nervous tissue diseases ( 26.9% ) , musculoskeletal and connective tissue diseases ( 18.3% ) , and congenital and chromosomal abnormalities ( 12.0% ) . however , the children s disability group did not statistically differ by caregivers depression status ( p=0.713 ) . other characteristics , including age , ethnicity , alcohol consumption , and employment status , did not statistically differ by depression status . table 2 shows differences in subjective and objective sleep parameters comparing depressed and nondepressed women as well as univariate and multivariable regression coefficients for the association between sleep and depression . compared with nondepressed women , depressed women self - reported shorter total time in bed ( 6.6 hours vs 7.3 hours ; p=0.014 ) , longer sleep latency ( 60.0 minutes vs 20.0 minutes ; p<0.001 ) , shorter sleep duration ( 5.8 hours vs 6.6 hours ; p=0.001 ) , and poorer overall sleep quality using the median global psqi score ( 11.0 vs 6.0 ; p<0.001 ) . no statistically significant differences were observed using subjective sleep efficiency ( 88.2% vs 91.7% ; p=0.570 ) . similar to subjective sleep duration , objective sleep duration differed comparing depressed and nondepressed women ( 6.4 hours vs 6.9 hours ; p=0.005 ) . depressed women did not statistically differ from non - depressed women based on objective total time in bed ( 7.7 hours vs 8.1 hours ; p=0.076 ) , objective sleep latency ( 7.2 minutes vs 11.1 minutes ; p=0.453 ) , objective sleep efficiency ( 86.2% vs 87.2% ; p=0.191 ) , objective waso ( 54.6 minutes vs 50.9 minutes ; p=0.361 ) , objective number of awakenings ( 14.4 vs 13.6 ; p=0.565 ) , and objective average awakening length ( 4.7 minutes vs 4.7 minutes ; p=0.921 ) . table 2 also shows differences between subjective and objective sleep parameters within depression status groups . among the depressed group , the mean subjective total time in bed ( 6.6 hours ) was lower than the mean objective total time in bed ( 7.7 hours ) and the difference statistically significant ( 1.1 hours , 95% confidence interval [ ci ] : 1.5 to 0.7 ; p<0.001 ) . among the nondepressed , the mean subjective total time in bed ( 7.3 hours ) was lower than the mean objective total time in bed ( 8.1 hours ) and the difference statistically significant ( 0.8 hours , 95% ci : 1.1 to 0.5 ; p<0.001 ) . among the depressed group , the mean subjective sleep duration ( 5.8 hours ) was lower than the mean objective sleep duration ( 6.4 hours ) and the difference statistically significant ( 0.6 hours , 95% ci : 1.0 to 0.2 ; p=0.005 ) . among the nondepressed , the mean subjective sleep duration ( 6.6 hours ) was lower than the mean objective sleep duration ( 6.9 hours ) ; however , the difference was not statistically significant ( 0.3 hours , 95% ci : 0.54 to 0.01 ; p=0.059 ) . among the depressed , the median subjective sleep latency ( 60.0 minutes ) was higher than the median objective sleep latency ( 7.2 minutes ) and difference statistically significant ( p<0.001 ) . among the nondepressed , the median subjective sleep latency ( 20.0 minutes ) was higher than the median objective sleep latency ( 11.1 minutes ) and the difference statistically significant ( p<0.001 ) . among the depressed group , the median subjective sleep efficiency ( 88.2% ) was higher than the median objective sleep efficiency ( 86.2% ) ; however , the difference was not statistically significant ( p=0.451 ) . among the nondepressed group , the median subjective sleep efficiency ( 91.7% ) was higher than the median objective sleep efficiency ( 87.2% ) and the difference was statistically significant ( p=0.019 ) . all multivariable models were adjusted for bmi ( continuous ) , marital status , current smoking status , education level , and children s disability group . subjective multivariable models show depression to be negatively associated with total time in bed ( =0.61 , se = 0.29 ; p=0.033 ) and sleep duration ( =0.71 , se = 0.25 ; p=0.006 ) and positively associated with subjective sleep latency ( =0.58 , se = 0.20 ; p=0.003 ) and subjective overall poor sleep quality using the global psqi score ( =0.53 , se = 0.08 ; p<0.001 ) . depression was also negatively associated with objective sleep duration ( =0.42 , se = 0.19 ; p=0.026 ) . no statistically significant associations were found for subjective sleep efficiency ( =0.01 , se = 0.03 ; p=0.743 ) , objective total time in bed ( =0.32 , se = 0.23 ; p=0.174 ) , objective sleep latency ( =0.19 , se = 0.23 ; p=0.408 ) , objective sleep efficiency ( =0.02 , se = 0.01 ; p=0.210 ) , objective waso ( =0.11 , se = 0.10 ; p=0.274 ) , objective average awakening length ( =0.06 , se = 0.09 ; p=0.531 ) , and objective number of awakenings ( =0.08 , se = 0.08 ; p=0.366 ) . table 3 shows the results from the sensitivity analyses that excluded the trouble sleeping item from the phq-8 . results from the sensitivity analyses did not substantially change the subjective associations previously reported in table 2 . notably , subjective sleep latency was no longer statistically significant in multivariable models ( = 0.30 , se = 0.22 ; p=0.184 ) . our study found an association between some sleep traits and depression and a difference in the extent of the association when comparing subjective and objective sleep measures . our study also observed an overall underestimation of total time in bed and sleep duration , although only the underestimation among the depressed group was statistically significant . we also report a robust negative association between depression and sleep duration using both subjective and objective sleep measures , and that the magnitude of association was strongest using subjective sleep measures . in addition , we found that the prevalence of depression among caregivers in our study ( 26.3% ) was within the range of depression estimates reported in other studies of caregivers for children with disabilities ( 15%32%).30,43 our finding of an association between some sleep traits and depression corroborates with the literature relating to this topic.1,37 our finding of differences comparing subjective and objective sleep measures is also supported by much of the literature,1019 despite limited findings to the contrary.44 among studies that have looked at such differences among the depressed patients , they have reported that depressed patients are more likely to poorly recall aspects of sleep compared to the nondepressed patients.1214 however , the direction of the inconsistency is mixed . for example , although we observed that depressed women significantly underreported sleep duration while nondepressed women did not , others have reported that depressed individuals may in fact be more likely to over report sleep duration.12 finally , our finding of differences in the association between sleep and depression comparing subjective and objective sleep measures has also been previously reported . for example , in a recent study of pregnant women with and without a confirmed mental disorder , daily reported ( subjective ) sleep variables showed significant associations with patient status in multivariable models , while wrist actigraphy ( objective ) sleep did not.16 results from a recent meta - analysis reported that when compared to normal sleep duration , short sleep duration is significantly associated with depression in adults ( relative risk = 1.31 , 95% ci : 1.04 to 1.64).4 in our study , a robust negative association between depression and sleep duration was found using both subjective and objective sleep measures . similar associations between mental health outcomes and sleep duration have been observed in other studies of caregivers.19,25 for example , in a sample of male and female caregivers , sleep duration was significantly associated with higher levels of depression using the hospital anxiety and depression scale.25 it is important to note that the association between sleep and depression is multifaceted and is impacted not only by changes in sleep features , such as shortened sleep duration , but also by changes in social and environmental factors.45,46 interestingly , in a study among young adult women , perceived stress was associated with poorer subjective sleep quality but not with actigraphic ( objective ) sleep measures.47 although perceived ( subjective ) and actual ( objective ) sleep measures share commonalities , it is not surprising that the two differ . buysse et al48 and vitiello et al49 have previously demonstrated that individuals adapt perception of acceptable sleep . for example , in a study of healthy older individuals , although participants reported no perceptions of poor sleep using subjective sleep measures , objective sleep measures showed significant sleep disturbances.49 in our case , the opposite may be occurring . depressed individuals may have had altered perceptions of acceptable sleep in that they perceived and self - reported sleep disturbances , despite few observed differences from nondepressed individuals using objective sleep measures . it may also be the case that perception of sleep among individuals with a comorbid mood disorder is a function of cognition , severity of depression , and actual sleep , rather than actual sleep alone , as suggested in previous studies.13,14 furthermore , shorter sleep duration interferes with memory consolidation,50 which may influence perception of sleep.51 perceptions of poor sleep may then go on to influence sleep - disruptive compensatory behaviors , such as increased anxiety during times of rest,52 thereby perpetuating poor sleep among individuals who perceive that they sleep poorly . our study had several strengths including the use of validated sleep and depression instruments and analytical handling of potential confounders . this exclusion criterion reduced the potential confounding influence that caregivers disability status might have had on both sleep and depression . second , we adjusted for covariates associated with sleep and depression in our study population in multivariable models . trouble sleeping did not substantially influence the observed associations between subjective sleep and depression . despite these strengths , our study had some limitations . our relatively small sample size might have affected our power to detect an association between sleep and depression in some instances . additionally , our study was cross - sectional and thus was unable to determine the temporal association between sleep and depression . we could not distinguish whether perception of acceptable sleep was influenced by depression or whether depression was influenced by perception of acceptable sleep . we were also limited in our ability to report on other comorbid features associated with caregiving , including anxiety and perceived stress . furthermore , the generalizability of our findings may also be limited to our specific study population and setting . the health of caregivers is a twofold concern , in our case affecting both caregivers and children with disabilities . rehabilitation providers for children with disabilities are well positioned to monitor the sleep and mental health of caregivers and provide the necessary resources , referrals , and interventions . our study shows that the association between sleep and depression differs comparing subjective and objective methods of assessment and that short sleep duration is associated with depression using both subjective and objective sleep measures . therefore , sleep intervention strategies for caregivers may benefit from a focus on sleep duration , and research strategies allowing for the integration of both perceived and objective measures of sleep traits are encouraged .

objectivethe objective of this study was to determine the association between sleep and depression using both self - reported ( subjective ) and actigraphic ( objective ) sleep traits.methodsa cross - sectional study was conducted among 175 female primary caregivers of children with disabilities receiving care at a rehabilitation center in punta arenas , chile . the eight - item patient health questionnaire was used to ascertain participants depression status . the pittsburgh sleep quality index was used to define subjective , or perceived , sleep quality . wrist - worn actigraph monitors , worn for seven consecutive nights , were used to characterize objective sleep quality and disturbances . interviewer - administered questionnaires were used to collect information on sociodemographic and lifestyle factors . linear regression models were fit using continuous sleep parameters as the dependent variables and depression status as the independent variable . multivariable models were adjusted for body mass index , marital status , smoking status , education level , and children s disabilities.resultsusing an eight - item patient health questionnaire score 10 , 26.3% of participants presented with depression . depressed women were more likely to self - report overall poorer ( subjective ) sleep compared to non - depressed women ; however , differences in sleep were not consistently noted using actigraphic ( objective ) sleep traits . among the depressed , both sleep duration and total time in bed were significantly underestimated . in multivariable models , depression was negatively associated with sleep duration using both subjective ( =0.71 , standard error [ se ] = 0.25 ; p=0.006 ) and objective sleep ( =0.42 , se = 0.19 ; p=0.026).conclusionthe association between sleep and depression differed comparing subjective and objective methods of assessment . research strategies allowing for the integration of both perceived and objective measures of sleep traits are encouraged .

Introduction Methods Study participants Study procedures Data collection instruments and covariates Pittsburgh Sleep Quality Index (PSQI) Wrist actigraphy Patient Health Questionnaire Covariates Statistical analysis Ethical consideration Results Discussion Conclusion

the international prevalence of poor sleep is estimated to range from 17% to 26%13 and is associated with depression using different study designs and populations.1,37 however one example of a subjective sleep instrument is the pittsburgh sleep quality index ( psqi).8 rather than self - reports , objective sleep parameters rely on monitoring of sleep wake activity using electronic instrumentation . these studies have included healthy men and women,10,11 the depressed,1214 pregnant women,15,16 women with severe premenstrual syndrome,17 the elderly,18 and caregivers.19 understanding the differences between subjective and objective sleep measures among caregivers is important because these populations are often at high risk for poor sleep and other comorbidities , which can have deleterious consequences on both the caregiver and care recipient.2024 caregivers report poorer sleep outcomes , greater anxiety , and greater depression when compared to noncaregivers,19,2528 and caregiver focus groups report that poor sleep has had negative impacts on health.20,29 caregivers of children with chronic health conditions show greater depressive symptoms compared to caregivers of children without chronic health conditions.26 in addition , a graded relationship between care - giving burden and depression was observed when comparing caregivers of children with both neurodevelopmental and behavioral disorders to caregivers of children with either one or none of these disorders.30 therefore , we determined whether self - reported ( subjective ) sleep and wrist actigraphy ( objective ) sleep traits were each associated with depression and investigated the extent to which these associations differed among a sample of female caregivers of children with disabilities . the chile pediatric and adult sleep study ( cpass ) is a cross - sectional study conducted at the centro de rehabilitacin club de leones cruz del sur in punta arenas , chile that was designed to investigate the relationship between the sleep quality of children with disabilities and that of their primary caregivers with cardiometabolic and psychological health . this study was conducted among female primary caregivers of children with disabilities enrolled between september 2012 and july 2013 in two waves . the second wave ( cpass ii ) was conducted between april 2013 and july 2013 among caregivers of children with disabilities aged 1021 years at the center . after obtaining written informed consent , a research psychologist administered structured questionnaires to women in order to collect information on sociodemographic and lifestyle factors and sleep characteristics . a global psqi score > 5 has been shown to yield a sensitivity of 89.6% and a specificity of 86.5% in distinguishing good ( score 5 ) and poor sleepers ( score>5).8 our analysis explored the following psqi component scores : total time in bed ( hours ) , sleep latency ( minutes ) , sleep duration ( hours ) , and sleep efficiency ( % ) . reported actigraphy - measured parameters include total time in bed ( hours ) , sleep latency ( minutes ) , sleep duration ( hours ) , sleep efficiency ( % ) , wake after sleep onset ( waso ; minutes ) ; average awakening length ( minutes ) , and number of awakenings . depression was assessed using the eight - item patient health questionnaire ( phq-8 ) , which uses a self - administered module for unipolar depression that asks the participant over the last 2 weeks , how often have you been bothered by the following problems ? also , in other studies , the modification of the nine - item patient health questionnaire to the phq-8 depression questionnaire has been demonstrated to minimally influence overall scale performance , mean scores , or diagnostic cut points.38,39 the eight items of the phq-8 include feigned interest , hopelessness , trouble sleeping , lack of energy , changes in appetite , self - deprecation , trouble concentrating , and changes in physical behavior . validated cutoffs were used to categorize patients into either depressed ( phq-8 score 10 ) or non - depressed ( phq-8 score < 10 ) categories for comparison.38 using the diagnostic statistical manual , fourth edition , a score 10 has been shown to be associated with 88% sensitivity and 88% specificity in diagnosing major depressive disorder38 and validated for use in spanish - speaking populations.40,41 structured interviewer - administered questionnaires collected data on caregivers age , body mass index ( bmi ) , ethnicity , marital status , current smoking status , alcohol consumption , employment status , and education level . the fisher s exact and chi - square tests were used to evaluate differences in sociodemographic and lifestyle characteristics by caregiver depression status ( phq-8 score of 10 vs phq-8 score < 10 ) . differences in sleep between depressed women and nondepressed women were analyzed for both subjective and objective sleep parameters using either student s t - test or kruskal wallis test . among depressed and non - depressed groups , overlapping objective and subjective sleep parameters multivariable models were adjusted for bmi ( continuous ) , marital status ( married or living with a partner vs divorced / separated / widowed / never married ) , current smoking status ( yes or no ) , education level ( < high school , high school , or > high school ) , and children s disability group ( mental and behavioral disorders , diseases of musculoskeletal system and connective / skin tissue , diseases of the nervous system , or congenital malformations and chromosomal abnormalities ) . the chile pediatric and adult sleep study ( cpass ) is a cross - sectional study conducted at the centro de rehabilitacin club de leones cruz del sur in punta arenas , chile that was designed to investigate the relationship between the sleep quality of children with disabilities and that of their primary caregivers with cardiometabolic and psychological health . this study was conducted among female primary caregivers of children with disabilities enrolled between september 2012 and july 2013 in two waves . after obtaining written informed consent , a research psychologist administered structured questionnaires to women in order to collect information on sociodemographic and lifestyle factors and sleep characteristics . a global psqi score > 5 has been shown to yield a sensitivity of 89.6% and a specificity of 86.5% in distinguishing good ( score 5 ) and poor sleepers ( score>5).8 our analysis explored the following psqi component scores : total time in bed ( hours ) , sleep latency ( minutes ) , sleep duration ( hours ) , and sleep efficiency ( % ) . reported actigraphy - measured parameters include total time in bed ( hours ) , sleep latency ( minutes ) , sleep duration ( hours ) , sleep efficiency ( % ) , wake after sleep onset ( waso ; minutes ) ; average awakening length ( minutes ) , and number of awakenings . depression was assessed using the eight - item patient health questionnaire ( phq-8 ) , which uses a self - administered module for unipolar depression that asks the participant over the last 2 weeks , how often have you been bothered by the following problems ? also , in other studies , the modification of the nine - item patient health questionnaire to the phq-8 depression questionnaire has been demonstrated to minimally influence overall scale performance , mean scores , or diagnostic cut points.38,39 the eight items of the phq-8 include feigned interest , hopelessness , trouble sleeping , lack of energy , changes in appetite , self - deprecation , trouble concentrating , and changes in physical behavior . validated cutoffs were used to categorize patients into either depressed ( phq-8 score 10 ) or non - depressed ( phq-8 score < 10 ) categories for comparison.38 using the diagnostic statistical manual , fourth edition , a score 10 has been shown to be associated with 88% sensitivity and 88% specificity in diagnosing major depressive disorder38 and validated for use in spanish - speaking populations.40,41 structured interviewer - administered questionnaires collected data on caregivers age , body mass index ( bmi ) , ethnicity , marital status , current smoking status , alcohol consumption , employment status , and education level . a global psqi score > 5 has been shown to yield a sensitivity of 89.6% and a specificity of 86.5% in distinguishing good ( score 5 ) and poor sleepers ( score>5).8 our analysis explored the following psqi component scores : total time in bed ( hours ) , sleep latency ( minutes ) , sleep duration ( hours ) , and sleep efficiency ( % ) . reported actigraphy - measured parameters include total time in bed ( hours ) , sleep latency ( minutes ) , sleep duration ( hours ) , sleep efficiency ( % ) , wake after sleep onset ( waso ; minutes ) ; average awakening length ( minutes ) , and number of awakenings . depression was assessed using the eight - item patient health questionnaire ( phq-8 ) , which uses a self - administered module for unipolar depression that asks the participant over the last 2 weeks , how often have you been bothered by the following problems ? also , in other studies , the modification of the nine - item patient health questionnaire to the phq-8 depression questionnaire has been demonstrated to minimally influence overall scale performance , mean scores , or diagnostic cut points.38,39 the eight items of the phq-8 include feigned interest , hopelessness , trouble sleeping , lack of energy , changes in appetite , self - deprecation , trouble concentrating , and changes in physical behavior . validated cutoffs were used to categorize patients into either depressed ( phq-8 score 10 ) or non - depressed ( phq-8 score < 10 ) categories for comparison.38 using the diagnostic statistical manual , fourth edition , a score 10 has been shown to be associated with 88% sensitivity and 88% specificity in diagnosing major depressive disorder38 and validated for use in spanish - speaking populations.40,41 structured interviewer - administered questionnaires collected data on caregivers age , body mass index ( bmi ) , ethnicity , marital status , current smoking status , alcohol consumption , employment status , and education level . the fisher s exact and chi - square tests were used to evaluate differences in sociodemographic and lifestyle characteristics by caregiver depression status ( phq-8 score of 10 vs phq-8 score < 10 ) . differences in sleep between depressed women and nondepressed women were analyzed for both subjective and objective sleep parameters using either student s t - test or kruskal wallis test . multivariable models were adjusted for bmi ( continuous ) , marital status ( married or living with a partner vs divorced / separated / widowed / never married ) , current smoking status ( yes or no ) , education level ( < high school , high school , or > high school ) , and children s disability group ( mental and behavioral disorders , diseases of musculoskeletal system and connective / skin tissue , diseases of the nervous system , or congenital malformations and chromosomal abnormalities ) . a sensitivity analysis excluding the trouble sleeping item from the phq-8 was performed for the association between subjective sleep and depression . using a phq-8 score 10 , 26.3% of participants presented with depression . when compared with nondepressed women , depressed women had higher mean bmi ( 31.0 kg / m vs 29.0 kg / m ; p=0.037 ) , were less likely to report being married or living with a partner ( 50.0% vs 72.1% ; p=0.007 ) , were more likely to report being a current smoker ( 63.0% vs 41.1% ; p=0.010 ) , and were less likely to report a completed education beyond high school ( 17.4% vs 37.2% ; p=0.013 ) . table 2 shows differences in subjective and objective sleep parameters comparing depressed and nondepressed women as well as univariate and multivariable regression coefficients for the association between sleep and depression . compared with nondepressed women , depressed women self - reported shorter total time in bed ( 6.6 hours vs 7.3 hours ; p=0.014 ) , longer sleep latency ( 60.0 minutes vs 20.0 minutes ; p<0.001 ) , shorter sleep duration ( 5.8 hours vs 6.6 hours ; p=0.001 ) , and poorer overall sleep quality using the median global psqi score ( 11.0 vs 6.0 ; p<0.001 ) . depressed women did not statistically differ from non - depressed women based on objective total time in bed ( 7.7 hours vs 8.1 hours ; p=0.076 ) , objective sleep latency ( 7.2 minutes vs 11.1 minutes ; p=0.453 ) , objective sleep efficiency ( 86.2% vs 87.2% ; p=0.191 ) , objective waso ( 54.6 minutes vs 50.9 minutes ; p=0.361 ) , objective number of awakenings ( 14.4 vs 13.6 ; p=0.565 ) , and objective average awakening length ( 4.7 minutes vs 4.7 minutes ; p=0.921 ) . table 2 also shows differences between subjective and objective sleep parameters within depression status groups . among the depressed group , the mean subjective total time in bed ( 6.6 hours ) was lower than the mean objective total time in bed ( 7.7 hours ) and the difference statistically significant ( 1.1 hours , 95% confidence interval [ ci ] : 1.5 to 0.7 ; p<0.001 ) . among the nondepressed , the mean subjective total time in bed ( 7.3 hours ) was lower than the mean objective total time in bed ( 8.1 hours ) and the difference statistically significant ( 0.8 hours , 95% ci : 1.1 to 0.5 ; p<0.001 ) . among the depressed group , the mean subjective sleep duration ( 5.8 hours ) was lower than the mean objective sleep duration ( 6.4 hours ) and the difference statistically significant ( 0.6 hours , 95% ci : 1.0 to 0.2 ; p=0.005 ) . among the nondepressed , the mean subjective sleep duration ( 6.6 hours ) was lower than the mean objective sleep duration ( 6.9 hours ) ; however , the difference was not statistically significant ( 0.3 hours , 95% ci : 0.54 to 0.01 ; p=0.059 ) . among the depressed , the median subjective sleep latency ( 60.0 minutes ) was higher than the median objective sleep latency ( 7.2 minutes ) and difference statistically significant ( p<0.001 ) . among the depressed group , the median subjective sleep efficiency ( 88.2% ) was higher than the median objective sleep efficiency ( 86.2% ) ; however , the difference was not statistically significant ( p=0.451 ) . all multivariable models were adjusted for bmi ( continuous ) , marital status , current smoking status , education level , and children s disability group . subjective multivariable models show depression to be negatively associated with total time in bed ( =0.61 , se = 0.29 ; p=0.033 ) and sleep duration ( =0.71 , se = 0.25 ; p=0.006 ) and positively associated with subjective sleep latency ( =0.58 , se = 0.20 ; p=0.003 ) and subjective overall poor sleep quality using the global psqi score ( =0.53 , se = 0.08 ; p<0.001 ) . depression was also negatively associated with objective sleep duration ( =0.42 , se = 0.19 ; p=0.026 ) . no statistically significant associations were found for subjective sleep efficiency ( =0.01 , se = 0.03 ; p=0.743 ) , objective total time in bed ( =0.32 , se = 0.23 ; p=0.174 ) , objective sleep latency ( =0.19 , se = 0.23 ; p=0.408 ) , objective sleep efficiency ( =0.02 , se = 0.01 ; p=0.210 ) , objective waso ( =0.11 , se = 0.10 ; p=0.274 ) , objective average awakening length ( =0.06 , se = 0.09 ; p=0.531 ) , and objective number of awakenings ( =0.08 , se = 0.08 ; p=0.366 ) . notably , subjective sleep latency was no longer statistically significant in multivariable models ( = 0.30 , se = 0.22 ; p=0.184 ) . our study found an association between some sleep traits and depression and a difference in the extent of the association when comparing subjective and objective sleep measures . our study also observed an overall underestimation of total time in bed and sleep duration , although only the underestimation among the depressed group was statistically significant . we also report a robust negative association between depression and sleep duration using both subjective and objective sleep measures , and that the magnitude of association was strongest using subjective sleep measures . in addition , we found that the prevalence of depression among caregivers in our study ( 26.3% ) was within the range of depression estimates reported in other studies of caregivers for children with disabilities ( 15%32%).30,43 our finding of an association between some sleep traits and depression corroborates with the literature relating to this topic.1,37 our finding of differences comparing subjective and objective sleep measures is also supported by much of the literature,1019 despite limited findings to the contrary.44 among studies that have looked at such differences among the depressed patients , they have reported that depressed patients are more likely to poorly recall aspects of sleep compared to the nondepressed patients.1214 however , the direction of the inconsistency is mixed . for example , although we observed that depressed women significantly underreported sleep duration while nondepressed women did not , others have reported that depressed individuals may in fact be more likely to over report sleep duration.12 finally , our finding of differences in the association between sleep and depression comparing subjective and objective sleep measures has also been previously reported . for example , in a recent study of pregnant women with and without a confirmed mental disorder , daily reported ( subjective ) sleep variables showed significant associations with patient status in multivariable models , while wrist actigraphy ( objective ) sleep did not.16 results from a recent meta - analysis reported that when compared to normal sleep duration , short sleep duration is significantly associated with depression in adults ( relative risk = 1.31 , 95% ci : 1.04 to 1.64).4 in our study , a robust negative association between depression and sleep duration was found using both subjective and objective sleep measures . similar associations between mental health outcomes and sleep duration have been observed in other studies of caregivers.19,25 for example , in a sample of male and female caregivers , sleep duration was significantly associated with higher levels of depression using the hospital anxiety and depression scale.25 it is important to note that the association between sleep and depression is multifaceted and is impacted not only by changes in sleep features , such as shortened sleep duration , but also by changes in social and environmental factors.45,46 interestingly , in a study among young adult women , perceived stress was associated with poorer subjective sleep quality but not with actigraphic ( objective ) sleep measures.47 although perceived ( subjective ) and actual ( objective ) sleep measures share commonalities , it is not surprising that the two differ . second , we adjusted for covariates associated with sleep and depression in our study population in multivariable models . additionally , our study was cross - sectional and thus was unable to determine the temporal association between sleep and depression . our study shows that the association between sleep and depression differs comparing subjective and objective methods of assessment and that short sleep duration is associated with depression using both subjective and objective sleep measures . therefore , sleep intervention strategies for caregivers may benefit from a focus on sleep duration , and research strategies allowing for the integration of both perceived and objective measures of sleep traits are encouraged .

to characterize both the extent and origin of variation , we posed 2 analytic aims . first , we assessed the extent to which variation exists in rates of positive angiograms among all referring providers . second , to explain the sources of variation , we evaluated selected physician characteristics and individual physicians as predictors of positive angiograms while controlling for patient factors that are known to predict positive angiograms . massachusetts general hospital is the largest hospital affiliated with harvard medical school and the largest volume center for diagnostic coronary angiography and percutaneous coronary intervention in new england . for each diagnostic coronary angiogram , physicians enter patient data into an electronic database , including demographic information , the clinical indication for the procedure , and the names of the referring physician and the physicians performing the procedure . of physicians who request angiograms , the majority are staff cardiologists at the hospital ( 74 of 117 , 63% ) . from the hospital 's catheterization database , we identified all cases of diagnostic coronary angiography performed at massachusetts general hospital from january 1 , 2012 , to june 30 , 2013 . we defined the outcome as a positive coronary angiogram with at least 1 lesion 50% narrowing of an epicardial coronary artery . to determine characteristics of the referring cardiologist , we linked angiogram data to administrative data about each physician , including age , physician gender , clinical fulltime equivalent , volume of catheterizations requested , and academic rank at harvard medical school ( instructor , assistant professor , associate professor , or professor ) . we identified patient characteristics such as indication for catheterization ( stsegment elevation myocardial infarction [ stemi ] , nonstemi , unstable angina , stable angina , atypical chest pain , or no symptoms ) , history of cardiac transplantation , cardiac valvular disease , cardiomyopathy , previous percutaneous coronary intervention , previous coronary artery bypass grafting , previous myocardial infarction , cardiogenic shock , congestive heart failure , diabetes , renal insufficiency , and peripheral vascular disease . we also defined a variable , called patient practice category , based on the requesting cardiologist 's primary affiliation within cardiology ( invasive / interventional , electrophysiology , heart failure , noninvasive ) . practice category was considered a physicianlevel variable because it reflected the practice group of the cardiologist . as a sensitivity analysis , we considered the patient practice category as a patientlevel variable because it might also have reflected differences among patients . we also linked patient records to hospital billing data identifying patient clinical characteristics including hypothyroidism , liver disease , solid tumor without metastasis , collagen vascular disease , obesity , weight loss , fluid and electrolyte disorders , drug abuse , psychosis , depression , and hypertension . we chose these variables because they are known to affect either coronary disease or health status generally . we explored the relative effects of physician characteristics and patient characteristics in determining the results of diagnostic angiograms . as such , we excluded angiograms for the indications of stemi , nonstemi , and unstable angina because we assumed that the decision to pursue coronary angiography would vary less by individual referring physicians in the setting of acute coronary syndrome . consequently , all angiograms included were requested by staff cardiologists at massachusetts general hospital the faculty of harvard medical school . using the binary outcome of diagnostic catheterization as either positive or negative , we sought to explain the extent and source of variance in the results of diagnostic catheterization using a series of mixedeffects models . the first , the unadjusted model , included only ordering physician as a random effect . this model was designed to measure the extent of crude variation before adjusting for patient and physician factors . the third model , the clinical and nonclinical model , added physician factors : age , physician gender , clinical fulltime equivalent , patient practice category , and harvard academic ranking . the difference in variation between models 2 and 3 can be considered variance due to style or practice of physicians . in all 3 models , for each of the 3 mixedeffects models , we calculated the median odds ratio ( mor ) to measure variance among ordering physicians after adjustment for variables in the sequential models . the mor is a measure of intraphysician variance in mixedeffect logistic models that estimates the difference in likelihood of a positive angiogram for 2 randomly selected physicians.10 by definition , the mor is always 1.0 . an mor of 1.0 would suggest no variation among physicians , and a greater mor would suggest the presence of variation among individual physicians . an mor > 1.2 has been recognized as a marker of clinically significant variation.11 , 12 to explore the validity of our primary findings , we performed 3 sensitivity analyses . practice category reflects differences between patients rather than differences between physicians , we considered patient practice category as a patientlevel variable instead of a physicianlevel variable . the institutional review board at partners healthcare waived the need for formal review because this work was performed for administrative purposes . in that context , the need for informed consent was waived . to characterize both the extent and origin of variation , we posed 2 analytic aims . first , we assessed the extent to which variation exists in rates of positive angiograms among all referring providers . second , to explain the sources of variation , we evaluated selected physician characteristics and individual physicians as predictors of positive angiograms while controlling for patient factors that are known to predict positive angiograms . massachusetts general hospital is the largest hospital affiliated with harvard medical school and the largest volume center for diagnostic coronary angiography and percutaneous coronary intervention in new england . for each diagnostic coronary angiogram , physicians enter patient data into an electronic database , including demographic information , the clinical indication for the procedure , and the names of the referring physician and the physicians performing the procedure . of physicians who request angiograms , the majority are staff cardiologists at the hospital ( 74 of 117 , 63% ) . from the hospital 's catheterization database , we identified all cases of diagnostic coronary angiography performed at massachusetts general hospital from january 1 , 2012 , to june 30 , 2013 . we defined the outcome as a positive coronary angiogram with at least 1 lesion 50% narrowing of an epicardial coronary artery . to determine characteristics of the referring cardiologist , we linked angiogram data to administrative data about each physician , including age , physician gender , clinical fulltime equivalent , volume of catheterizations requested , and academic rank at harvard medical school ( instructor , assistant professor , associate professor , or professor ) . we identified patient characteristics such as indication for catheterization ( stsegment elevation myocardial infarction [ stemi ] , nonstemi , unstable angina , stable angina , atypical chest pain , or no symptoms ) , history of cardiac transplantation , cardiac valvular disease , cardiomyopathy , previous percutaneous coronary intervention , previous coronary artery bypass grafting , previous myocardial infarction , cardiogenic shock , congestive heart failure , diabetes , renal insufficiency , and peripheral vascular disease . we also defined a variable , called patient practice category , based on the requesting cardiologist 's primary affiliation within cardiology ( invasive / interventional , electrophysiology , heart failure , noninvasive ) . patient practice category was considered a physicianlevel variable because it reflected the practice group of the cardiologist . as a sensitivity analysis , we considered the patient practice category as a patientlevel variable because it might also have reflected differences among patients . we also linked patient records to hospital billing data identifying patient clinical characteristics including hypothyroidism , liver disease , solid tumor without metastasis , collagen vascular disease , obesity , weight loss , fluid and electrolyte disorders , drug abuse , psychosis , depression , and hypertension . we chose these variables because they are known to affect either coronary disease or health status generally . we explored the relative effects of physician characteristics and patient characteristics in determining the results of diagnostic angiograms . as such , we excluded angiograms for the indications of stemi , nonstemi , and unstable angina because we assumed that the decision to pursue coronary angiography would vary less by individual referring physicians in the setting of acute coronary syndrome . consequently , all angiograms included were requested by staff cardiologists at massachusetts general hospital the faculty of harvard medical school . using the binary outcome of diagnostic catheterization as either positive or negative , we sought to explain the extent and source of variance in the results of diagnostic catheterization using a series of mixedeffects models . the first , the unadjusted model , included only ordering physician as a random effect . this model was designed to measure the extent of crude variation before adjusting for patient and physician factors . the third model , the clinical and nonclinical model , added physician factors : age , physician gender , clinical fulltime equivalent , patient practice category , and harvard academic ranking . the difference in variation between models 2 and 3 can be considered variance due to style or practice of physicians . in all 3 models , the ordering physician is a cluster variable with patients clustered among individual physicians . for each of the 3 mixedeffects models , we calculated the median odds ratio ( mor ) to measure variance among ordering physicians after adjustment for variables in the sequential models . the mor is a measure of intraphysician variance in mixedeffect logistic models that estimates the difference in likelihood of a positive angiogram for 2 randomly selected physicians.10 by definition , the mor is always 1.0 . an mor of 1.0 would suggest no variation among physicians , and a greater mor would suggest the presence of variation among individual physicians . an mor > 1.2 has been recognized as a marker of clinically significant variation.11 , 12 to explore the validity of our primary findings , we performed 3 sensitivity analyses . practice category reflects differences between patients rather than differences between physicians , we considered patient practice category as a patientlevel variable instead of a physicianlevel variable . the institutional review board at partners healthcare waived the need for formal review because this work was performed for administrative purposes . in that context , the need for informed consent was waived . over the time period of this study , 5015 total coronary angiograms were performed at massachusetts general hospital . for the variance analysis , we excluded angiograms ordered by physicians who requested < 10 angiograms . we also excluded angiograms for the indications of stemi , nonstemi , and unstable angina . after application of the exclusion criteria , 2925 angiograms ( 58.3% ) remained in the analysis . characteristics of the angiograms appear in table 1 . among the 2925 angiograms , 1450 ( 49.6% ) catheterizations were more likely to be positive for patients with stable angina , cardiomyopathy , previous percutaneous coronary intervention , or previous coronary artery bypass grafting and were more likely to be negative for patients with atypical chest pain , female patient gender , valvular disease , chronic pulmonary disease , and liver disease . characteristics of positive and negative angiograms cabg indicates coronary artery bypass grafting ; ep , electrophysiologist ; mi , myocardial infarction ; pci , percutaneous coronary intervention . after exclusions , 49 ordering physicians were represented in 4 patient practice categories . of those , 24 were noninvasive cardiologists ( 49% ) , 9 were electrophysiologists ( 18% ) , 11 were interventional or invasive cardiologists ( 22% ) , and 5 were heart failure cardiologists ( 10% ) . the mean ages for instructors , assistant professors , associate professors , and professors , respectively , were 44.4 , 49.6 , 54.9 , and 66.4 years ( p=0.0006 ) . before statistical adjustment , catheterizations requested by invasive / interventional cardiologists were more likely to be positive , and catheterizations requested by heart failure cardiologists were more likely to be negative ( p<0.01 for both ) . catheterizations requested by physicians did not differ by level of harvard academic rank in the unadjusted analyses ( figure 2 ) . tables 2 and 3 present the estimates of the clinical and nonclinical model . including both patient and physician variables as fixed effects and individual physicians as random effects , catheterizations requested by professors at harvard medical school ( relative to instructors , the lowest academic rank ) were more likely to be negative ( odds ratio [ or ] 0.51 , p<0.01 ) . catheterizations requested by associate professors were also more likely to be negative , although this finding was of marginal statistical significance ( or 0.77 , p=0.11 ) . catheterizations requested by assistant professors were positive at similar rates to catheterizations requested by instructors ( or 1.13 , p=0.31 ) . catheterizations requested by male and female cardiologists did not have different proportions of positive catheterizations after multivariate adjustment ( or for female cardiologists 1.02 , p=0.92 ) . with respect to patient practice category , angiograms for patients requested by noninvasive cardiologists , electrophysiologists ( p=0.51 ) , heart failure cardiologists ( p=0.79 ) , and invasive / interventional cardiologists ( p=0.93 ) had similar positivity rates . ors for positive angiograms ( patient characteristics ) cabg indicates coronary artery bypass grafting ; or , odds ratio ; pci , percutaneous coronary intervention . ors for positive angiograms ( provider characteristics ) ep indicates electrophysiologist ; fte , fulltime equivalent ; or , odds ratio . with respect to patient variables in the clinical and nonclinical model , angiograms were more likely to be positive for older patients ; for patients with stable angina , previous myocardial infarction , previous coronary artery bypass grafting , or previous percutaneous coronary intervention ; or for patients with peripheral artery disease , diabetes with complications , and weight loss . angiograms were more likely to be negative for female patients , patients with atypical chest pain , patients with no symptoms , and patients with a history of cardiac transplantation . full results of the clinical and nonclinical model , including both patient and physician variables , are shown in figure 3 . odds ratios for positive catheterizations in the full model , including both ( a ) patient and ( b ) physician variables . cabg indicates coronary artery bypass grafting ; fte , fulltime equivalent ; ior 80 , 80% interval odds ratio ; or , odds ratio ; pci , percutaneous coronary intervention . in the unadjusted model , the mor was 1.47 ( 95% ci 1.321.60 ) , consistent with clinically significant variation . in the clinical model , which included patient variables but not nonclinical provider variables such as physician age , physician gender , clinical fulltime equivalent , cardiology subspecialty , and harvard rank , the mor was 1.23 ( 95% ci 1.01.36 ) , suggesting borderline significant variation . in the full clinical and nonclinical model , the mor was 1.07 ( 95% ci 1.01.20 ) , also consistent with clinically insignificant variation . the mor and the associated variance of the 3 models are shown in table 4 . the ors for positive catheterizations by referring cardiologists according to the 3 models appear together in figure 4 . variance in angiogram positivity among physicians in the unadjusted model , the clinical model , and the clinical and nonclinical model odds ratios for positive catheterizations , by referring cardiologist , with and without statistical adjustment . a , unadjusted model . c , clinical and nonclinical model . given the association of harvard academic rank with test outcome , we performed a sensitivity analysis to test the strength of our findings . we recategorized the harvard academic rank variable such that associate professors and full professors were combined into 1 category . the new combined variable was significant ( or 0.63 , p=0.008 ) , with a magnitude in between the ors of the 2 independent categories . because the patient practice category might reflect differences among either patients or cardiologists , we also performed a sensitivity analysis considering this variable as a patientlevel variable . this sensitivity analysis did not change the value of the variable for any particular catheterization but characterized the variable among the patient attributes rather than the physician attributes . consequently , the sensitivity analysis included this variable in both the clinical model and the clinical and nonclinical model , whereas the primary analysis included the variable only in the clinical and nonclinical model . the mor for the models did not substantially change . finally , including patients with unstable angina in the analysis did not substantially change the mors of the 3 models . because all of these sensitivity analyses demonstrated the robustness of the primary analysis , only the results of the primary analysis are presented . tables 2 and 3 present the estimates of the clinical and nonclinical model . including both patient and physician variables as fixed effects and individual physicians as random effects , catheterizations requested by professors at harvard medical school ( relative to instructors , the lowest academic rank ) were more likely to be negative ( odds ratio [ or ] 0.51 , p<0.01 ) . catheterizations requested by associate professors were also more likely to be negative , although this finding was of marginal statistical significance ( or 0.77 , p=0.11 ) . catheterizations requested by assistant professors were positive at similar rates to catheterizations requested by instructors ( or 1.13 , p=0.31 ) . catheterizations requested by male and female cardiologists did not have different proportions of positive catheterizations after multivariate adjustment ( or for female cardiologists 1.02 , p=0.92 ) . practice category , angiograms for patients requested by noninvasive cardiologists , electrophysiologists ( p=0.51 ) , heart failure cardiologists ( p=0.79 ) , and invasive / interventional cardiologists ( p=0.93 ) had similar positivity rates . ors for positive angiograms ( patient characteristics ) cabg indicates coronary artery bypass grafting ; or , odds ratio ; pci , percutaneous coronary intervention . ors for positive angiograms ( provider characteristics ) ep indicates electrophysiologist ; fte , fulltime equivalent ; or , odds ratio . with respect to patient variables in the clinical and nonclinical model , angiograms were more likely to be positive for older patients ; for patients with stable angina , previous myocardial infarction , previous coronary artery bypass grafting , or previous percutaneous coronary intervention ; or for patients with peripheral artery disease , diabetes with complications , and weight loss . angiograms were more likely to be negative for female patients , patients with atypical chest pain , patients with no symptoms , and patients with a history of cardiac transplantation . full results of the clinical and nonclinical model , including both patient and physician variables , are shown in figure 3 . odds ratios for positive catheterizations in the full model , including both ( a ) patient and ( b ) physician variables . cabg indicates coronary artery bypass grafting ; fte , fulltime equivalent ; ior 80 , 80% interval odds ratio ; or , odds ratio ; pci , percutaneous coronary intervention . in the unadjusted model , the mor was 1.47 ( 95% ci 1.321.60 ) , consistent with clinically significant variation . in the clinical model , which included patient variables but not nonclinical provider variables such as physician age , physician gender , clinical fulltime equivalent , cardiology subspecialty , and harvard rank , the mor was 1.23 ( 95% ci 1.01.36 ) , suggesting borderline significant variation . in the full clinical and nonclinical model , the mor was 1.07 ( 95% ci 1.01.20 ) , also consistent with clinically insignificant variation . the mor and the associated variance of the 3 models are shown in table 4 . the ors for positive catheterizations by referring cardiologists according to the 3 models appear together in figure 4 . variance in angiogram positivity among physicians in the unadjusted model , the clinical model , and the clinical and nonclinical model odds ratios for positive catheterizations , by referring cardiologist , with and without statistical adjustment . a , unadjusted model . given the association of harvard academic rank with test outcome , we performed a sensitivity analysis to test the strength of our findings . we recategorized the harvard academic rank variable such that associate professors and full professors were combined into 1 category . the new combined variable was significant ( or 0.63 , p=0.008 ) , with a magnitude in between the ors of the 2 independent categories . because the patient practice category might reflect differences among either patients or cardiologists , we also performed a sensitivity analysis considering this variable as a patientlevel variable . this sensitivity analysis did not change the value of the variable for any particular catheterization but characterized the variable among the patient attributes rather than the physician attributes . consequently , the sensitivity analysis included this variable in both the clinical model and the clinical and nonclinical model , whereas the primary analysis included the variable only in the clinical and nonclinical model . the mor for the models did not substantially change . finally , including patients with unstable angina in the analysis did not substantially change the mors of the 3 models . because all of these sensitivity analyses demonstrated the robustness of the primary analysis , only the results of the primary analysis are presented . in this study , we found substantial variance in results of diagnostic coronary angiography among cardiologists . after adjustment for clinical variables , variation by individual requesting physician was of only borderline clinically significance ( mor 1.23 , 95% ci 1.01.36 ) . these results emphasize that in measuring the performance of individual physicians , controlling for patient characteristics is essential . our findings on patient characteristics are consistent with findings from the national cardiovascular data registry , which also found that patientlevel variables including higher age , male patient gender , peripheral arterial disease , renal failure , and typical angina are associated with positive angiograms.7 variance in rates of normal coronary angiography among hospitals has been previously found to be substantial.13 we have extended those results to apply to individual referring cardiologists . we found , for example , that catheterizations requested by full professors and associate professors were more likely to be negative than catheterizations requested by assistant professors and instructors . the effect was graded by academic rank , with an or for professors relative to instructors of 0.51 ( p=0.001 ) and an or for associate professors relative to instructors of 0.77 ( borderline significance , p=0.11 ) . although physician age is correlated with harvard rank , the model included both variables so the reported effect of harvard rank reflects adjustment for age . the strong relationship between incremental increases in harvard academic rank and negative angiograms has multiple plausible explanations . first , harvard academic rank may be associated with an unmeasured confounder . our full patient and physician model adjusted for many of these plausible confounders , including patient age , aspects of patient history ( previous percutaneous coronary intervention , previous coronary artery bypass grafting , previous myocardial infarction , valvular disease ) , type of physician practice , and physician age and gender . second , harvard academic rank may be associated with treatment selection bias insofar as different types of patients are referred to senior and junior clinicians . in particular , patients with challenging symptoms or previous inconclusive evaluations may be more likely to seek the care of senior staff . in fact , the unadjusted positivity rate did not differ among clinicians divided by harvard academic rank . the fact that higher levels of academic rank were associated with more negative angiograms after statistical adjustment suggests that patient case mix differs for senior and junior physicians . the linkage between angiogram outcome and angiogram decision is important because it establishes physician variance at the level of the individual clinician who actually makes the decision to pursue angiography . in national surveys , cardiologists have been shown to vary substantially in propensity to request cardiac catheterization for other than purely clinical reasons , including meeting patient expectations , meeting peer expectations , and malpractice concerns.14 physician practice style may also influence individual physicians propensity to pursue coronary angiography . this has important implications for improving both quality and value because these results suggest that feedback to physicians that have low proportions of positive catheterizations may decrease the overall negative rate , potentially reducing costs . at the same time , by including both patient and physicianlevel variables in a mixedeffects regression model , we demonstrated that a substantial proportion of the explained variation is related to patient variables . any attempt to provide feedback to clinicians should acknowledge and incorporate patientlevel variables and report riskstandardized odds of positive angiograms . importantly , although invasive / interventional cardiologists were more likely to obtain positive angiograms and heart failure cardiologists were more likely to obtain negative angiograms in unadjusted bivariate analysis , after adjusting for confounding with the full model , cardiology subspecialty was not significant . we believe this result is probably related to confounding between patient factors and the types of cardiologists that care for them . consequently , improperly adjusted analyses judging cardiologists on metrics of utilization used for either public reporting or pay for performance could unfairly designate groups of cardiologists as either positive or negative outliers . because the 95% ci of the mor in the clinical model encompasses the threshold of clinical significance , it is also possible that our study was underpowered to detect clinically significant variation attributable to nonclinical variables , including academic rank . if clinical variation exists in the clinical model and is reduced by variables in the clinical and nonclinical model in ways that do not reflect differences in case mix , this would suggest potential opportunities for actionable quality improvement . in particular , groups of clinicians associated with high negative rates could review practice patterns associated with negative catheterizations . the hypothesis that some existing variation is associated with specific physician groups could be tested in a larger data set . first , as a singlecenter study , the extent to which we can generalize our findings to other health care settings is unclear . in particular , as the largest general hospital in new england and as a teaching hospital , the extent to which we can generalize these results to smaller and nonteaching hospitals is unclear . the challenge with studying this question through national databases remains that those databases do not have granular information about physicians referring for procedures . we believe , however , that these results emphasize the importance of risk adjustment in variation analyses , no matter the hospital setting . second , we did not capture the true denominator , the number of patients evaluated for coronary angiography , because many do not ultimately receive the procedure ; therefore , we can not draw precise conclusions about propensity to test . we used the positivity ratio as a proxy for propensity to test , with the implication that providers with more negative angiograms have more propensity ( a lower threshold ) to test . third , the true optimal positivity rate or riskadjusted positivity rate is uncertain , so we can not make prescriptive judgments about an individual provider 's positivity rate . nevertheless , in the context of high negative rates both locally and nationally , we believe that this information should encourage providers with lower riskadjusted positivity rates to review their referral patterns . in particular , the strong presence of patientlevel factors in predicting positivity rates encourages proper risk stratification before referral . fourth , by excluding providers with < 10 requested catheterizations , we may have introduced selection bias by excluding cases referred to lowvolume or primarily researchoriented cardiologists . fifth , we were not able to distinguish between inpatient and outpatient referrals , and that may have been a potential source of unmeasured confounding . finally , our database did not include information about appropriate use criteria , which was not available , so that does not appear as a variable in our analysis . we believe that following of appropriate use criteria may vary between individual physicians , so including appropriateness as a fixed effect may have disguised variation between individual physicians . in conclusion , we demonstrated that there is substantial variance among referring providers at a large academic medical center with respect to the proportion of positive results in requested coronary angiograms . these findings underscore the importance of accounting for risk factors in analyzing physician performance . this work was funded by the massachusetts general hospital physicians organization fellowship in health policy and management . hidrue , pomerantsev , armstrong , dec , jr , fifer , ferris have no relevant disclosures . yeh : research grant : national heart lung and blood institute , amount : $10 000 . massachusetts general hospital , amount $10 000 . other research support : harvard clinical research institute ,

backgroundunderstanding the sources of variation for highcost services has the potential to improve both patient outcomes and value in health care delivery . nationally , the overall diagnostic yield of coronary angiography is relatively low , suggesting overutilization . understanding how individual cardiologists request catheterization may suggest opportunities for improving quality and value . we aimed to assess and explain variation in positive angiograms among referring cardiologists.methods and resultswe identified all cases of diagnostic coronary angiography at massachusetts general hospital from january 1 , 2012 , to june 30 , 2013 . we excluded angiograms for acute coronary syndrome . for each angiogram , we identified clinical features of the patients and characteristics of the requesting cardiologists . we also identified angiogram positivity , defined as at least 1 epicardial coronary stenosis 50% luminal narrowing . we then constructed a series of mixedeffects logistic regression models to analyze predictors of positive coronary angiograms . we assessed variation by physician in the models with median odds ratios . over this time period , 5015 angiograms were identified . we excluded angiograms ordered by cardiologists requesting < 10 angiograms . among the remaining 2925 angiograms , 1450 ( 49.6% ) were positive . significant predictors of positive angiograms included age , male patients , and peripheral arterial disease . after adjustment for clinical variables only , the median odds ratio was 1.23 ( 95% ci 1.01.36 ) , consistent with only borderline clinical variation after adjustment . in the full clinical and nonclinical model , the median odds ratio was 1.07 ( 95% ci 1.071.20 ) , also consistent with clinically insignificant variation.conclusionssubstantial variation exists among requesting cardiologists with respect to positive and negative coronary angiograms . after adjustment for clinical variables , there was only borderline clinically significant variation . these results emphasize the importance of risk adjustment in reporting related to quality and value .

Methods Analytic Aims Study Population Outcomes and Covariates Statistical Analysis Results Adjusted Results Variation by Individual Physicians After Adjustment Sensitivity Analyses Discussion Sources of Funding Disclosures

to characterize both the extent and origin of variation , we posed 2 analytic aims . first , we assessed the extent to which variation exists in rates of positive angiograms among all referring providers . second , to explain the sources of variation , we evaluated selected physician characteristics and individual physicians as predictors of positive angiograms while controlling for patient factors that are known to predict positive angiograms . massachusetts general hospital is the largest hospital affiliated with harvard medical school and the largest volume center for diagnostic coronary angiography and percutaneous coronary intervention in new england . for each diagnostic coronary angiogram , physicians enter patient data into an electronic database , including demographic information , the clinical indication for the procedure , and the names of the referring physician and the physicians performing the procedure . of physicians who request angiograms , the majority are staff cardiologists at the hospital ( 74 of 117 , 63% ) . from the hospital 's catheterization database , we identified all cases of diagnostic coronary angiography performed at massachusetts general hospital from january 1 , 2012 , to june 30 , 2013 . we defined the outcome as a positive coronary angiogram with at least 1 lesion 50% narrowing of an epicardial coronary artery . to determine characteristics of the referring cardiologist , we linked angiogram data to administrative data about each physician , including age , physician gender , clinical fulltime equivalent , volume of catheterizations requested , and academic rank at harvard medical school ( instructor , assistant professor , associate professor , or professor ) . we identified patient characteristics such as indication for catheterization ( stsegment elevation myocardial infarction [ stemi ] , nonstemi , unstable angina , stable angina , atypical chest pain , or no symptoms ) , history of cardiac transplantation , cardiac valvular disease , cardiomyopathy , previous percutaneous coronary intervention , previous coronary artery bypass grafting , previous myocardial infarction , cardiogenic shock , congestive heart failure , diabetes , renal insufficiency , and peripheral vascular disease . we also defined a variable , called patient practice category , based on the requesting cardiologist 's primary affiliation within cardiology ( invasive / interventional , electrophysiology , heart failure , noninvasive ) . we also linked patient records to hospital billing data identifying patient clinical characteristics including hypothyroidism , liver disease , solid tumor without metastasis , collagen vascular disease , obesity , weight loss , fluid and electrolyte disorders , drug abuse , psychosis , depression , and hypertension . as such , we excluded angiograms for the indications of stemi , nonstemi , and unstable angina because we assumed that the decision to pursue coronary angiography would vary less by individual referring physicians in the setting of acute coronary syndrome . consequently , all angiograms included were requested by staff cardiologists at massachusetts general hospital the faculty of harvard medical school . using the binary outcome of diagnostic catheterization as either positive or negative , we sought to explain the extent and source of variance in the results of diagnostic catheterization using a series of mixedeffects models . the first , the unadjusted model , included only ordering physician as a random effect . the third model , the clinical and nonclinical model , added physician factors : age , physician gender , clinical fulltime equivalent , patient practice category , and harvard academic ranking . in all 3 models , for each of the 3 mixedeffects models , we calculated the median odds ratio ( mor ) to measure variance among ordering physicians after adjustment for variables in the sequential models . an mor of 1.0 would suggest no variation among physicians , and a greater mor would suggest the presence of variation among individual physicians . an mor > 1.2 has been recognized as a marker of clinically significant variation.11 , 12 to explore the validity of our primary findings , we performed 3 sensitivity analyses . to characterize both the extent and origin of variation , we posed 2 analytic aims . first , we assessed the extent to which variation exists in rates of positive angiograms among all referring providers . second , to explain the sources of variation , we evaluated selected physician characteristics and individual physicians as predictors of positive angiograms while controlling for patient factors that are known to predict positive angiograms . massachusetts general hospital is the largest hospital affiliated with harvard medical school and the largest volume center for diagnostic coronary angiography and percutaneous coronary intervention in new england . for each diagnostic coronary angiogram , physicians enter patient data into an electronic database , including demographic information , the clinical indication for the procedure , and the names of the referring physician and the physicians performing the procedure . of physicians who request angiograms , the majority are staff cardiologists at the hospital ( 74 of 117 , 63% ) . from the hospital 's catheterization database , we identified all cases of diagnostic coronary angiography performed at massachusetts general hospital from january 1 , 2012 , to june 30 , 2013 . we defined the outcome as a positive coronary angiogram with at least 1 lesion 50% narrowing of an epicardial coronary artery . to determine characteristics of the referring cardiologist , we linked angiogram data to administrative data about each physician , including age , physician gender , clinical fulltime equivalent , volume of catheterizations requested , and academic rank at harvard medical school ( instructor , assistant professor , associate professor , or professor ) . we identified patient characteristics such as indication for catheterization ( stsegment elevation myocardial infarction [ stemi ] , nonstemi , unstable angina , stable angina , atypical chest pain , or no symptoms ) , history of cardiac transplantation , cardiac valvular disease , cardiomyopathy , previous percutaneous coronary intervention , previous coronary artery bypass grafting , previous myocardial infarction , cardiogenic shock , congestive heart failure , diabetes , renal insufficiency , and peripheral vascular disease . we also defined a variable , called patient practice category , based on the requesting cardiologist 's primary affiliation within cardiology ( invasive / interventional , electrophysiology , heart failure , noninvasive ) . we also linked patient records to hospital billing data identifying patient clinical characteristics including hypothyroidism , liver disease , solid tumor without metastasis , collagen vascular disease , obesity , weight loss , fluid and electrolyte disorders , drug abuse , psychosis , depression , and hypertension . as such , we excluded angiograms for the indications of stemi , nonstemi , and unstable angina because we assumed that the decision to pursue coronary angiography would vary less by individual referring physicians in the setting of acute coronary syndrome . consequently , all angiograms included were requested by staff cardiologists at massachusetts general hospital the faculty of harvard medical school . using the binary outcome of diagnostic catheterization as either positive or negative , we sought to explain the extent and source of variance in the results of diagnostic catheterization using a series of mixedeffects models . the third model , the clinical and nonclinical model , added physician factors : age , physician gender , clinical fulltime equivalent , patient practice category , and harvard academic ranking . for each of the 3 mixedeffects models , we calculated the median odds ratio ( mor ) to measure variance among ordering physicians after adjustment for variables in the sequential models . an mor of 1.0 would suggest no variation among physicians , and a greater mor would suggest the presence of variation among individual physicians . an mor > 1.2 has been recognized as a marker of clinically significant variation.11 , 12 to explore the validity of our primary findings , we performed 3 sensitivity analyses . over the time period of this study , 5015 total coronary angiograms were performed at massachusetts general hospital . for the variance analysis , we excluded angiograms ordered by physicians who requested < 10 angiograms . we also excluded angiograms for the indications of stemi , nonstemi , and unstable angina . after application of the exclusion criteria , 2925 angiograms ( 58.3% ) remained in the analysis . characteristics of the angiograms appear in table 1 . among the 2925 angiograms , 1450 ( 49.6% ) catheterizations were more likely to be positive for patients with stable angina , cardiomyopathy , previous percutaneous coronary intervention , or previous coronary artery bypass grafting and were more likely to be negative for patients with atypical chest pain , female patient gender , valvular disease , chronic pulmonary disease , and liver disease . characteristics of positive and negative angiograms cabg indicates coronary artery bypass grafting ; ep , electrophysiologist ; mi , myocardial infarction ; pci , percutaneous coronary intervention . of those , 24 were noninvasive cardiologists ( 49% ) , 9 were electrophysiologists ( 18% ) , 11 were interventional or invasive cardiologists ( 22% ) , and 5 were heart failure cardiologists ( 10% ) . tables 2 and 3 present the estimates of the clinical and nonclinical model . including both patient and physician variables as fixed effects and individual physicians as random effects , catheterizations requested by professors at harvard medical school ( relative to instructors , the lowest academic rank ) were more likely to be negative ( odds ratio [ or ] 0.51 , p<0.01 ) . with respect to patient practice category , angiograms for patients requested by noninvasive cardiologists , electrophysiologists ( p=0.51 ) , heart failure cardiologists ( p=0.79 ) , and invasive / interventional cardiologists ( p=0.93 ) had similar positivity rates . ors for positive angiograms ( patient characteristics ) cabg indicates coronary artery bypass grafting ; or , odds ratio ; pci , percutaneous coronary intervention . ors for positive angiograms ( provider characteristics ) ep indicates electrophysiologist ; fte , fulltime equivalent ; or , odds ratio . with respect to patient variables in the clinical and nonclinical model , angiograms were more likely to be positive for older patients ; for patients with stable angina , previous myocardial infarction , previous coronary artery bypass grafting , or previous percutaneous coronary intervention ; or for patients with peripheral artery disease , diabetes with complications , and weight loss . angiograms were more likely to be negative for female patients , patients with atypical chest pain , patients with no symptoms , and patients with a history of cardiac transplantation . full results of the clinical and nonclinical model , including both patient and physician variables , are shown in figure 3 . odds ratios for positive catheterizations in the full model , including both ( a ) patient and ( b ) physician variables . in the unadjusted model , the mor was 1.47 ( 95% ci 1.321.60 ) , consistent with clinically significant variation . in the clinical model , which included patient variables but not nonclinical provider variables such as physician age , physician gender , clinical fulltime equivalent , cardiology subspecialty , and harvard rank , the mor was 1.23 ( 95% ci 1.01.36 ) , suggesting borderline significant variation . in the full clinical and nonclinical model , the mor was 1.07 ( 95% ci 1.01.20 ) , also consistent with clinically insignificant variation . variance in angiogram positivity among physicians in the unadjusted model , the clinical model , and the clinical and nonclinical model odds ratios for positive catheterizations , by referring cardiologist , with and without statistical adjustment . c , clinical and nonclinical model . the new combined variable was significant ( or 0.63 , p=0.008 ) , with a magnitude in between the ors of the 2 independent categories . because the patient practice category might reflect differences among either patients or cardiologists , we also performed a sensitivity analysis considering this variable as a patientlevel variable . this sensitivity analysis did not change the value of the variable for any particular catheterization but characterized the variable among the patient attributes rather than the physician attributes . consequently , the sensitivity analysis included this variable in both the clinical model and the clinical and nonclinical model , whereas the primary analysis included the variable only in the clinical and nonclinical model . finally , including patients with unstable angina in the analysis did not substantially change the mors of the 3 models . tables 2 and 3 present the estimates of the clinical and nonclinical model . including both patient and physician variables as fixed effects and individual physicians as random effects , catheterizations requested by professors at harvard medical school ( relative to instructors , the lowest academic rank ) were more likely to be negative ( odds ratio [ or ] 0.51 , p<0.01 ) . practice category , angiograms for patients requested by noninvasive cardiologists , electrophysiologists ( p=0.51 ) , heart failure cardiologists ( p=0.79 ) , and invasive / interventional cardiologists ( p=0.93 ) had similar positivity rates . ors for positive angiograms ( patient characteristics ) cabg indicates coronary artery bypass grafting ; or , odds ratio ; pci , percutaneous coronary intervention . ors for positive angiograms ( provider characteristics ) ep indicates electrophysiologist ; fte , fulltime equivalent ; or , odds ratio . with respect to patient variables in the clinical and nonclinical model , angiograms were more likely to be positive for older patients ; for patients with stable angina , previous myocardial infarction , previous coronary artery bypass grafting , or previous percutaneous coronary intervention ; or for patients with peripheral artery disease , diabetes with complications , and weight loss . angiograms were more likely to be negative for female patients , patients with atypical chest pain , patients with no symptoms , and patients with a history of cardiac transplantation . full results of the clinical and nonclinical model , including both patient and physician variables , are shown in figure 3 . odds ratios for positive catheterizations in the full model , including both ( a ) patient and ( b ) physician variables . in the unadjusted model , the mor was 1.47 ( 95% ci 1.321.60 ) , consistent with clinically significant variation . in the clinical model , which included patient variables but not nonclinical provider variables such as physician age , physician gender , clinical fulltime equivalent , cardiology subspecialty , and harvard rank , the mor was 1.23 ( 95% ci 1.01.36 ) , suggesting borderline significant variation . in the full clinical and nonclinical model , the mor was 1.07 ( 95% ci 1.01.20 ) , also consistent with clinically insignificant variation . variance in angiogram positivity among physicians in the unadjusted model , the clinical model , and the clinical and nonclinical model odds ratios for positive catheterizations , by referring cardiologist , with and without statistical adjustment . the new combined variable was significant ( or 0.63 , p=0.008 ) , with a magnitude in between the ors of the 2 independent categories . because the patient practice category might reflect differences among either patients or cardiologists , we also performed a sensitivity analysis considering this variable as a patientlevel variable . this sensitivity analysis did not change the value of the variable for any particular catheterization but characterized the variable among the patient attributes rather than the physician attributes . consequently , the sensitivity analysis included this variable in both the clinical model and the clinical and nonclinical model , whereas the primary analysis included the variable only in the clinical and nonclinical model . finally , including patients with unstable angina in the analysis did not substantially change the mors of the 3 models . in this study , we found substantial variance in results of diagnostic coronary angiography among cardiologists . after adjustment for clinical variables , variation by individual requesting physician was of only borderline clinically significance ( mor 1.23 , 95% ci 1.01.36 ) . these results emphasize that in measuring the performance of individual physicians , controlling for patient characteristics is essential . our findings on patient characteristics are consistent with findings from the national cardiovascular data registry , which also found that patientlevel variables including higher age , male patient gender , peripheral arterial disease , renal failure , and typical angina are associated with positive angiograms.7 variance in rates of normal coronary angiography among hospitals has been previously found to be substantial.13 we have extended those results to apply to individual referring cardiologists . although physician age is correlated with harvard rank , the model included both variables so the reported effect of harvard rank reflects adjustment for age . our full patient and physician model adjusted for many of these plausible confounders , including patient age , aspects of patient history ( previous percutaneous coronary intervention , previous coronary artery bypass grafting , previous myocardial infarction , valvular disease ) , type of physician practice , and physician age and gender . in national surveys , cardiologists have been shown to vary substantially in propensity to request cardiac catheterization for other than purely clinical reasons , including meeting patient expectations , meeting peer expectations , and malpractice concerns.14 physician practice style may also influence individual physicians propensity to pursue coronary angiography . this has important implications for improving both quality and value because these results suggest that feedback to physicians that have low proportions of positive catheterizations may decrease the overall negative rate , potentially reducing costs . at the same time , by including both patient and physicianlevel variables in a mixedeffects regression model , we demonstrated that a substantial proportion of the explained variation is related to patient variables . importantly , although invasive / interventional cardiologists were more likely to obtain positive angiograms and heart failure cardiologists were more likely to obtain negative angiograms in unadjusted bivariate analysis , after adjusting for confounding with the full model , cardiology subspecialty was not significant . because the 95% ci of the mor in the clinical model encompasses the threshold of clinical significance , it is also possible that our study was underpowered to detect clinically significant variation attributable to nonclinical variables , including academic rank . if clinical variation exists in the clinical model and is reduced by variables in the clinical and nonclinical model in ways that do not reflect differences in case mix , this would suggest potential opportunities for actionable quality improvement . first , as a singlecenter study , the extent to which we can generalize our findings to other health care settings is unclear . in particular , as the largest general hospital in new england and as a teaching hospital , the extent to which we can generalize these results to smaller and nonteaching hospitals is unclear . we believe , however , that these results emphasize the importance of risk adjustment in variation analyses , no matter the hospital setting . second , we did not capture the true denominator , the number of patients evaluated for coronary angiography , because many do not ultimately receive the procedure ; therefore , we can not draw precise conclusions about propensity to test . nevertheless , in the context of high negative rates both locally and nationally , we believe that this information should encourage providers with lower riskadjusted positivity rates to review their referral patterns . fourth , by excluding providers with < 10 requested catheterizations , we may have introduced selection bias by excluding cases referred to lowvolume or primarily researchoriented cardiologists . fifth , we were not able to distinguish between inpatient and outpatient referrals , and that may have been a potential source of unmeasured confounding . in conclusion , we demonstrated that there is substantial variance among referring providers at a large academic medical center with respect to the proportion of positive results in requested coronary angiograms . these findings underscore the importance of accounting for risk factors in analyzing physician performance . this work was funded by the massachusetts general hospital physicians organization fellowship in health policy and management . massachusetts general hospital , amount $10 000 .

during the last two decades , effects of immunotherapy and autologous stem cell transplantation have been extensively studied in the treatment of human cancer . immunotherapy often includes cancer vaccines , but vaccine - induced anticancer reactivity is often not associated with significant clinical responses [ 13 ] . similarly , high - dose chemotherapy combined with autotransplantation has become a part of routine clinical practice only for a minority of cancer patients due to limited clinical benefits [ 4 , 5 ] . anticancer immune reactivity is probably important in autotransplantation , because early lymphoid reconstitution is associated with prolonged progression- or disease - free survival in many malignancies [ 6 , 7 ] . this has been described in patients with b - cell malignancies , acute myeloid leukemia ( aml ) , and solid tumors , suggesting that early reconstitution represents a general anticancer effect [ 710 ] . even though cancer patients often have both disease - associated and treatment - induced immune defects that may persist for several months , the combined use of autotransplantation and anticancer vaccines should be considered to try to increase anticancer effects . in the present paper we review the experience with intensive chemotherapy and immunotherapy for patients receiving intensive chemotherapy for aggressive hematological malignancies . we focus on acute myeloid leukemia ( aml ) , one of the most aggressive human malignancies that is usually treated with very intensive therapy eventually in combination with stem cell transplantation . the experience from these patients is that anticancer immune reactivity is maintained and can be induced after the intensive treatment . it seems likely that similar therapeutic strategies should be possible also in other patients receiving less intensive chemotherapy for less aggressive malignancies . patients with acute myeloid leukemia receive intensive chemotherapy followed by a period of severe leukopenia , but even these patients have a functional t cell system , and rapid lymphoid reconstitution is associated with a decreased risk of aml relapse [ 11 , 12 ] . circulating t cells are mainly t cell receptor ( tcr) with only a minority of tcr cells . chemotherapy - induced lymphopenia is not a random process and these patients have a decreased percentage of circulating clonogenic t cells . their most important growth factors are il-2 and il-15 , but several other cytokines can also cause detectable t cell proliferation . t cell proliferation can be induced through the tcr - cd3 complex even in the presence of aml accessory cells , and responses are increased by cd28 mediated costimulation . activated t cells release several cytokines ; high levels are detected for ifn , il-6 and gm - csf and detectable release of il-2 , il-3 , il-4 , il-10 , il-13 , and tnf is often seen . local t cell recruitment to the cancer cell compartment is essential for antileukemic t cell reactivity , and the chemotactic gradients are then determined by the systemic serum levels and the local levels in the cancer cell compartment . the systemic levels vary between patients and can be influenced by several factors , including the type of chemotherapy , patient age , and complicating febrile neutropenia . constitutive release of t cell chemotactic chemokines by the cancer cells may be an important determinant of the tissue chemokine levels . altogether , these observations clearly demonstrate that a functional t cell system remains during the severe posttreatment leukopenia even for patients receiving the most intensive conventional chemotherapy , and t cell targeting therapy may be possible even during this period . studies of immunological reconstitution after conventional intensive chemotherapy has been carried out for patients with nonhodgkin 's lymphoma , sarcomas and brain tumors , but these patients generally received less intensive chemotherapy than patients with aml [ 1923 ] . these studies have described a decrease in circulating cd4 cells that may last for several months , and there seems to be a predominance of memory - type ( cd4cd45rocd45ra ) cells . this defect seems to be at least partly age - dependent and is less pronounced in children . the number of cd3cd16cd56 nk lymphocytes is usually normalised within 6 weeks . it seems likely that similar defects are also seen after conventional chemotherapy for other malignancies , including the most intensive aml therapy . certain drugs seem to cause more severe cd4 t cell defects ; this is especially true for fludarabine that can be used as a part of conventional chemotherapy and in the reduced intensity conditioning before allogeneic stem cell transplantation . finally , the effect of chemotherapy on treg cells seem to differ between cytotoxic drugs as reviewed recently , myeloid suppressor cells can be transiently increased early after recovery from chemotherapy and the heterogeneity within the dendritic cell population varies during the recovery phase after chemotherapy . the status of all these three cell types may influence the response to anticancer vaccines . hematopoietic stem cells ( hsc ) are now preferably harvested from peripheral blood after growth factor mobilisation [ 28 , 29 ] , and this method is associated with earlier engraftment than hsc aspired from bone marrow . neutrophil engraftment with peripheral blood neutrophils > 0.1 10/l is usually seen within 1012 days and platelet engraftment with thrombocytes > 20 10/l within 1214 days [ 30 , 31 ] . the conclusion from the overall results is therefore very clear : ( i ) mobilisation of peripheral blood stem cells is safe and effective ; and ( ii ) the short time until hematopoietic reconstitution when using mobilised cells has increased the safety of the procedure . g - csf is the most widely used drug for stem cell mobilisation and is thought to stimulate immature stem cells to produce progenitors of all classes . in addition g - csf indirectly stimulates the production of progenitor cells by increasing the production of hematopoietic growth factors and it promotes the release of progenitors into the general circulation . when administered early after chemotherapy , g - csf acts synergistically with the natural increase in hsc that is seen during the recovery phase . the morbidity during and after g - csf mobilisation is very low , and the most frequent side effects are bone pain , fever , and nausea . successful mobilisation can also be achieved with longer - acting g - csf ( pegylated g - csf ) its main advantage is less frequent administration . a combination of g - csf plus stem cell factor ( scf ) will often more than double the yield of cd34 cells compared to g - csf alone [ 39 , 40 ] . the cxcr4 antagonist amd3100 can also be combined with g - csf to improve hsc mobilisation [ 4143 ] . finally , even though mobilisation regimens that include chemotherapy have more side effects than g - csf alone , the use of chemotherapy is appealing because insufficient mobilisation of cd34 cells is more common in cancer patients with g - csf alone , and the disease - specific chemotherapy in such regimens can have additional anticancer effects . autologous stem cell grafts are usually cryopreserved , and the protocols are generally based on the use of the cryoprotectant dimethyl sulphoxide ( dmso ) in the freezing medium [ 4547 ] . after harvesting , the final product is often diluted with autologous plasma if the nucleated cell concentration is higher than 200 10 cells / ml to improve cell viability . most centers will use a controlled programmed freezer and storage in nitrogen at 160c . the grafts are usually prepared by apheresis procedures alone without further enrichment of cd34 stem cells , and large numbers of immunocompetent cells are therefore reinfused together with the stem cells . early posttransplant lymphocyte reconstitution after both auto- and allotransplantation is associated with prolonged relapse - free survival in several malignancies [ 710 ] . for allotransplanted patients reconstitution of cd4 t cells seems particularly important , and infusion of a high number of cd4 t cells and nkt cells seems to be associated with a better prognosis . this may also be true for autografted patients , and these observations suggest that immunological events early after reinfusion are important for the risk of later relapse / progression . we investigated the viability of total lymphocytes and the distribution of various t cell subsets in peripheral blood stem cell autografts after long - term storage with 2% , 4% , 5% and 10% dmso . the viability of the total lymphocyte population was significantly higher for cells preserved in 4% and 5% dmso , but the dmso effect differed between t cell subsets ( table 1 ) . second , naive and central memory t cells usually express cd62l , late effector t cells show intermediate / low expression , while effector memory t cells do not express cd62l . third , the ccr7 chemokine receptor is expressed by naive and central memory t cells and directs migration to lymph nodes ; this homing process is important for initiation of immune responses . again finally , foxp3 positive t cells are referred to as thymus - derived natural t regulatory ( treg ) cells . the percentage of cd4cd25foxp3 cells among cd4 t cells was significantly lower after cryopreservation with 10% dmso . several nk cell subsets have now been characterised , including immunoregulatory ( cd3cd56cd16 ) and cytotoxic cells ( cd3cd56cd16 ) [ 5456 ] . cryopreservation with various dmso concentrations did not alter the percentages among viable graft lymphocytes of total nk cells ( cd3cd56 ) or the various nk cell subsets . taken together these results suggest that the procedures used for cryopreservation affect lymphocyte viability ; this is not a nonspecific effect but rather an effect that differs between various t cell subsets and it may thereby affect posttransplant t cell reconstitution and immunocompetence . the mobilisation and harvesting procedures would also be expected to influence the graft lymphocyte content but to the best of our knowledge this has not been investigated in clinical studies . the studies described above demonstrate that cytotoxic t cells are preserved and reinfused in autologous stem cell grafts . furthermore , recent studies have demonstrated that t cells specific for leukemia - associated antigens remain in the circulation after intensive aml chemotherapy ( see section 5 ) , and for this reason one would expect such cells also to be present in the autografts . at present it is not known whether reinfusion of such cells as a part of the transplantation procedure will have any clinical impact , or whether the immunocompetent cells in the graft will differ between various mobilisation procedures . it is possible to separate graft cells into cd34 and cd34 cells before freezing , and the cd34 subset could then be used for ex vivo enrichment of cancer - reactive t cells before reinfusion together with the cd34 cells . although techniques for enrichment of leukemia - reactive t cells are available , they have not been used in a large - scale clinical setting in combination with stem cell transplantation . as stated above , in vitro this strategy has not been widely used in the aggressive hematological malignancies , but the experiences from solid tumors ( e.g. , malignant melanoma ) suggest that such cells can induce clinically relevant antitumor activity [ 27 , 58 ] . in a recent study a comparable strategy was also used as a posttransplant treatment in allotransplanted patient ; donor - derived leukemia - reactive t cells were then reinfused in patients with leukemia relapse after allotransplantation . taken together these studies suggest that infusion of ex vivo generated cancer - reactive t cells can be combined with high - dose chemotherapy and possible also with vaccination strategies . t cell functions during the early posttransplant period with severe treatment - induced leukopenia seem to be very similar to aml patients receiving intensive conventional chemotherapy , and the later reconstitution also shows many similarities ( table 2 ) . however , after transplantation the absolute number of circulating cd3 cells usually remains decreased for three to five months . low levels of circulating cd4 cells have been reported for 1218 months , whereas cd8 t cells usually recover within 312 months . defective proliferation of both cd4 and cd8 t cells in response to anticd3 and anticd2 persists for at least for 24 months , and this seems to be caused by defective il-2 responsiveness [ 64 , 66 ] . furthermore , the specific cytotoxic t cell response against epstein - barr virus is significantly impaired for 25 months , whereas the frequency of circulating cytokine - secreting t helper cells and il-2 responding t cells can be decreased for up to 5 years posttransplant . peripheral blood mobilized stem cells ( pbmsc ) are now used for most cancer patients treated with autologous stem cell transplantation . the immunological reconstitution differs between patients receiving peripheral blood and bone marrow autografts , and the following data on t cell subset reconstitution refers to patients transplanted with mobilized stem cells . pbmsc autografted patients show early recovery of cd14 monocytes and cd56 nk cells during the first month after autotransplantation [ 64 , 69 , 70 ] . the homeostasis of total circulating dendritic cells is usually achieved relatively early after transplantation , although differences in dendritic cell subset composition may be detected for several months . however , as pointed out by these authors the kinetics of dendritic cell reconstitution may differ between patients and also depend on the chemotherapy regimen . in contrast , a long - lasting t cell defect similar to chemotherapy - treated patients is also observed after autotransplantation [ 69 , 70 ] . this defect is detected after 6 months for most patients , and for a minority the defect will last for more than a year . the total levels of cd8 t cells seem to normalize within a few months , whereas total cd4 t cell counts remain decreased for several months . the cd4 defect is mainly due to a reduction of naive cd3cd4cd45ra t cells , and there seems to be a reduction even of cd8 naive t cells [ 69 , 70 ] . immunogenetic factors may also be important for posttransplant t cell functions ; single nucleotide polimorphisms in immunoregulatory chemokine / cytokine genes seem to influence the risk of infections , graft versus host disease and leukemia relapse after allogeneic stem cell transplantation . such immunogenetic influences may also be important for the response to posttransplant vaccination therapy after conventional chemotherapy or stem cell transplantation . the number of mature b cells is markedly decreased during the first 3 months posttransplant , but will thereafter gradually increase although complete normalisation may take up to 18 months . both t cell - dependent and independent b cell response are decreased for 1218 months , and the igm production will normalise earlier ( often within 6 months ) than the corresponding igg response . the nk cell number is often increased on day 15 posttransplant compared with normal individuals , but by day 75 nk cell activity has usually returned to a normal level . nk cells are important for rejection of malignant cells , and pharmacological agents that increase cancer cell susceptibility to nk cell mediated lysis are now being developed . as described in a recent review the immunological reconstitution after allogeneic stem cell transplantation differ between patients receiving myeloablative and reduced intensity conditioning therapy . briefly , allotransplantation is also associated with a similar quantitative defect in cd4 t cells , this defect may last for several months but early normalization seems to be more common for patients receiving reduced intensity conditioning ( for detailed discussion and additional references see . a major question is whether it is possible to induce anticancer t cell reactivity and especially cytotoxicity in patients who have recently received intensive chemotherapy . t cells specific for cancer - associated antigens can be detected in healthy individuals and also in untreated cancer patients despite their disease - induced immunosuppression , and many of these cells seem to be cd8 cytotoxic effector memory cells . however , if it is possible to induce anticancer t cell reactivity by vaccination in aml patients who have a very aggressive disease and receive the most intensive chemotherapy , one would expect immunotherapy to be effective also in other cancer patients . several strategies for immunotherapy in aml have been tried ( table 3 ) . in this paper aml patients are very heterogeneous with regard to genetic abnormalities encoding leukemia - specific antigens . to find a common vaccination strategy the available studies have therefore focused on the use of either whole aml cells ( cell lysates or modulated aml cells ) or peptides derived from aml - associated proteins . wt1 is a zinc finger transcription factor that is expressed in normal cd34 hematopoietic cells , myoepithelal progenitors , renal podocytes , and some cells in testis and ovary . antibodies against this molecule have been detected in cancer patients and several immunogenic peptides have been identified . briefly , both cd4 and cd8 t cell epitopes have been identified , and hla - a0201 and hla - a24-restricted cd8 t cell cytotoxicity against wt1 expressing cancer cells have been detected . the cd4 peptides bind to different hla - class ii molecules and induce cd4 t cell responses that enhance cytotoxic t cell reactivity either through induction of cd4 cytotoxic t cells or through induction of cd4 th1 helper cells . finally , a recent study demonstrated that wt1-specific cytotoxic t cells remain even after remission - inducing intensive aml chemotherapy . a proteinase 3-derived peptide named pr1 has been identified by screening for binding avidity to hla - a0201 . in vitro studies suggest that pr1-specific t cells can kill leukemic cells , including the more immature clonogenic subsets , but not normal hematopoietic stem cells [ 78 , 80 , 81 ] . however , it should be emphasized that these data are mainly based on studies of healthy individuals and cancer patients receiving low - toxicity chemotherapy . untreated aml patients have also been investigated and circulating pr1 specific t cells could not be detected then . this may be due to apoptosis of high - avidity t cells induced by exposure to high peptide concentrations or leukemic cells overexpressing the proteinase 3 . however , pr1 specific t cells can be detected later after remission - inducing aml chemotherapy . the receptor for hyaluronic - acid - mediated motility ( rhamm ) is overexpressed in leukemic blasts from aml and cml patients but not in normal cd34 hematopoietic cells . greiner et al . identified a rhamm - derived peptide ( referred to as the rhamm - r3 peptide ) that could be presented by hla - a2 and recognised by cd8 t cells [ 78 , 83 ] . this peptide was a naturally processed t cell epitope , specific t cells were detected in aml patients even following intensive chemotherapy , and in vitro primed t cells could lyse human aml blasts . taken together these studies clearly demonstrate that cancer - specific , hla - restricted t cell reactivity , including specific cytotoxicity , is maintained even after the most intensive chemotherapy . oka et al . investigated the effect of wt1 peptide vaccination in 26 cancer patients , including 13 patients with de novo aml . they used intradermal injection of a modified 9-mer wt1 peptide emulsified in montanide isa51 adjuvant ; 18 of the 26 patients completed the vaccination protocol with 3 or more injections every second week and most patients were vaccinated with a modified peptide that gave stronger cytotoxic t cell responses than the natural peptide . all patients were hla - a2402 positive and their malignant cells showed high wt1 expression . tetramer flow cytometry of circulating cells showed an increase in specific t cells during vaccination for 9 of the 13 aml patients . an increase in antigen - specific induction of ifn expression was also observed for 6 of the patients . only 10 of these patients could be evaluated with regard to clinical responses : ( i ) 2 patients showed decreased residual aml ; ( ii ) stable disease was seen for 2 patients ; ( iii ) bone marrow expression of wt1 was used as a surrogate marker of residual disease for those patients without detectable aml blasts , and for 5 of these patients decreased expression was detected following vaccination ; ( iv ) 1 patient showed progressive disease . these observations suggest that wt1 vaccination can induce a specific t cell response ; these t cells can locate to the bone marrow compartment and they mediate wt1-specific antileukemic effects . another study examined a regimen with gm - csf therapy on days 14 and vaccination with antigenic peptide + keyhole limpet hemocyanine on day 3 . all patients were hla - a2 positive and had high expression of wt1 in their leukemia cells , 17 out of the 19 included patients had received previous chemotherapy and all patients had detectable aml with increased bone marrow blasts . the vaccines induced immunological responses judged from tetramer analyses of peripheral blood , and a significant increase of these cells was also seen in the bone marrow . importantly , responses were recorded especially in patients that had received previous chemotherapy and showed relatively low levels of bone marrow blasts ( < 50% ) . reduction of bone marrow wt1 levels was observed for a subset of patients following vaccination . thus , previous intensive chemotherapy does not eradicate leukemia - reactive t cells ; the chemotherapy - induced reduction of the aml cell burden rather seems to reduce disease - induced immune defects and thereby increase the efficiency of the vaccination . pr1 responses have been investigated in a study that combined vaccination with wt1 and pr1 peptides . this vaccine was also based on concomitant gm - csf administration and subcutaneous administration of peptides in montanide adjuvant . responses were evaluated by tetramer staining , and immunological responses were detected for one or both peptides in most patients . bone marrow expression of wt1 was used as a surrogate marker for residual disease , and these levels decreased when immunological responses became detectable . thus , even though in vitro exposure of pr1-specific t cells to aml cells with high antigen levels causes apoptosis of these cells ( see section 5.1 ) , detectable pr1 t cell responses could be induced early after induction chemotherapy . the rhamm - r3 peptide identified in previous in vitro studies has been tried for vaccination in hla - a2 patients with hematological malignancies . this study included only 10 patients with aml , mds , or multiple myeloma ; all 3 aml patients had received intensive chemotherapy before vaccination and the 4 myeloma patients had received autologous stem cell transplantation . the vaccine consisted of injection of 300 g peptide in incomplete freund adjuvant subcutaneously on day 3 , gm - csf was administered on days 1 and 5 , and this cycle was repeated 4 times with 2-weeks intervals . immunological responses were evaluated by tetramer flowcytometry and elispot analysis for ifn and granzyme b. an immunological response was detected by at least one of these methods for 9 patients , only 1 aml patient in relapse did not respond . vaccination - induced t cell cytotoxicity towards autologous aml cells or hla - a2rhamm target cells could also be detected . a clinical response with further reduction of bone marrow blasts this study illustrates that t cell reactivity against leukemia - associated antigenic epitopes is maintained after intensive conventional chemotherapy as well as autologous stem cell transplantation , and this reactivity can be enhanced by peptide vaccination . the clinical toxicity of vaccination was generally low , the most common side effect being grade 1 - 2 reactions with pain and erythema at the injection site . one study observed progressing leukopenia in two patients with mds and mds - aml , respectively ; this may be caused by immunological reactivity against normal stem cells in patients with disease - induced reduction of normal hematopoiesis . oka et al . also described a patient with a febrile reaction during the first injection . a recent article described two patients vaccinated with ex vivo generated monocytic dendritic cells that had been incubated with leukemic cell lysates and keyhole limpet hemocyanine . another study also investigated preparation of monocyte - derived dendritic cells in aml ; these authors combined ex vivo generation of the cells followed by cryopreservation before transfection of wt1 mrna by electroporation . it is difficult to see from these articles whether immunological responses were induced by the vaccination . an additional advantage with this approach could also be activation of nk cells and not only specific t cells and thereby induction of an additional anticancer effect . primary human aml cells can be induced to differentiate in the direction of a dendritic cell phenotype by exposure to various cytokines or cytokine combinations . these cells show dendritic morphology , increased expression of t cell costimulatory molecule , increased antigen - presenting capacity and a constitutive chemokine release profile consistent with a dendritic cell phenotype [ 89 , 90 ] . one study has reported the efficiency and toxicity when using a vaccine based on subcutaneous injection of dendritic aml cells . five patients treated in a palliative setting were included , and the authors observed increased immune responses towards a peptide derived from the leukemia - associated antigen prame . however , care should be taken when injecting ex vivo expanded cells , and possibly the cells should be irradiated before injection . even though the experience with dendritic cell vaccines in aggressive hematological malignancies is limited , the experience from other malignancies is promising . the dendritic cells orchestrate a repertoire of immune responses , but various dendritic cell subsets differ in their immunoregulatory characteristics . dendritic cell vaccines can thereby be used for cross - presentation of cancer - associated antigens ; a possible approach then being to load the cells with autologous cancer cell lysates . , a recent study in patients with lung cell cancer showed no serious side effects and increased t cell responsiveness to cancer - associated antigens for more than half of the patients vaccinated with antigen - loaded autologous dendritic cells . another strategy is viral transfection of cancer - associated antigens alone or antigens together with various immunostimulatory molecules [ 95 , 96 ] . the experiences from other cancers suggest that such therapeutic strategies should be further investigated also in hematological malignancies , and combination of chemotherapy and dendritic cell vaccination should then be possible . however , the optimal procedures for antigenic loading and dendritic cell preparation remain to be established . the general intensity of the chemotherapy has to be decided based on a clinical evaluation of the patients , and one has to take into consideration that there is evidence from clinical studies that a low cancer cell burden is associated with increased anticancer t cell reactivity [ 1 , 97 ] firstly , if possible one should use a regimen that induces immunogenic cancer cell apoptosis that will enhance anticancer immune reactivity ; this has been described especially for the anthracyclines ( figure 1 ) . second , if possible the chemotherapy should reduce the levels of regulatory t cells ( see section 6.3 ) . finally , the vaccine studies described above started at least 4 weeks after chemotherapy , but even patients with severe chemotherapy - induced cytopenia have an operative t cell system and immunotherapy can probably start even earlier after chemotherapy . firstly , additional disease - directed chemotherapy can be administered both for mobilisation and as high - dose intensive chemotherapy before transplantation ; this is safe and may further reduce the cancer cell burden . secondly , graft preparation offers the possibility to manipulate the immunocompetent graft cells and thereby combine chemotherapy and immunotherapy . cd34 enrichment is now possible as a part of routine therapy before cryopreservation , and by using similar methodological approaches as previously used in experimental studies , one could enrich anticancer cytotoxic t cells and reinfuse them as a part of the transplantation procedure . finally , even though both chemotherapy and autotransplantation seem to induce a general quantitative cd4 t cell defect , intensive chemotherapy does not eradicate cancer - specific t cells and enhancement of this reactivity in graft immunocompetent cells or posttransplant cells should therefore be possible . for example , animal experiments have shown that dendritic cells administered subcutaneously will localise to the draining lymph nodes , whereas intravenous administration will cause localisation to the spleen . several techniques are now available for preparation of the vaccines ; these strategies have been reviewed recently and are exemplified by the results summarized in figure 2 and tables 3 and 4 [ 82 , 104 ] . manipulation of immunocompetent cells through stimulation of toll - like receptors ( tlr ) seems to be of particular interest [ 108 , 109 ] . tlr9 is expressed by dendritic cells and b cells , and cpg oligonucleotides can be used as tlr9 agonists . these oligonucleotides will increase anticancer immune reactivity through several mechanisms , including increased presentation of cancer - associated antigens by dendritic cells [ 105107 , 110 ] ( figure 2 ) . these agents have been used alone , but they can also be used as adjuvants in cancer vaccines and be combined with the montanide adjuvant used in the peptide vaccines ( see section 5.2 ) . however , the overall results from experimental and clinical studies with regard to effects of adjuvants on t regulator cells ( treg ) are conflicting and require further studies . other adjuvants should also be tried in vaccination therapy , especially chemokines that are now considered as possible therapeutic targets in proinflammatory conditions . these mediators can be released by malignant cells and have several biological effects including chemotactic , growth - stimulatory , immunomodulatory , and angioregulatory effects [ 26 , 43 , 112 ] . other cytokines than gm - csf are now investigated as vaccine adjuvants , including flts - ligand as well as the chemokine ccl5 and cxcl9 [ 113115 ] . these experiences from animal cancer models suggest that such approaches should be tried also in the treatment of human cancers , although the possible roles of chemokine decoy receptors have to be explored . an alternative to in vivo expansion of antigen - specific t cells through vaccination would be ex vivo enrichment of specific cells followed by infusion of these cells . based on experimental observations various strategies may be possible , including ( i ) stimulation and thereby in vitro prokiferation / expansion of antigen - specific t cells ; or ( ii ) ex vivo generation of antigen - specific t cell reactivity through viral transduction of specific t cell receptor genes . first , antigen - loaded dendritic cells can be used to stimulate proliferation of cancer - specific cells , and this expansion can be increased by subsequent costimulation through cd28 [ 117 , 118 ] . second , t cell receptor gene - modified lymphocytes can be generated , such cells persist in patients after infusion and reduction of tumor cell burden has been described [ 120 , 121 ] . whether in vivo ( vaccination ) and ex vivo expansion ( in vitro culture ) of cancer - reactive t cells can be combined in cancer patients has not been clarified , and future studies also have to clarify whether these therapeutic strategies will be effective in hematologic malignancies . il-17 is a family of t cell derived cytokines that triggers the production of proinflammatory cytokines and chemokines by a wide range of cells , including epithelial cells , endothelial cells and macrophages [ 122 , 123 ] . in healthy individuals circulating th17 cells constitute less than 1.01.5% of total circulating t cells , increased levels are observed in patients with advanced cancers , and tumor infiltrating th17 cells have been detected in ovarian , pancreatic and renal cell cancer . several experimental observations suggest that th17 cells can increase specific antitumor immune activity as well as anticancer nk cell reactivity . these animal studies suggest that the role of th17 cells in human cancer should be further investigated , including the cryopreservation of th17 cells in autologous stem cell grafts and the possibility to enhance anticancer reactivity through ex vivo enrichment of th17 cells in the autografts before reinfusion . immunosuppressive treg cells comprise 515% of peripheral cd4 t cells [ 128 , 129 ] . in animal models these cells prevent autoimmune diseases , graft rejection and anticancer reactivity [ 128 , 130 ] . fludarabine therapy in patients with chronic lymphocytic leukemia often causes a decrease or abrogation of the activity of treg cells . suppression of treg cells by cyclophosphamide may allow immunotherapy of established tumors to be curative in animal models , but no effect is observed after a single cyclophosphamide infusion combined with nonspecific immunotherapy in patients with metastatic carcinoma . the mtor antagonist rapamycin that is used in anticancer therapy , increases the number of treg cells , whereas docetaxel does not seem to have any effect . thus , the effects of anticancer therapy on treg levels seem to differ between therapeutic agents . previous studies in aml have demonstrated that increased treg cells are detected in patients with newly diagnosed aml , and these high levels persist even after intensive chemotherapy and induction of disease control with hematological remission . thus , the overall intensity of the chemotherapy is not decisive for elimination of these cells ; rather the design and use of specific drugs seem to be essential . experimental studies have demonstrated that suppressive treg cells can be stimulated to develop into proinflammatory th17 cells . the possibility to use this approach instead of chemotherapy for elimination of treg cells and enhancement of immunoreactivity should be explored both with regard to ex vivo manipulation of stem cell grafts and in vivo immunomodulation . many of the vaccination studies reviewed above included only patients with certain hla - types known to bind and present the vaccine peptides . future studies have to consider how vaccination strategies should be designed to include all patients and not only selected subsets . other immunogenetic factors also need to be considered then , for example , genetic polymorphisms in the chemokine system or in t cell regulatory molecules . this is possible even for patients with the most aggressive hematological malignancies that are treated with very intensive chemotherapy , and it should therefore be possible also in other malignancies . the future challenge will now be to design optimal combinations of conventional disease - reducing therapy ( chemotherapy , surgery , and irradiation ) , induction of antigen - specific immunity through vaccination and antigen - nonspecific immunomodulation ( e.g. , targeting of treg and th17 cells as well as nk cells ) to enhance anticancer reactivity .

in vitro studies have demonstrated that cancer - specific t cell cytotoxicity can be induced both ex vivo and in vivo , but this therapeutic strategy should probably be used as an integrated part of a cancer treatment regimen . initial chemotherapy should be administered to reduce the cancer cell burden and disease - induced immune defects . this could be followed by autologous stem cell transplantation that is a safe procedure including both high - dose disease - directed chemotherapy and the possibility for ex vivo enrichment of the immunocompetent graft cells . the most intensive conventional chemotherapy and stem cell transplantation are used especially in the treatment of aggressive hematologic malignancies ; both strategies induce t cell defects that may last for several months but cancer - specific t cell reactivity is maintained after both procedures . enhancement of anticancer t cell cytotoxicity is possible but posttransplant vaccination therapy should probably be combined with optimalisation of immunoregulatory networks . such combinatory regimens should be suitable for patients with aggressive hematological malignancies and probably also for other cancer patients .

1. Introduction 2. Effects of Conventional Intensive Chemotherapy on T Cells 3. Immunocompetent Cells in Autologous Stem Cell Grafts 4. T Cell Reconstitution after Autotransplantation 5. Induction of Anticancer Reactivity by Vaccination Is Possible in Heavily Pre-Treated Patients 6. Future Directions

during the last two decades , effects of immunotherapy and autologous stem cell transplantation have been extensively studied in the treatment of human cancer . immunotherapy often includes cancer vaccines , but vaccine - induced anticancer reactivity is often not associated with significant clinical responses [ 13 ] . similarly , high - dose chemotherapy combined with autotransplantation has become a part of routine clinical practice only for a minority of cancer patients due to limited clinical benefits [ 4 , 5 ] . anticancer immune reactivity is probably important in autotransplantation , because early lymphoid reconstitution is associated with prolonged progression- or disease - free survival in many malignancies [ 6 , 7 ] . even though cancer patients often have both disease - associated and treatment - induced immune defects that may persist for several months , the combined use of autotransplantation and anticancer vaccines should be considered to try to increase anticancer effects . in the present paper we review the experience with intensive chemotherapy and immunotherapy for patients receiving intensive chemotherapy for aggressive hematological malignancies . we focus on acute myeloid leukemia ( aml ) , one of the most aggressive human malignancies that is usually treated with very intensive therapy eventually in combination with stem cell transplantation . the experience from these patients is that anticancer immune reactivity is maintained and can be induced after the intensive treatment . patients with acute myeloid leukemia receive intensive chemotherapy followed by a period of severe leukopenia , but even these patients have a functional t cell system , and rapid lymphoid reconstitution is associated with a decreased risk of aml relapse [ 11 , 12 ] . their most important growth factors are il-2 and il-15 , but several other cytokines can also cause detectable t cell proliferation . t cell proliferation can be induced through the tcr - cd3 complex even in the presence of aml accessory cells , and responses are increased by cd28 mediated costimulation . local t cell recruitment to the cancer cell compartment is essential for antileukemic t cell reactivity , and the chemotactic gradients are then determined by the systemic serum levels and the local levels in the cancer cell compartment . constitutive release of t cell chemotactic chemokines by the cancer cells may be an important determinant of the tissue chemokine levels . altogether , these observations clearly demonstrate that a functional t cell system remains during the severe posttreatment leukopenia even for patients receiving the most intensive conventional chemotherapy , and t cell targeting therapy may be possible even during this period . studies of immunological reconstitution after conventional intensive chemotherapy has been carried out for patients with nonhodgkin 's lymphoma , sarcomas and brain tumors , but these patients generally received less intensive chemotherapy than patients with aml [ 1923 ] . these studies have described a decrease in circulating cd4 cells that may last for several months , and there seems to be a predominance of memory - type ( cd4cd45rocd45ra ) cells . it seems likely that similar defects are also seen after conventional chemotherapy for other malignancies , including the most intensive aml therapy . certain drugs seem to cause more severe cd4 t cell defects ; this is especially true for fludarabine that can be used as a part of conventional chemotherapy and in the reduced intensity conditioning before allogeneic stem cell transplantation . finally , the effect of chemotherapy on treg cells seem to differ between cytotoxic drugs as reviewed recently , myeloid suppressor cells can be transiently increased early after recovery from chemotherapy and the heterogeneity within the dendritic cell population varies during the recovery phase after chemotherapy . g - csf is the most widely used drug for stem cell mobilisation and is thought to stimulate immature stem cells to produce progenitors of all classes . the morbidity during and after g - csf mobilisation is very low , and the most frequent side effects are bone pain , fever , and nausea . the cxcr4 antagonist amd3100 can also be combined with g - csf to improve hsc mobilisation [ 4143 ] . finally , even though mobilisation regimens that include chemotherapy have more side effects than g - csf alone , the use of chemotherapy is appealing because insufficient mobilisation of cd34 cells is more common in cancer patients with g - csf alone , and the disease - specific chemotherapy in such regimens can have additional anticancer effects . autologous stem cell grafts are usually cryopreserved , and the protocols are generally based on the use of the cryoprotectant dimethyl sulphoxide ( dmso ) in the freezing medium [ 4547 ] . we investigated the viability of total lymphocytes and the distribution of various t cell subsets in peripheral blood stem cell autografts after long - term storage with 2% , 4% , 5% and 10% dmso . the viability of the total lymphocyte population was significantly higher for cells preserved in 4% and 5% dmso , but the dmso effect differed between t cell subsets ( table 1 ) . the studies described above demonstrate that cytotoxic t cells are preserved and reinfused in autologous stem cell grafts . furthermore , recent studies have demonstrated that t cells specific for leukemia - associated antigens remain in the circulation after intensive aml chemotherapy ( see section 5 ) , and for this reason one would expect such cells also to be present in the autografts . at present it is not known whether reinfusion of such cells as a part of the transplantation procedure will have any clinical impact , or whether the immunocompetent cells in the graft will differ between various mobilisation procedures . it is possible to separate graft cells into cd34 and cd34 cells before freezing , and the cd34 subset could then be used for ex vivo enrichment of cancer - reactive t cells before reinfusion together with the cd34 cells . although techniques for enrichment of leukemia - reactive t cells are available , they have not been used in a large - scale clinical setting in combination with stem cell transplantation . as stated above , in vitro this strategy has not been widely used in the aggressive hematological malignancies , but the experiences from solid tumors ( e.g. in a recent study a comparable strategy was also used as a posttransplant treatment in allotransplanted patient ; donor - derived leukemia - reactive t cells were then reinfused in patients with leukemia relapse after allotransplantation . taken together these studies suggest that infusion of ex vivo generated cancer - reactive t cells can be combined with high - dose chemotherapy and possible also with vaccination strategies . t cell functions during the early posttransplant period with severe treatment - induced leukopenia seem to be very similar to aml patients receiving intensive conventional chemotherapy , and the later reconstitution also shows many similarities ( table 2 ) . furthermore , the specific cytotoxic t cell response against epstein - barr virus is significantly impaired for 25 months , whereas the frequency of circulating cytokine - secreting t helper cells and il-2 responding t cells can be decreased for up to 5 years posttransplant . peripheral blood mobilized stem cells ( pbmsc ) are now used for most cancer patients treated with autologous stem cell transplantation . the immunological reconstitution differs between patients receiving peripheral blood and bone marrow autografts , and the following data on t cell subset reconstitution refers to patients transplanted with mobilized stem cells . the homeostasis of total circulating dendritic cells is usually achieved relatively early after transplantation , although differences in dendritic cell subset composition may be detected for several months . the total levels of cd8 t cells seem to normalize within a few months , whereas total cd4 t cell counts remain decreased for several months . immunogenetic factors may also be important for posttransplant t cell functions ; single nucleotide polimorphisms in immunoregulatory chemokine / cytokine genes seem to influence the risk of infections , graft versus host disease and leukemia relapse after allogeneic stem cell transplantation . such immunogenetic influences may also be important for the response to posttransplant vaccination therapy after conventional chemotherapy or stem cell transplantation . as described in a recent review the immunological reconstitution after allogeneic stem cell transplantation differ between patients receiving myeloablative and reduced intensity conditioning therapy . briefly , allotransplantation is also associated with a similar quantitative defect in cd4 t cells , this defect may last for several months but early normalization seems to be more common for patients receiving reduced intensity conditioning ( for detailed discussion and additional references see . a major question is whether it is possible to induce anticancer t cell reactivity and especially cytotoxicity in patients who have recently received intensive chemotherapy . t cells specific for cancer - associated antigens can be detected in healthy individuals and also in untreated cancer patients despite their disease - induced immunosuppression , and many of these cells seem to be cd8 cytotoxic effector memory cells . however , if it is possible to induce anticancer t cell reactivity by vaccination in aml patients who have a very aggressive disease and receive the most intensive chemotherapy , one would expect immunotherapy to be effective also in other cancer patients . briefly , both cd4 and cd8 t cell epitopes have been identified , and hla - a0201 and hla - a24-restricted cd8 t cell cytotoxicity against wt1 expressing cancer cells have been detected . the cd4 peptides bind to different hla - class ii molecules and induce cd4 t cell responses that enhance cytotoxic t cell reactivity either through induction of cd4 cytotoxic t cells or through induction of cd4 th1 helper cells . in vitro studies suggest that pr1-specific t cells can kill leukemic cells , including the more immature clonogenic subsets , but not normal hematopoietic stem cells [ 78 , 80 , 81 ] . however , it should be emphasized that these data are mainly based on studies of healthy individuals and cancer patients receiving low - toxicity chemotherapy . however , pr1 specific t cells can be detected later after remission - inducing aml chemotherapy . this peptide was a naturally processed t cell epitope , specific t cells were detected in aml patients even following intensive chemotherapy , and in vitro primed t cells could lyse human aml blasts . taken together these studies clearly demonstrate that cancer - specific , hla - restricted t cell reactivity , including specific cytotoxicity , is maintained even after the most intensive chemotherapy . they used intradermal injection of a modified 9-mer wt1 peptide emulsified in montanide isa51 adjuvant ; 18 of the 26 patients completed the vaccination protocol with 3 or more injections every second week and most patients were vaccinated with a modified peptide that gave stronger cytotoxic t cell responses than the natural peptide . these observations suggest that wt1 vaccination can induce a specific t cell response ; these t cells can locate to the bone marrow compartment and they mediate wt1-specific antileukemic effects . all patients were hla - a2 positive and had high expression of wt1 in their leukemia cells , 17 out of the 19 included patients had received previous chemotherapy and all patients had detectable aml with increased bone marrow blasts . thus , previous intensive chemotherapy does not eradicate leukemia - reactive t cells ; the chemotherapy - induced reduction of the aml cell burden rather seems to reduce disease - induced immune defects and thereby increase the efficiency of the vaccination . thus , even though in vitro exposure of pr1-specific t cells to aml cells with high antigen levels causes apoptosis of these cells ( see section 5.1 ) , detectable pr1 t cell responses could be induced early after induction chemotherapy . the rhamm - r3 peptide identified in previous in vitro studies has been tried for vaccination in hla - a2 patients with hematological malignancies . this study included only 10 patients with aml , mds , or multiple myeloma ; all 3 aml patients had received intensive chemotherapy before vaccination and the 4 myeloma patients had received autologous stem cell transplantation . vaccination - induced t cell cytotoxicity towards autologous aml cells or hla - a2rhamm target cells could also be detected . a clinical response with further reduction of bone marrow blasts this study illustrates that t cell reactivity against leukemia - associated antigenic epitopes is maintained after intensive conventional chemotherapy as well as autologous stem cell transplantation , and this reactivity can be enhanced by peptide vaccination . one study observed progressing leukopenia in two patients with mds and mds - aml , respectively ; this may be caused by immunological reactivity against normal stem cells in patients with disease - induced reduction of normal hematopoiesis . another study also investigated preparation of monocyte - derived dendritic cells in aml ; these authors combined ex vivo generation of the cells followed by cryopreservation before transfection of wt1 mrna by electroporation . primary human aml cells can be induced to differentiate in the direction of a dendritic cell phenotype by exposure to various cytokines or cytokine combinations . however , care should be taken when injecting ex vivo expanded cells , and possibly the cells should be irradiated before injection . even though the experience with dendritic cell vaccines in aggressive hematological malignancies is limited , the experience from other malignancies is promising . dendritic cell vaccines can thereby be used for cross - presentation of cancer - associated antigens ; a possible approach then being to load the cells with autologous cancer cell lysates . , a recent study in patients with lung cell cancer showed no serious side effects and increased t cell responsiveness to cancer - associated antigens for more than half of the patients vaccinated with antigen - loaded autologous dendritic cells . the experiences from other cancers suggest that such therapeutic strategies should be further investigated also in hematological malignancies , and combination of chemotherapy and dendritic cell vaccination should then be possible . the general intensity of the chemotherapy has to be decided based on a clinical evaluation of the patients , and one has to take into consideration that there is evidence from clinical studies that a low cancer cell burden is associated with increased anticancer t cell reactivity [ 1 , 97 ] firstly , if possible one should use a regimen that induces immunogenic cancer cell apoptosis that will enhance anticancer immune reactivity ; this has been described especially for the anthracyclines ( figure 1 ) . second , if possible the chemotherapy should reduce the levels of regulatory t cells ( see section 6.3 ) . finally , the vaccine studies described above started at least 4 weeks after chemotherapy , but even patients with severe chemotherapy - induced cytopenia have an operative t cell system and immunotherapy can probably start even earlier after chemotherapy . firstly , additional disease - directed chemotherapy can be administered both for mobilisation and as high - dose intensive chemotherapy before transplantation ; this is safe and may further reduce the cancer cell burden . secondly , graft preparation offers the possibility to manipulate the immunocompetent graft cells and thereby combine chemotherapy and immunotherapy . finally , even though both chemotherapy and autotransplantation seem to induce a general quantitative cd4 t cell defect , intensive chemotherapy does not eradicate cancer - specific t cells and enhancement of this reactivity in graft immunocompetent cells or posttransplant cells should therefore be possible . tlr9 is expressed by dendritic cells and b cells , and cpg oligonucleotides can be used as tlr9 agonists . these agents have been used alone , but they can also be used as adjuvants in cancer vaccines and be combined with the montanide adjuvant used in the peptide vaccines ( see section 5.2 ) . these experiences from animal cancer models suggest that such approaches should be tried also in the treatment of human cancers , although the possible roles of chemokine decoy receptors have to be explored . an alternative to in vivo expansion of antigen - specific t cells through vaccination would be ex vivo enrichment of specific cells followed by infusion of these cells . based on experimental observations various strategies may be possible , including ( i ) stimulation and thereby in vitro prokiferation / expansion of antigen - specific t cells ; or ( ii ) ex vivo generation of antigen - specific t cell reactivity through viral transduction of specific t cell receptor genes . first , antigen - loaded dendritic cells can be used to stimulate proliferation of cancer - specific cells , and this expansion can be increased by subsequent costimulation through cd28 [ 117 , 118 ] . second , t cell receptor gene - modified lymphocytes can be generated , such cells persist in patients after infusion and reduction of tumor cell burden has been described [ 120 , 121 ] . whether in vivo ( vaccination ) and ex vivo expansion ( in vitro culture ) of cancer - reactive t cells can be combined in cancer patients has not been clarified , and future studies also have to clarify whether these therapeutic strategies will be effective in hematologic malignancies . il-17 is a family of t cell derived cytokines that triggers the production of proinflammatory cytokines and chemokines by a wide range of cells , including epithelial cells , endothelial cells and macrophages [ 122 , 123 ] . these animal studies suggest that the role of th17 cells in human cancer should be further investigated , including the cryopreservation of th17 cells in autologous stem cell grafts and the possibility to enhance anticancer reactivity through ex vivo enrichment of th17 cells in the autografts before reinfusion . fludarabine therapy in patients with chronic lymphocytic leukemia often causes a decrease or abrogation of the activity of treg cells . suppression of treg cells by cyclophosphamide may allow immunotherapy of established tumors to be curative in animal models , but no effect is observed after a single cyclophosphamide infusion combined with nonspecific immunotherapy in patients with metastatic carcinoma . previous studies in aml have demonstrated that increased treg cells are detected in patients with newly diagnosed aml , and these high levels persist even after intensive chemotherapy and induction of disease control with hematological remission . experimental studies have demonstrated that suppressive treg cells can be stimulated to develop into proinflammatory th17 cells . the possibility to use this approach instead of chemotherapy for elimination of treg cells and enhancement of immunoreactivity should be explored both with regard to ex vivo manipulation of stem cell grafts and in vivo immunomodulation . many of the vaccination studies reviewed above included only patients with certain hla - types known to bind and present the vaccine peptides . other immunogenetic factors also need to be considered then , for example , genetic polymorphisms in the chemokine system or in t cell regulatory molecules . this is possible even for patients with the most aggressive hematological malignancies that are treated with very intensive chemotherapy , and it should therefore be possible also in other malignancies . the future challenge will now be to design optimal combinations of conventional disease - reducing therapy ( chemotherapy , surgery , and irradiation ) , induction of antigen - specific immunity through vaccination and antigen - nonspecific immunomodulation ( e.g.

leptospirosis is an emerging zoonotic disease of global importance and is now the leading cause of hemorrhagic disease . humans are usually infected following contact with the urine of reservoir animals via contaminated soil or water . while infection is asymptomatic in wild rodents and other reservoirs , in humans and other accidental hosts , it can cause severe clinical disease : hepato - renal failure ( weil s disease ) , pulmonary hemorrhage syndrome ( lphs ) , and even death . lphs and weil s disease have case fatalities of > 50% and > 10% , respectively . the development of disease complications is thought to depend on infectious dose or host immunity . protective immunity during naturally acquired infection is associated with a humoral immune response , indicated by increased production of agglutinating antibodies following infection and animal passive transfer studies . a major hurdle in the development of effective vaccines and serodiagnostic against complex microorganisms that encode thousands of proteins is the identification of a limited number of antigens that will induce a protective immune response . the advent of high - throughput sequencing during the past decade has made possible an approach called reverse vaccinology , which takes advantage of bioinformatics to narrow down the number of potential vaccine antigen candidates . some proteomic features possibly associated with antigenicity and vaccine efficacy , such as extracellular location , outer - membrane proteins , signal peptides , and b- and t - cell epitopes , can be computationally predicted based on the amino acid sequence alone , reducing the number of candidates to several hundred . although bioinformatic approaches can be used to remove groups of antigens from consideration as vaccine and serodiagnostic candidates , it tends to produce large lists of potential candidates that require additional expensive and time - consuming laboratory investigations to further narrow down the candidates . bioinformatic approaches to antigen prediction are also inherently oversimplistic , and bona fide antigens can be missed . more recently , whole proteome microarray chips , developed in our lab and by others , have provided an empirical approach to interrogate the entire proteome of any microorganism and to efficiently determine antibody profiles associated with acute or chronic infection or with vaccine mediated protection from infection . proteomic studies are a powerful tool for elucidating the molecular mechanisms involved in cellular functioning as well as disease development and progression . protein microarray chips are particularly effective for the study of human serum samples , defining the antibody immune response against infectious agents on a proteomic scale and enabling the access to the complete antibody repertoire produced during an infection . additionally , the protein array usually encompasses thousands of proteins , representing the entire encoded proteome . the individual proteins printed on these chips capture antibodies in serum from infected individuals , which can be quantified using a fluorescent secondary antibody . in this fashion , antibodies produced after infection can be identified , characterizing the type of infection . the objective of proteomic studies is to provide a more complete understanding of the immune response to infection , also allowing the identification of novel serodiagnostic and prognostic markers as well as potential subunit vaccines . the mechanism for host recognition remains unclear , and the ability to predict antigenicity in silico is imperfect . usually only a relatively small subset of the proteome of infectious agents , such as l. interrogans , is recognized by the immune system , and little is known about the characteristics of these antigens . consequently , our group has used proteome microarray data and enrichment analyses to identify proteomic features observed in the immunodominant and serodiagnostic antigen sets for more than 25 medically important agents and has successfully identified antigens that were later employed in a variety of diagnostic platforms . among the organisms studied we have continued this iterative approach , first using a proteome microarray to identify the spectrum of immunoreactive l. interrogans antigens recognized in human leptospirosis cases and then classifying their reactivity according to annotated functional and computationally predicted features . these results inform us about the benefits and limitations of antigen prediction and provide a framework for future studies to improve predictive capability for serodominant antigens . the institutional review board committees of yale university and oswaldo cruz foundation approved the study protocol . samples from infected patients came from the following projects : natural history of urban leptospirosis ( r01ai052473 ) , disease determinants of urban leptospirosis ( u01ai088752 ) , and ecoepidemiology of leptospirosis ( r01tw009504 ) . after collection , a unique code identifier was assigned to each sample so that all samples were deidentified for researchers before their use . the study was conducted with a group of 90 laboratory - confirmed leptospirosis patients from the state of bahia , brazil , including 30 patients with mild clinical presentations , 31 patients with severe disease , and 30 patients who died due to leptospiral infection . laboratory confirmation was defined based on the results of the microagglutination test ( mat ) and according to the criteria of seroconversion or a four - fold rise in titer in patients with paired serum sample or a single titer of 1:800 in patients with only one serum sample . sera samples from patients with severe leptospirosis were collected at three different time points and are designated as follows : ( i ) early acute sample , collected at patient admittance at the health care unit , ( ii ) late acute sample , collected 2 to 3 days after early acute sample collection , and ( iii ) convalescent sample , collected at least 14 days after the first sampling . for patients with mild leptospirosis , no late acute sample was collected ; that is , only early acute and convalescent samples were provided . an early acute sample was collected from all deceased patients , but only five patients from this group survived through the late acute sampling . samples were organized in groups , separated by clinical presentation ( mild , severe , or deceased ) and by time point ( early acute , late acute , or convalescent ) so that a total of 188 samples were categorized into seven groups . the complete orfeome of leptospira interrogans serovar copenhageni strain fiocruz l1 - 130 was amplified by pcr and cloned into pxi vector using a high - throughput pcr recombination cloning method described elsewhere . the cloning strategy allows the expression of recombinant proteins containing an n - terminal hemaglutinin ( ha ) tag and a c - terminal poly histidine ( his ) tag . genes larger than 3 kb were cloned as smaller segments as previously described , and the liga and ligb genes ( lic10465 and lic10464 , respectively ) were fragmented according to the repeated big domains present in the structure of each protein ( ligb repeats 712 , liga repeats 713 , and liga / b repeats 16 ) , which are recognized by human sera as previously described . recombinant plasmids were confirmed by pcr using the insert specific primers for amplification . after identifying the seroreactive antigens on the microarrays , the inserts in the corresponding plasmids microarray fabrication was performed as previously described . in brief , purified mini - preparations of dna were used for expression in a 10 l e. coli in - vitro - based transcription - translation ( ivtt ) reaction system ( rts kit , roche ) for 16 h at 26 c with shaking ( 300 rpm ) according to the manufacturer s instructions . negative control reactions were those performed in the absence of dna template ( nodna controls ) . a protease inhibitor mixture ( complete , roche ) and tween-20 ( 0.5% v / v final concentration ) were added to the reactions to minimize protein degradation and improve protein solubilization . unpurified supernatants were immediately printed onto nitrocellulose - coated glass fast slides using an omni grid 100 microarray printer ( genomic solutions ) together with multiple negative control reactions and positive control spots of an igg mix containing mouse , rat , and human igg and igm ( jackson immuno research ) . protein expression was verified by probing the array with monoclonal antipolyhistidine ( sigma - aldrich ) and antihemaglutinin ( roche applied science ) diluted 1/400 in protein array blocking buffer ( whatman ) , as previously described . probing with human sera samples was performed with samples diluted 1/100 in protein array blocking buffer ( whatman ) supplemented with e. coli lysate 10 mg / ml ( mclab ) at a final concentration of 10% v / v and incubated 30 min at room temperature with constant mixing . antibodies bound to e. coli proteins were removed by centrifugation prior to addition to the microarray . arrays were blocked for 30 min with protein array blocking buffer and then incubated with diluted samples overnight at 4 c with gentle rocking . washes and incubation with conjugate antibodies slides were scanned in a perkinelmer scanarray confocal laser and intensities were quantified using quantarray package . enrichment analysis was performed based on leptospira interrogans serovar copenhageni l1 - 130 strain genome annotations available in the national center for biotechnology information ( ncbi ) and john craig venter institute ( jcvi ) databases . the antigens were classified according to annotated functional features and computationally predicted features . the clusters of orthologous groups ( cog ) information and the mainrole classification utilized can be found at ncbi and jcvi web sites . the following programs were utilized for computational prediction : tmhmm v2.0 for trans - membrane domains prediction ( http://www.cbs.dtu.dk/services/tmhmm/ ) ; signalp v3.0 for signal peptide prediction ( http://www.cbs.dtu.dk/services/signalp/ ) ; and psortb v3.0 software for subcellular location prediction ( http://www.psort.org/psortb/ ) . proteins with potentially biological importance , such as mass spectrometry ( ms ) evidence of in vitro expression or in vivo mrna up - regulation , were also submitted to enrichment analysis . spot intensity raw data were obtained as the mean pixel signal intensity with automatic correction for spot - specific background . for each array , the average of control ivtt reactions ( nodna controls ) was subtracted from spot signal intensities to minimize background reactivity . positive expression of proteins was determined by a signal intensity of 2.5 standard deviation ( sd ) above the mean of nodna control reactions for either the his or ha tags . the same cutoff was applied to identify the reactive proteins using the sera collection . antigens were classified as seroreactive if ( i ) the average signal intensity of a sera group was above the established cutoff or ( ii ) at least 33% of the samples within a group showed individual signal intensity above the cutoff . our rationale for using this second criterion was to include antigens that may not be strongly recognized by all of the patients but are recognized by a significant proportion of them . when segments of fragmented proteins showed reactivity , enrichment analysis was performed considering the annotation / predictions of the original protein . we amplified the 3667 predicted orfs from l. interrogans serovar copenhageni from genomic dna and cloned them into the pxi expression vector , as full or partial length proteins , using the high - throughput recombination cloning method developed by our group . cloning efficiency was 94% , and all cloned orfs were expressed under a t7 promoter in the e. coli in vitro transcription / translation system . microarray probing with anti - his and anti - ha antibodies revealed an expression level of 91% of all 3819 proteins and fragments printed on the array ( 3359 of 3667 total orfs ) . a list of the proteins not represented on the microarrays is provided in table s1 in the supporting information . we probed leptospiral protein arrays with a collection of 188 serum samples , composed of longitudinal samples from patients with mild and severe clinical presentations of leptospirosis , at different phases of the disease , as well as patients that died from acute leptospirosis infection . analyzing longitudinal samples increased the likelihood of detecting an antigen with transient seroreactivity . in figure 1 , we show representative histograms of the number of reactive antigens selected from the convalescent time point for the severe patient group ( n = 30 ) using the inclusion criteria ( described in the materials and methods ) . for some antigens , the high average signal intensity is due to the strong reactivity of a few patients , as observed by the five orange dots below the orange dotted line in figure 1b . we also observed that antigens with lower average signal intensity show positive reactivity in a few patients , as observed by the last 17 antigens in figure 1a . an antigen was considered to be reactive if either the group average signal intensity or at least 33% of the samples within a group showed signal intensity above 2.5 standard deviations of the nodna control reactions . the histogram plots the average signal intensity ( y axis ) and the number of responsive individuals ( secondary y axis ) for each reactive antigen selected ( x axis ) for igm ( a ) or igg ( b ) probing of convalescent - phase samples from severe patients . dotted lines correspond to the control reactions cutoff ( black ) or the minimum number of responsive individuals ( orange ) included using this criteria . by applying these criteria to all groups of samples separately of these , 49 were reactive for both igm and igg antibodies , 61 were reactive only for igm and , 81 were only for igg ( figure 2 ) . in this work , we investigated the proteomic features shared by antigens that are recognized by human sera because these features might increase their likelihood to be seroreactive . the aim is to identify all leptosprial antigens that are reactive in humans , without excluding cross - reactive antigens reactive in healthy unexposed individuals . a complete evaluation of the antibody immune response during naturally acquired leptospirosis compared with healthy controls , along with the analysis of new diagnostic and vaccine candidates , will be the focus of a separate study . heat map showing the overall igm and igg reactivity detected for mild , severe , and deceased patients . antigens are shown in rows , grouped as reactive for both igm and igg antibodies , or for igm- or igg - only antibodies ; patients are in columns , organized from left to right by the increasing average reactivity detected for the reactive antigens . samples from patients with mild leptospirosis are shown in green , patients with severe presentations are in yellow , and deceased patients are in blue . l. interrogans serovar copenhageni proteins are annotated with ncbi clusters of orthologous groups ( cog ) functional categories ( table s2 in the supporting information ) . each cog is defined by a group of three or more proteins that are inferred to be orthologs , that is , they are direct evolutionary counterparts and represent a conserved functional category . of the total 3667 leptospiral proteins , 1313 are not associated with cogs , whereas 500 proteins are associated with cogs but have not been assigned to a specific function , which are shown as general function prediction only ( category r ) or function unknown ( category s ) . proteins with predicted cogs n ( cell motility and secretion ) and u ( intracellular trafficking and secretion ) were significantly enriched for both igm and igg reactivity , with significant fold increases of 4.4 ( n ) and 5.9 ( u ) for igm and 3.8 ( n ) and 5.1 ( u ) for igg , respectively ( table 1 ) . we also found that proteins predicted as cog d ( cell division and chromosome partitioning ) were 4.5 times enriched in igm responses . interestingly , we determined that the igg response to cogs s ( unknown function ) and v ( defense mechanism ) was 1.9- and 3.7-fold enriched , respectively , in igg response , although these enrichments were less significant than cogs n and u. cog e ( amino acid transport and metabolism ) was lacking for igg reactivity despite 4% of the l. interrogans orfeome being classified in this category ( table 1 ) . leptospiral proteins are also annotated with jcvi mainrole classification functional categories ( table s3 in the supporting information ) . a total of 305 leptospiral proteins are not classified in any category , whereas 1257 proteins are assigned to a category but have no specific function prediction , shown as unknown function or hypothetical protein . because some proteins have multiple functional assignments , we identified 3212 proteins predicted with jcvi mainrole classification in total for the 3667 proteins represented on the chip . similar to the cog categories enriched in patient sera , we found that proteins predicted as cell envelope and cellular processes were significantly enriched for both igm and igg reactivity , with fold increases of 2.2 and 3.7 for igm and 2.3 and 3.0 for igg , respectively ( table 1 ) . we observed that antigens classified in the protein fate category in igm responses , which is related to protein folding , stabilization , and degradation , were enriched 2.7-fold . similarly to the cogs classifications , we found that proteins categorized as enzymes involved in energy metabolism were under - represented in the igm response , with a fold decrease of 0.3 ( table 1 ) . interestingly , the jcvi class of proteins that comprised most of the reactive antigens was the hypothetical proteins category , with 23 and 29% of igm- and igg - reactive antigens , respectively . we also analyzed the enrichment of antigens using computationally predicted features to identify motifs that are enriched in the humoral immune response to leptospira infection ( tables s4s6 in the supporting information ) . as shown in table 1 , we found that proteins containing 1 transmembrane domain were highly significantly enriched for both igm and igg antibodies reactivity , with fold increases of 2.0 and 1.7 , respectively . indeed , we observed that proteins lacking transmembrane domains were an under - represented feature in both responses , with fold decreases of 0.8 ( igm ) and 0.4 ( igg ) . interestingly , we observed that proteins with more than one predicted transmembrane domain were under - represented in igg responses ( 0.3 fold change ) . conversely , we determined that proteins with predicted signal peptides were significantly enriched in both igm and igg responses , with fold increases of 2.3 and 2.4 , respectively . accordingly , outer membrane proteins were also significantly enriched in both responses ( 3.2 and 4.1 fold increase ) , and proteins predicted as cytoplasmic localization were significantly under - represented ( 0.8 and 0.6 fold change ) . finally , prediction of subcellular localization as extracellular was enriched for igg reactivity , with a fold increase of 2.7 . although the majority of the antibody reactive antigens lack both a signal peptide and a transmembrane domain or are classified as cytoplasmic by psort , these groups account for a larger proportion of all of the computationally classified proteins in the l. interrogans proteome . another potential characteristic that could influence protein antigenicity is the in vivo expression level because leptospiral proteins expressed at higher levels have a higher probability of being recognized by the host immune system . therefore , we interfaced our seroreactive data with two existing data sets : ( i ) ms data obtained from in vitro cultured bacteria and ( ii ) a transcriptome study that identified differentially expressed genes when the bacteria was grown in vivo in dialysis membrane chambers ( dmcs ) implanted in rats . more than 50% ( 102/191 ) of the seroreactive antigens identified in our study were identified in vitro by ms ( figure 3a ) , making this method an enriching feature for both igm and igg seroreactivity ( table 1 ) . the up - regulated leptospiral genes identified in vivo were also enriched among the seroreactive antigens , with a fold change of 2.5 and 2.9 for igm and igg , respectively ( table 1 ) . ( a ) venn diagram of the leptospiral proteins identified by mass spectrometry when the bacteria were grown in vitro ( red ) ; proteins for which the corresponding mrna was up - regulated when the bacteria were cultivated in dialysis membrane chambers in vivo ( green ) ; and antibody reactivity in leptospirosis patients detected by protein microarray ( blue ) . ( b ) scatter plots with the antibody reactivity and number of protein copies / cell detected by mass spectrometry . array average signal intensity for igm or igg probing is plotted on the x axis ; the number of protein copies per cell detected in vitro by mass spectrometry is shown on the y axis . even though we observed a significant overlap between the number of seroreactive antigens identified in this study and the number of proteins detected in vitro by ms , there was no linear correlation between the number of protein copies per bacterium cell and the intensity of antibody reactivity detected on the protein arrays ( figure 3b ) . few proteins had both high cellular concentrations and showed strong antibody response , as exemplified by lic12966 , lic13050 , lic10191 , and the lig proteins ( lic10464 and lic10465 ) in figure 3b . notably , > 60% ( 67/108 ) of the up - regulated genes in vivo were not detected by ms under regular cultivation conditions , and only 15/108 up - regulated genes were also serodominant . a major component of the adaptive immune response to infection is the generation of protective and long - lasting humoral immunity , but factors governing selection of the antigens recognized by the immune system are largely unknown . it is not uncommon for viruses encoding a small number of proteins to elicit antibody responses against each structural protein . in contrast , for infectious bacteria or parasites that encode hundreds or thousands of proteins , only a subset of the proteome is recognized by the humoral immune response . here we describe utilization of a leptospira interrogans proteome microarray to determine empirically the entire antibody repertoire of infected individuals . this technique allows the identification of the types of proteomic structural , physical chemical , and functional features that are recognized more frequently by the human immune system . we previously identified a set of serodiagnostic antigens from a partial l. interrogans proteome . an important difference between the present study and this previous report is that analysis of the complete proteome not only has allowed us to delineate additional serodiagnostic antigens but , more fundamentally , also has provided the basis for a rigorous , comprehensive , and quantitative determination of the protein characteristics of the entire set of the serodominant antigens on a genomic scale . our results represent a large - scale evaluation of l. interrogans proteins that are antigenic in the context of naturally acquired human infection . our group has previously shown that > 90% of the urban leptospirosis cases in brazil are caused by l. interrogans serovar copenhageni and has isolated strain l1 - 130 from an infected patient from the city of salvador , brazil . the homogeneity of pathogen exposure and the use of a bacterial strain isolated from our study site minimize possible errors due to the natural diversity of leptospira strains . additionally , the availability of strain l1 - 130 complete genome sequence makes it ideal for this proteomic study . we identified 191 immunodominant protein antigens with either igm or igg recognition , which correspond to 6% of the leptospiral coding genome . this relatively small set of antigens represents the complete repertoire of antibodies that are generated during symptomatic leptospiral infections . mounting an immune response against a limited set of antigens may have the advantage of minimizing energy consumption and avoiding an excessive innate inflammatory reaction or cross - reactive autoimmune responses while still controlling infection effectively . we classified the annotated functional proteomic features that are associated with antigenicity in humans . enrichment analysis found 14 protein features that were enriched in the seroreactive antigens , of which 9 had both igm and igg responses ( table 1 ) . interestingly , we identified features that were significantly enriched exclusively in igm or in igg responses . accounting for these differences in enrichment categories between igg and igm antigen targets may be related to class switching from igm to igg and affinity maturation of the igg . the most significant enriched or under - represented features , however , were the ones detected for both igm and igg antibodies . similar studies from our group with brucella melitensis and burkholderia pseudomallei corroborate the enriching features identified for leptospira ( table 1 ) . the most significant positive predictive features identified in this study , a signal peptide , 1 transmembrane domain , cog u ( intracellular trafficking and secretion ) , and cog n ( cell motility and secretion ) , were also enriched for b. melitensis . accordingly , artemis - defined surface proteins ( outer membrane , inner membrane , secreted , and surface structures ) were enriched for b. pseudomallei . the enriched ncbi cog categories n and u are both related to protein secretion , and the externalization of such proteins can easily explain their accessibility to the host immune system . the presence of a signal peptide is a cellular tag for secretion or anchoring a protein on membranes , as we also found that proteins with one or more transmembrane domains , as well as outer - membrane subcellular location , were enriched . these studies demonstrate that antigens are not randomly recognized by the host immune response . rather , the immune system focuses on specific types of antigens . armed with this information and the increasing availability of genome sequences , we can begin to make generalizations to make a priori predictions about immunoreactivity of antigens based on amino acid sequence data alone . sequence analysis and bioinformatics can be used to increase the odds of predicting immunoreactivity , but each has limitations that can be addressed by proteomic studies . for example , many reactive antigens can not yet be predicted using bioinformatic methods because they do not contain features of an enrichment category , decreasing prediction sensitivity . in this study , we observed that 25% percent of the proteome is represented by enrichment categories , but only 12% of these proteins are seroreactive antigens . furthermore , 50% of the reactive antigens identified here do not fall into any enrichment category and therefore can not be predicted using this method . use of the proteome microarray therefore allows the identification of a broader spectrum of reactive antigens that can not be predicted by current methods ; by selecting 25% of the proteome based on enriched categories , we would identify 50% of the immunoreactive antigens . proteomics is a powerful method for identifying seroreactive antigens but does have several limitations . for studies of this kind , we take advantage of bioinformatics tools to characterize the complete orfeome of l. interrogans regarding proteomic features that help predict protein antigenicity in humans . our findings are dependent on computational predictions and genome annotations . even though different versions of the signal peptide predictor used here ( signalp v3 and v4 ) led to the same conclusions about enrichment ( data not shown ) , we recognize that the use of different bioinformatics tools or possible errors and biases in the algorithms used can significantly alter the enrichment analysis . it is also possible that incorrect folding of the proteins printed on the microarrays may interfere with antigen antibody recognition , either positively or negatively , and the accurate identification of immune - reactive proteins . moreover , 9% of the leptospiral orfeome were not represented on the microarrays , and we can not affirm that those proteins are not seroreactive . our results correlate antigenicity with in vivo and in vitro expression of individual proteins allowing comparison of microarray data with data from previous protein expression studies . we found that > 60% of the antigenic proteins were also detected by ms , indicating a large overlap in proteins expressed during both infection and in vitro growth . only 102 of the 1574 proteins identified by ms were found to be antigenic , suggesting that antigenicity involves structural features in addition to protein abundance . this discrepancy may be partially explained by differences between the level and identity of proteins in vitro and in vivo in the human host . a similar argument can be made for the 93/108 differentially expressed leptospiral genes identified during cultivation in dmcs in rats that were not seroreactive on the array . for example , they identified eight up - regulated genes associated with virulence , of which four were identified as seroreactive in our study : lic12631 and lic12632 ( hemolysins ) , lic10465 ( liga ) , and lic11219 ( peroxiredoxin ) . additionally , we identified 18 antigens associated with secretion ( cog u ) that could be involved in virulence . of these , only lic10053 was up - regulated in leptospira in the rat reservoir . the profile of up - regulated leptospiral proteins may differ significantly between chronic carriage in an animal reservoir and the human host because of different host environments and immune responses . this lack of correlation between protein concentration and antigenicity supports the observation that up - regulated genes are not necessarily antigenic and that not all virulence factors elicit an antibody response . seroreactive antigens were selected based on a limited sera collection that comprised leptospirosis patients with different clinical outcomes from the city of salvador in bahia , brazil . expanding this collection or studying individuals from different areas , infected with different species or serovars , this study of specimens derived from one clinical site in salvador involving exposure to one leptospira interrogans strain has identified a reference set of antigens that are important in the humoral response to leptospira infection in humans , including hypothetical proteins that would be missed by other bioinformatics approaches . future studies comparing humoral responses among disease outcomes and against healthy , uninfected controls may identify prognostic markers and promising subunit vaccine candidates .

with increasing efficiency , accuracy , and speed we can access complete genome sequences from thousands of infectious microorganisms ; however , the ability to predict antigenic targets of the immune system based on amino acid sequence alone is still needed . here we use a leptospira interrogans microarray expressing 91% ( 3359 ) of all leptospiral predicted orfs ( 3667 ) and make an empirical accounting of all antibody reactive antigens recognized in sera from naturally infected humans ; 191 antigens elicited an igm or igg response , representing 5% of the whole proteome . we classified the reactive antigens into 26 annotated cogs ( clusters of orthologous groups ) , 26 jcvi mainrole annotations , and 11 computationally predicted proteomic features . altogether , 14 significantly enriched categories were identified , which are associated with immune recognition including mass spectrometry evidence of in vitro expression and in vivo mrna up - regulation . together , this group of 14 enriched categories accounts for just 25% of the leptospiral proteome but contains 50% of the immunoreactive antigens . these findings are consistent with our previous studies of other gram - negative bacteria . this genome - wide approach provides an empirical basis to predict and classify antibody reactive antigens based on structural , physical chemical , and functional proteomic features and a framework for understanding the breadth and specificity of the immune response to l. interrogans .

Introduction Material and Methods Results Discussion

while infection is asymptomatic in wild rodents and other reservoirs , in humans and other accidental hosts , it can cause severe clinical disease : hepato - renal failure ( weil s disease ) , pulmonary hemorrhage syndrome ( lphs ) , and even death . protective immunity during naturally acquired infection is associated with a humoral immune response , indicated by increased production of agglutinating antibodies following infection and animal passive transfer studies . a major hurdle in the development of effective vaccines and serodiagnostic against complex microorganisms that encode thousands of proteins is the identification of a limited number of antigens that will induce a protective immune response . some proteomic features possibly associated with antigenicity and vaccine efficacy , such as extracellular location , outer - membrane proteins , signal peptides , and b- and t - cell epitopes , can be computationally predicted based on the amino acid sequence alone , reducing the number of candidates to several hundred . more recently , whole proteome microarray chips , developed in our lab and by others , have provided an empirical approach to interrogate the entire proteome of any microorganism and to efficiently determine antibody profiles associated with acute or chronic infection or with vaccine mediated protection from infection . protein microarray chips are particularly effective for the study of human serum samples , defining the antibody immune response against infectious agents on a proteomic scale and enabling the access to the complete antibody repertoire produced during an infection . additionally , the protein array usually encompasses thousands of proteins , representing the entire encoded proteome . the objective of proteomic studies is to provide a more complete understanding of the immune response to infection , also allowing the identification of novel serodiagnostic and prognostic markers as well as potential subunit vaccines . the mechanism for host recognition remains unclear , and the ability to predict antigenicity in silico is imperfect . usually only a relatively small subset of the proteome of infectious agents , such as l. interrogans , is recognized by the immune system , and little is known about the characteristics of these antigens . among the organisms studied we have continued this iterative approach , first using a proteome microarray to identify the spectrum of immunoreactive l. interrogans antigens recognized in human leptospirosis cases and then classifying their reactivity according to annotated functional and computationally predicted features . these results inform us about the benefits and limitations of antigen prediction and provide a framework for future studies to improve predictive capability for serodominant antigens . samples from infected patients came from the following projects : natural history of urban leptospirosis ( r01ai052473 ) , disease determinants of urban leptospirosis ( u01ai088752 ) , and ecoepidemiology of leptospirosis ( r01tw009504 ) . the study was conducted with a group of 90 laboratory - confirmed leptospirosis patients from the state of bahia , brazil , including 30 patients with mild clinical presentations , 31 patients with severe disease , and 30 patients who died due to leptospiral infection . laboratory confirmation was defined based on the results of the microagglutination test ( mat ) and according to the criteria of seroconversion or a four - fold rise in titer in patients with paired serum sample or a single titer of 1:800 in patients with only one serum sample . genes larger than 3 kb were cloned as smaller segments as previously described , and the liga and ligb genes ( lic10465 and lic10464 , respectively ) were fragmented according to the repeated big domains present in the structure of each protein ( ligb repeats 712 , liga repeats 713 , and liga / b repeats 16 ) , which are recognized by human sera as previously described . after identifying the seroreactive antigens on the microarrays , the inserts in the corresponding plasmids microarray fabrication was performed as previously described . unpurified supernatants were immediately printed onto nitrocellulose - coated glass fast slides using an omni grid 100 microarray printer ( genomic solutions ) together with multiple negative control reactions and positive control spots of an igg mix containing mouse , rat , and human igg and igm ( jackson immuno research ) . protein expression was verified by probing the array with monoclonal antipolyhistidine ( sigma - aldrich ) and antihemaglutinin ( roche applied science ) diluted 1/400 in protein array blocking buffer ( whatman ) , as previously described . enrichment analysis was performed based on leptospira interrogans serovar copenhageni l1 - 130 strain genome annotations available in the national center for biotechnology information ( ncbi ) and john craig venter institute ( jcvi ) databases . the antigens were classified according to annotated functional features and computationally predicted features . the clusters of orthologous groups ( cog ) information and the mainrole classification utilized can be found at ncbi and jcvi web sites . proteins with potentially biological importance , such as mass spectrometry ( ms ) evidence of in vitro expression or in vivo mrna up - regulation , were also submitted to enrichment analysis . for each array , the average of control ivtt reactions ( nodna controls ) was subtracted from spot signal intensities to minimize background reactivity . we amplified the 3667 predicted orfs from l. interrogans serovar copenhageni from genomic dna and cloned them into the pxi expression vector , as full or partial length proteins , using the high - throughput recombination cloning method developed by our group . cloning efficiency was 94% , and all cloned orfs were expressed under a t7 promoter in the e. coli in vitro transcription / translation system . microarray probing with anti - his and anti - ha antibodies revealed an expression level of 91% of all 3819 proteins and fragments printed on the array ( 3359 of 3667 total orfs ) . a list of the proteins not represented on the microarrays is provided in table s1 in the supporting information . in figure 1 , we show representative histograms of the number of reactive antigens selected from the convalescent time point for the severe patient group ( n = 30 ) using the inclusion criteria ( described in the materials and methods ) . the histogram plots the average signal intensity ( y axis ) and the number of responsive individuals ( secondary y axis ) for each reactive antigen selected ( x axis ) for igm ( a ) or igg ( b ) probing of convalescent - phase samples from severe patients . the aim is to identify all leptosprial antigens that are reactive in humans , without excluding cross - reactive antigens reactive in healthy unexposed individuals . a complete evaluation of the antibody immune response during naturally acquired leptospirosis compared with healthy controls , along with the analysis of new diagnostic and vaccine candidates , will be the focus of a separate study . antigens are shown in rows , grouped as reactive for both igm and igg antibodies , or for igm- or igg - only antibodies ; patients are in columns , organized from left to right by the increasing average reactivity detected for the reactive antigens . l. interrogans serovar copenhageni proteins are annotated with ncbi clusters of orthologous groups ( cog ) functional categories ( table s2 in the supporting information ) . of the total 3667 leptospiral proteins , 1313 are not associated with cogs , whereas 500 proteins are associated with cogs but have not been assigned to a specific function , which are shown as general function prediction only ( category r ) or function unknown ( category s ) . proteins with predicted cogs n ( cell motility and secretion ) and u ( intracellular trafficking and secretion ) were significantly enriched for both igm and igg reactivity , with significant fold increases of 4.4 ( n ) and 5.9 ( u ) for igm and 3.8 ( n ) and 5.1 ( u ) for igg , respectively ( table 1 ) . interestingly , we determined that the igg response to cogs s ( unknown function ) and v ( defense mechanism ) was 1.9- and 3.7-fold enriched , respectively , in igg response , although these enrichments were less significant than cogs n and u. cog e ( amino acid transport and metabolism ) was lacking for igg reactivity despite 4% of the l. interrogans orfeome being classified in this category ( table 1 ) . leptospiral proteins are also annotated with jcvi mainrole classification functional categories ( table s3 in the supporting information ) . we observed that antigens classified in the protein fate category in igm responses , which is related to protein folding , stabilization , and degradation , were enriched 2.7-fold . similarly to the cogs classifications , we found that proteins categorized as enzymes involved in energy metabolism were under - represented in the igm response , with a fold decrease of 0.3 ( table 1 ) . interestingly , the jcvi class of proteins that comprised most of the reactive antigens was the hypothetical proteins category , with 23 and 29% of igm- and igg - reactive antigens , respectively . we also analyzed the enrichment of antigens using computationally predicted features to identify motifs that are enriched in the humoral immune response to leptospira infection ( tables s4s6 in the supporting information ) . accordingly , outer membrane proteins were also significantly enriched in both responses ( 3.2 and 4.1 fold increase ) , and proteins predicted as cytoplasmic localization were significantly under - represented ( 0.8 and 0.6 fold change ) . although the majority of the antibody reactive antigens lack both a signal peptide and a transmembrane domain or are classified as cytoplasmic by psort , these groups account for a larger proportion of all of the computationally classified proteins in the l. interrogans proteome . another potential characteristic that could influence protein antigenicity is the in vivo expression level because leptospiral proteins expressed at higher levels have a higher probability of being recognized by the host immune system . therefore , we interfaced our seroreactive data with two existing data sets : ( i ) ms data obtained from in vitro cultured bacteria and ( ii ) a transcriptome study that identified differentially expressed genes when the bacteria was grown in vivo in dialysis membrane chambers ( dmcs ) implanted in rats . more than 50% ( 102/191 ) of the seroreactive antigens identified in our study were identified in vitro by ms ( figure 3a ) , making this method an enriching feature for both igm and igg seroreactivity ( table 1 ) . the up - regulated leptospiral genes identified in vivo were also enriched among the seroreactive antigens , with a fold change of 2.5 and 2.9 for igm and igg , respectively ( table 1 ) . ( a ) venn diagram of the leptospiral proteins identified by mass spectrometry when the bacteria were grown in vitro ( red ) ; proteins for which the corresponding mrna was up - regulated when the bacteria were cultivated in dialysis membrane chambers in vivo ( green ) ; and antibody reactivity in leptospirosis patients detected by protein microarray ( blue ) . array average signal intensity for igm or igg probing is plotted on the x axis ; the number of protein copies per cell detected in vitro by mass spectrometry is shown on the y axis . even though we observed a significant overlap between the number of seroreactive antigens identified in this study and the number of proteins detected in vitro by ms , there was no linear correlation between the number of protein copies per bacterium cell and the intensity of antibody reactivity detected on the protein arrays ( figure 3b ) . few proteins had both high cellular concentrations and showed strong antibody response , as exemplified by lic12966 , lic13050 , lic10191 , and the lig proteins ( lic10464 and lic10465 ) in figure 3b . notably , > 60% ( 67/108 ) of the up - regulated genes in vivo were not detected by ms under regular cultivation conditions , and only 15/108 up - regulated genes were also serodominant . a major component of the adaptive immune response to infection is the generation of protective and long - lasting humoral immunity , but factors governing selection of the antigens recognized by the immune system are largely unknown . in contrast , for infectious bacteria or parasites that encode hundreds or thousands of proteins , only a subset of the proteome is recognized by the humoral immune response . here we describe utilization of a leptospira interrogans proteome microarray to determine empirically the entire antibody repertoire of infected individuals . this technique allows the identification of the types of proteomic structural , physical chemical , and functional features that are recognized more frequently by the human immune system . an important difference between the present study and this previous report is that analysis of the complete proteome not only has allowed us to delineate additional serodiagnostic antigens but , more fundamentally , also has provided the basis for a rigorous , comprehensive , and quantitative determination of the protein characteristics of the entire set of the serodominant antigens on a genomic scale . our results represent a large - scale evaluation of l. interrogans proteins that are antigenic in the context of naturally acquired human infection . our group has previously shown that > 90% of the urban leptospirosis cases in brazil are caused by l. interrogans serovar copenhageni and has isolated strain l1 - 130 from an infected patient from the city of salvador , brazil . additionally , the availability of strain l1 - 130 complete genome sequence makes it ideal for this proteomic study . we identified 191 immunodominant protein antigens with either igm or igg recognition , which correspond to 6% of the leptospiral coding genome . we classified the annotated functional proteomic features that are associated with antigenicity in humans . interestingly , we identified features that were significantly enriched exclusively in igm or in igg responses . the most significant positive predictive features identified in this study , a signal peptide , 1 transmembrane domain , cog u ( intracellular trafficking and secretion ) , and cog n ( cell motility and secretion ) , were also enriched for b. melitensis . accordingly , artemis - defined surface proteins ( outer membrane , inner membrane , secreted , and surface structures ) were enriched for b. pseudomallei . the enriched ncbi cog categories n and u are both related to protein secretion , and the externalization of such proteins can easily explain their accessibility to the host immune system . rather , the immune system focuses on specific types of antigens . armed with this information and the increasing availability of genome sequences , we can begin to make generalizations to make a priori predictions about immunoreactivity of antigens based on amino acid sequence data alone . furthermore , 50% of the reactive antigens identified here do not fall into any enrichment category and therefore can not be predicted using this method . use of the proteome microarray therefore allows the identification of a broader spectrum of reactive antigens that can not be predicted by current methods ; by selecting 25% of the proteome based on enriched categories , we would identify 50% of the immunoreactive antigens . for studies of this kind , we take advantage of bioinformatics tools to characterize the complete orfeome of l. interrogans regarding proteomic features that help predict protein antigenicity in humans . even though different versions of the signal peptide predictor used here ( signalp v3 and v4 ) led to the same conclusions about enrichment ( data not shown ) , we recognize that the use of different bioinformatics tools or possible errors and biases in the algorithms used can significantly alter the enrichment analysis . it is also possible that incorrect folding of the proteins printed on the microarrays may interfere with antigen antibody recognition , either positively or negatively , and the accurate identification of immune - reactive proteins . moreover , 9% of the leptospiral orfeome were not represented on the microarrays , and we can not affirm that those proteins are not seroreactive . our results correlate antigenicity with in vivo and in vitro expression of individual proteins allowing comparison of microarray data with data from previous protein expression studies . we found that > 60% of the antigenic proteins were also detected by ms , indicating a large overlap in proteins expressed during both infection and in vitro growth . this discrepancy may be partially explained by differences between the level and identity of proteins in vitro and in vivo in the human host . for example , they identified eight up - regulated genes associated with virulence , of which four were identified as seroreactive in our study : lic12631 and lic12632 ( hemolysins ) , lic10465 ( liga ) , and lic11219 ( peroxiredoxin ) . expanding this collection or studying individuals from different areas , infected with different species or serovars , this study of specimens derived from one clinical site in salvador involving exposure to one leptospira interrogans strain has identified a reference set of antigens that are important in the humoral response to leptospira infection in humans , including hypothetical proteins that would be missed by other bioinformatics approaches .

accurate assessment of genetic heterogeneity is relevant to manifold fields , ranging from clinical research , pharmacogenetics , and statistical genetics , over forensic sciences up to evolution ( for a review , cf . ) . in planning genetic epidemiological studies or the collection of control cohorts for prospective studies it is crucial to prevent confounding effects due to undetected or disregarded population structure [ 2 , 3 ] . population - based association studies of unrelated individuals , involving case - control and cohort studies , are prone to population structure , which may lead to false positive results or to failure to reveal genuine associations [ 4 , 5 ] . in family - based linkage analysis great efforts are set to establish well - designed cohorts and large control samples , intended to serve as basis for genetic epidemiological studies . besides restricting recruitment to individuals of uniform ancestry , a common strategy applied to efficiently gain a representative sample of the inspected population and to control for potential unknown population substructure is to collect samples in smaller geographical areas , usually in medium to large urban centers ( e.g. , [ 79 ] ) . even well - characterized or supposedly homogeneous regions may still account for subtle genetic structure with potential geographical components [ 3 , 10 ] . shortly reviewed studies related to geographic genetic structure of human populations and pointed out clear lack of research focusing on genetic heterogeneity of smaller geographic regions or those focused on more urban , highly admixed populations . most available well - standardized methods in geographical genetics were developed for other research areas and may not be suited for assessing subtle genetic heterogeneity of modern populations inhabiting geographically restricted areas . modern humans account per se for the lowest species genetic diversity among primates . a typical western population inhabiting a geographically restricted area sets additional difficulties . such populations are typically outbred and account for a large degree of admixture , product of older and recent regional , interregional , and even international migration . it is to expect that genetic evolutionary forces , such as selection , mutation , drift , or barriers to gene flow , would play a relatively insignificant role in modeling fine - scale variation of genetic heterogeneity . at this geographical scale , it is more likely that neighborhood preferences and modern mating behavior would have a central role in modeling recent admixture , consequently , having strong influence on the observed pattern of genetic variation of modern small areas ( i.e. , ) . in other words , within modern western circumscribed areas , socio - demographic factors would probably explain a large proportion of the observed pattern of genetic heterogeneity . with the aim of unveiling modest amounts of population substructure in a small , admixed area we ( a ) searched for subtle patters of genetic heterogeneity and ( b ) explored potential predictors of the observed patterns . to this end , we combined statistical genetics with spatial statistics ( geostatistics ) within the framework of a geographic information system ( gis ) . a gis provides a computational environment designed for spatial analysis of geographic data , therefore the most suitable framework to detect , to model , and to analyze the geographic variation of genetic diversity . we analyzed a well - characterized cohort collected for prospective studies in a small area of southern germany . the sampling area included the middle - size city of augsburg , the surrounding suburban area , and the neighboring countryside . as previously reported by steffens et al . , the kora s4 sample shows a minimal but measurable increase of the inbreeding factor ( 8.4e 5% heterozygotes deficit ) measured in terms of fis values , that is , within - group deviation from expected heterozygosity but no indication of population substructure . despite extensive search of potential population stratification with the software package structure , in this cohort it estimates the proportion of individuals ' genome that may originate from differential populations , the probability that an individual belongs to a certain population as well as allele frequency differences in terms of wright 's fst statistics . regardless of intensive computations under several models , structure results did not provide any indication of a potential pattern of genetic heterogeneity in the kora s4 survey . our analysis is based on a subset of the kora cohort ( kooperative gesundheitsforschung in der region augsburg ; in english : cooperative health research in the region of augsburg ; [ 7 , 9 ] ) . the kora survey is an ongoing study , which takes place in a circumscribed region of southern germany : augsburg city and the two neighboring districts . the kora cohort was recruited for prospective studies . in 4 surveys ( s1s4 ) , a total of 18,000 participants were randomly selected from the adult population of resident german citizens ( 2574 years ) [ 7 , 9 ] . the analysis was conducted on a random set of the kora s4 survey ( n = 4261 ) , recruited in the period between the years 1999 and 2001 [ 7 , 9 ] . the data set consisted of 728 unrelated healthy german citizens , which included subjects born within and outside of germany as well . the graphical method grr ( graphical representation of relationships ; ) was used to exclude the presence of biological relationship of individuals based on genetic data ( see supplement 7 ) . in this paper the immigrant group ( n = 179 ) included subjects born in twenty worldwide distributed countries , half of these countries represented only once . four countries , czech republic , romania , poland , and ukraine , corresponded to the land of birth of 82 percent of all immigrants . the group of subjects born in one of these four countries was classified as major immigrant group . based on the information land of birth we differentiated between data sets : ( a ) all : the complete data set of 728 subjects ( resident german citizens ) , ( b ) ger : the total set of 549 natives , and ( c ) main_img : the subset of 146 immigrants , born in either czech republic , romania , poland , or ukraine . the kora s4 sample was genotyped for 212 single nucleotide polymorphisms ( snps ) ( supplementary table s6 in supplementary material available online at http://dx.doi.org/10.1155/2015/693193 ) . these snps either cause an amino acid exchange or an effective promotor alteration in respect to the resulting protein . assuming evolutionary times these snps may be subject to selective forces . genomic deserts , pursuing to achieve uniform distribution across the genome ( setting a minimum of 500 kbp intermarker distance ) . for this selection only snps presenting a minor allele frequency between 10 and 50% in caucasians were considered . the markers included in the final intergenic set were uniformly spaced and located > 100 kbp apart from any known genes and > 1 mbp apart from centromeres and telomeres . this procedure followed the set of rules proposed by devlin and roeder for genomic control markers . accordingly , these intergenic snps are assumed to be neutral to selection forces in the absence of any specific information . in this sense , these loci are expected to reflect the effects of demographic processes involving migration ( gene flow ) and even drift , if evolutionary times are considered . the averaged call rate over all samples was 97.3% ; intragenic snps achieved an average call rate of 96.2% and intergenic snps , an average call rate of 97.9% . details of the genetic properties of the full set of 212 snps are listed in supplementary table s6 . the study area comprised three administrative regions : the municipality of augsburg city and its two neighboring districts , aichach - friedberg district and augsburg district ( figure 1 ) . this is a surface comparable with the grand duchy of luxembourg ( figure 1 ) . the area is located in the swabia administrative region of bavaria , southern germany , between the coordinates 10.491e/48.091n and 11.310e/48.642n . the mean population density is 212 inhabitants / km , a figure that is comparable with the german average . the aichach - friedberg and augsburg districts include a suburban area neighboring augsburg city and a periurban area , a patchy pattern of smaller cities and villages widespread across a rural landscape . three regionalization methods were applied : ( a ) a subdivision of the total study area into minimal representative spatial units of analysis ; ( b ) a subdivision of the total study area into contiguous sampled units using a polygon - based method ; and ( c ) modification of the first regionalization in order to achieve a set of contiguous spatial analytical units while retaining the original geometry defined in ( a ) . genetic landscapes , in this work referred to matricial representations of genetic variation in the geographic space , were created with geostatistic methods of surface interpolation ( see section 2.7.1 ) . for this objective , it is convenient to define a minimal spatial unit of analysis which is representative of data coverage and it covers a spatial surface much smaller that the phenomenon of interest . the basic spatial unit of analysis of the genetic landscapes was the postal area . the postal area corresponds to the smallest district or region defined by the german postal system ( the german postal system divides germany in ca 28.700 postal areas ) . we considered this an appropriate analytical area because the german postal system divides the territory into spatial units with a similar number of inhabitants , independent of the extension of the spatial unit . postal areas include as well a population size large enough in order to guarantee subjects ' anonymity . a finer geographical reference of subjects , that is , postal address , was not available and it would not be in agreement with local official restrictions in respect to personal anonymity . we considered the subdivision of the study area into postal areas adequate to identify and to analyze fine - scale patterns of genetic variation . the spatial extension of the postal areas ranged from 1.8 km to 93 km , with an average of 26 km . the sampled area covered about 20% of the total study area , that is , approximately 600 km ( figure 1 ) . it included augsburg city and 15 settlements located in aichach - friedberg district and augsburg district . augsburg city itself contains 14 postal areas . in summary , out of a total of 64 postal areas , data was available in augsburg city ( including 14 postal areas ) and in another 15 postal areas . augsburg city samples were pooled together for frequency computations , since no information about postal area of residence was available for residents in this city . a subdivision of augsburg city into postal areas was only considered in the step of spatial interpolation to improve interpolation results ( see section 2.7.1 ) . postal areas with a very low number of samples were aggregated to neighboring sampled areas in order to exclude bias due to low number of samples per land unit . explicitly , the quarters stadtbergen ( n = 7 ) and gersthofen ( n = 6 ) were aggregated to neus ( n = 47 ) ; meitingen ( n = 12 ) was aggregated to langweid ( n = 22 ) ( figure 1 ) . in the final geostatistical analysis work analytical land units ( areal representing sampled data ) defined on the basis of the geographical coverage of postal areas are further referred to as land unit ( lu ) . lus were labeled with the sampling - location name ; aggregated lus were labeled with the name of the location accounting for the largest number of samples . the remaining 15 sampled postal areas ( aggregated into 12 lus ) included a total of 369 samples . letting aside augsburg city , sample size per lu ranged between n = 9 samples ( eurasburg ) and n = 60 samples ( neus ) . the mean sample size per lu was 30.8 and the standard deviation was 17.4 ( table 1 ) . the implementation of the spatial autocorrelation tests performed in this study ( see section 2.7.2 ) required to count with a set of adjacent spatial analytical units . this means that only spatial units with at least one contiguous neighbor could be included in the analysis . the total study area was divided into 13 thiessen polygons ( designation given to voronoi diagrams used to analyze spatially distributed data ) . each polygon corresponded to one lu defined in the first regionalization ( see section 2.3.1 ) . we chose this simple type of regionalization since many natural patterns may be closely approximated to this type of areal structure . thiessen polygons were delimited based on the centroids ( polygon geometrical center ) of the 13 sampled land units ( figure 2(a ) ) . the voronoi tessellation was created with the method v.voronoi of the open - source software package grass 6.4 ( geographic resources analysis support system , http://grass.osgeo.org/ ) . the implementation of the algorithms used in this study to search for best predictors of spatial variation fitting the data ( see section 2.7.3 ) required as well contiguous analytical units . for such more complex analysis , the coverage of each lu was retained . in this case four lus did not account for contiguous neighbors : aichach , pttmes , schwabmnchen , and altenmnster ( figure 1 ) . of these , the first four lus were not further than 5 km away from the next closest lu border . this geographical distance was considered negligible in the context of connectivity and human interaction between modern settlements . in order to get maximal information of the available data we modified slightly the geometry of these four lus and of their closest neighbors in order to meet the contiguity condition for at least 12 lus . schwabmnchen was connected to bobingen , aichach to friedberg , and pttmes to aichach ( figure 2(b ) ) . with this step , 12 lus conformed a continuous geographical space . the most peripheral sampled land unit , altenmnster , without a close sampled contiguous neighbors ( distance to the closest lu > 10 km ) , was not included in the computations ( figure 2(b ) ) . both implementations of spatial dependence analysis performed in this study required the definition of a matrix of spatial weights representing the interaction between lus ( see sections 2.7.2 and 2.7.3 ) . on the basis of the previously defined regionalization , either the thiessen polygons ( figure 2(a ) ) or the geometrically modified lus , for each analytical unit the geographical central point ( the centroid ) , were specified . these two tests were performed in this study based on a binary representation of the spatial weight matrix , which assigned a weight of unity for neighbors , and zero otherwise . a binary encoding was chosen since not enough information was available to set assumptions about the assumed spatial process . the function poly2nb was used to construct the neighbor list with default parameters and the function nb2listw to construct the weight matrix , setting the function parameter style = b for a binary system . pairs of lus assigned a spatial weight equal to unity are indicated in figure 2 with a vector net . socio - demographic information collected during recruitment included age , education years , degree of professional training and education , and place of birth . as described in the introduction the kora s4 survey mirrors the case of plenty of study designs in human genetic research , in which control cohorts are used for population studies . in the context of these studies one strategy is to collect samples in small areas and to restrict recruitment to individuals of same ancestry ( see section 1 ) . in the concrete case of the kora s4 it could be verified that the presence of immigrants introduced a small but significant effect on the total amount of genetic variation ( see supplement 4 ) . in this paper we focus on the case of a population that may be considered a priori to be genetically homogeneous and may account for subtle genetic substructure and if this is the case , which factors may be regarded as best predictors . to perform our study in accordance with these objectives and assumptions we worked with the two data sets . we used this set with the purpose of inspecting the effect of immigrants on the total genetic variation among other factors . on the other side we excluded immigrants and analyzed the subset of natives ( ger ) . this analysis is intended to specify best predictors of sublet genetic substructure and to estimate their effect in an admixed modern small population . the total set of immigrants did not include enough individuals of similar ancestry to perform further separated geostatistical analysis . measures related to age , education years , and education level were computed only on the native ger data set . variables related to immigrant representation in the total sample were computed for the total data set ( all ) . ( i ) variables related to age , education years , and education level , computed only for the ger data set : age25_39 : percentage of subjects in the age category of 25 to 39 years ; age40_54 : percentage of subjects in the age category of 40 to 54 years ; age55_74 : percentage of subjects in the age category of 55 to 74 years ; age_mean : mean age ; ey8_11 : percentage of subjects achieving a maximum of 11 school years ; this variable indicates the fraction of the sample which did not achieve the education level required to access to academic studies ; ey_mean : mean years of school attendance ; edu_mean : mean education level , scored according to the degree of professional training and education , ranging from 0 = no school degree up to 9 = graduate degree ( m.s . equivalent or higher ) . age25_39 : percentage of subjects in the age category of 25 to 39 years ; age40_54 : percentage of subjects in the age category of 40 to 54 years ; age55_74 : percentage of subjects in the age category of 55 to 74 years ; ey8_11 : percentage of subjects achieving a maximum of 11 school years ; this variable indicates the fraction of the sample which did not achieve the education level required to access to academic studies ; ey_mean : mean years of school attendance ; edu_mean : mean education level , scored according to the degree of professional training and education , ranging from 0 = no school degree up to 9 = graduate degree ( m.s . equivalent or higher ) . ( ii ) in order to inspect the effect of immigrant representation on total population from a model building perspective , variables related to birth land were included ; these variables were computed for the all data set . the representation of the total immigrant fraction in relation to the total sample was modeled with the variable : ger_p : percentage of natives over all subjects . ( iii ) on the same line and for purpose of ascertaining a potential effect related to the presence of major fraction of immigrants incoming from a reduced number of countries , which could be acting as a differentiated population within the migrant group , a further variable was included : main_imp : percentage of the major group of immigrants ( subjects born in czech republic , romania , poland , or ukraine ) over all immigrants ( german citizens born outside of germany ) . main_imp : percentage of the major group of immigrants ( subjects born in czech republic , romania , poland , or ukraine ) over all immigrants ( german citizens born outside of germany ) . we attempted to achieve a reduction in form of genetic landscapes of the complex georeferenced data available for this cohort ( genotype per snp and sample and geocoordinates of lus ) . these genetic landscapes should allow visualization of the estimated distribution of genetic diversity across geographic space and further assessment of associations between spatial patterns of genetic diversity and other georeferenced data , for example , average socio - demographic characteristics per lu . the reason for this is that we considered these types of genetic landscapes easier to interpret than those based on relative measures , for instance , the genetic differences between lus . for this step it was necessary to select a genetic measure of diversity referred to each single lu , in opposition to relative measures such as wright 's fst or alike , which would characterize variation of genetic diversity in terms of pair of lus . we chose this simple measure of genetic diversity , the observed heterozygosity ( ho ) , to summarize sample genetic attributes of each lu . ho , the observed frequency of heterozygotes averaged over loci , was estimated using(1)ho=jlhjl , where l is the number of loci and hj indicates the proportion of heterozygote individuals per locus jth . observed heterozygosity was computed separately for each land unit with the total data set ( ho(all ) ) and with the subset of natives ( ho(ger ) ) . in an attempt to frame the challenge embedded in this sample , first exploratory analysis with geostatistical methods indicated a potential differentiation of the periurban areas from augsburg city and its periphery ( see supplement 2 ) . potentially , the fine - scale patterns of genetic diversity observed in these first exploratory evaluations could be explained by various simple models of spatial variation . for instance , the observed pattern ( supplementary figure s2a - b ) may be the result of a simple process of isolation by distance . as a result , genetic diversity would follow a pattern of gradual variation ( e.g. , gradients of allele or genotype frequencies ) . it must be noted that in such case , the observed pattern would correspond to a small fraction of the geographical landscape where the process occurs . this is so because both geographic extension of the study area and evolutionary times of the study population ( here it refers to the number of generations necessary to fix the effects of any gene - flow process [ 23 , 24 ] ) are jointly , most probably , not large enough to have generated a local process of fixation of gradual variation of genetic features . spatial correlation methods ( e.g. , spatial autocorrelation ) and mantel tests would be the first methods of choice to detect spatial correlations of genetic distance with geographic distance . the observed pattern ( supplementary figure s2a - b ) could as well be the product of undetected population clustering . in this case , individuals of similar genetic features tend to reside in distinct areas than individuals less similar . clustering would also require that individuals of distinct groups present reduced interaction with individuals of other groups . at larger geographical scales this could be observed when cultural , linguistic , or political limits set a barrier to gene flow . for instance , it is improbable that a modern western population inhabiting such a small area would be composed of several groups with reduced exchange ( low migration rates among the subareas and low predisposition to mate with individuals of other groups ) . this situation is even less probable if considered that spatial patterns of genetic variation were even detected within the group of natives . the result is supported by previous analysis undertaken with this sample : an exhaustive evaluation of population clustering was conducted by steffens et al . with the well - known software structure . despite the large number of runs with varying models and parameters there was no indication of any population substructure . results indicated that the model assuming a number of populations equal to unity ( k = 1 ) showed the highest posterior probability for the kora s4 data . the following software packages were used in this step : ( a ) geneland [ 20 , 25 ] ; ( b ) eigenstrat | eigensoft [ 26 , 27 ] ; ( c ) plink , version v0.99s ( http://pngu.mgh.harvard.edu/purcell/plink ; ) , with additional multidimensional scaling using the r software package , version 2.12.1 ( r foundation for statistical computing , 2010 ) ; ( d ) spagedi . the wide - spread used software structure was not considered , since in a previous study no evidence for genetic substructure was found with this tool . methods ( a ) to ( c ) , as well as structure , share the possibility to search for groups of genetically similar individuals . spagedi ( d ) is a tool for detecting dependency between genetic and geographic distances among individuals or populations . geneland ( b ) and spagedi ( d ) are individual - based methods and require including in the computation the geographic reference of each individual . as mentioned above ( see section 2.3.1 ) , available data and anonymity restrictions did not allow a more precise georeference of subjects than sampling location . for these reasons , all individuals sampled in one location were georeferenced to the same geographical coordinates . therefore , in our case and as it most probably would occur in this type of human genetic studies , the full capabilities of software making use of individual geographical coordinates could not be exploited . with each tool ( a d ) following , multiple runs with varied parameter values and model assumptions were conducted . for computations demanding a priori definition of an assumed number of subpopulations geostatistical analysis was conducted using the open - source software package grass 6.4 and spatial packages contributed to r software package , version . in this framework , genetic landscapes were defined as matrix representations of genetic variation in the geographic space . spatial matrices were created by the transformation of sampling - point data to an elevation surface by spatial interpolation . an elevation surface is a 3d layer of continuous data ( grid or raster layer ) with elevation information at each point of the area . grass defines this type of spatial object as 2.5 dimensions ( 2.5d ) . as a simplification , the usual denomination for this type of spatial object : interpolation based on splines proved to be a better choice for phenomena which combine a random component as well as processes which minimize energy , as it could be considered socio - demographic processes . we chose the function regularized spline with tension implemented in the grass - method v.surf.rst . this method computes the continuous 3d layer ( raster data ) simulating a thin flexible plate passing through or close to the measured data points ; it is the most general and accurate method available in grass . in order to run v.surf.rst , we obtained one point - data layer for each measured statistic . in case of augsburg city , which contains 14 postal areas and covers a disproportionately large area , centroids of all postal areas computed values for augsburg city data were assigned to all its 14 centroids . with this step , we smoothed spatial interpolation results in the area of augsburg city and surroundings , while avoiding interpolation artifacts . we modeled genetic landscapes based on tuned values of v.surf.rst parameters . in order to be able to adequately execute the v.surf.rst procedure the ho values computed using the raw data ( ho raw ) were transformed into percentage as follows:(2)ho raw100=ho , where ho raw , ho . interpolation surfaces based on ho values were created for the following data sets : ho(all ) , and ho(ger ) . interestingly , since the kora s4 genotypes conform a control population pool for genetic studies [ 7 , 9 ] , ho(all ) landscape may be examined as a representative estimation of the spatial variation of genetic diversity of the extant population and ho(ger ) landscape of the native fraction in the region of augsburg . the presence of simple association between the variability of an attribute and the geographical space was tested by means of spatial autocorrelation . in this case , the null hypothesis is that the feature of interest is spatially distributed at random among other attributes within the study area . spatial correlation measured with the test statistic moran 's i is inferential , which implies that results must be interpreted in dependence of the null hypothesis . for this analysis we used a global moran 's i statistic , which means that we tested for spatial autocorrelation in the study area as a whole , assuming that the spatial process is the same everywhere . spatial autocorrelation of each of the genetic and socio - demographic variables defined in this study was tested with the r package spdep . moran 's i tests were performed using the function implementations moran.test and moran.mc . accounting for normality deviation of the data , moran.test was run under the specification of randomization assumption in computing the variance of the statistic . this test specification allows relaxing the simpler normality assumption by introducing a correction term based on the kurtosis of the inspected variable . the second implementation , moran.mc , is a permutation - based test . with this implementation spatial autocorrelation the function moran.mc uses a monte carlo test , based on a permutation bootstrap . observed values are randomly assigned to areal entities , and the value of the observed moran 's i is computed nsim times . these tests were run using a binary matrix of spatial weights ( see section 2.3.4 ) . both implementations , moran.test and moran.mc , were used to test for spatial autocorrelation in measures of genetic variation : ( ho(all ) , ho(ger ) ) , as well as on the socio - demographic variables : ger_p , main_imp , age25_39 , age40_54 , ea55_74 , age_mean , ey8_11 , ey_mean , and edu_mean . socio - demographic measures were inspected as predictors of the observed pattern of ho(ger ) under the assumption that socio - demography would provide useful indication of spatial arrangement of recent migration processes , specially regional and national migration , which we assumed that it must have had a strong influence on fine - scale genetic variation . the contribution of socio - demographic factors to explain the observed spatial pattern was analyzed under the assumption of spatial dependence . best - fit spatial autoregressive models ( sar ) predicting heterozygosity ( hm ) were selected . a stepwise forward search was conducted using the function spautolm of the package r spdep . the function spautolm computes a regression on the values from the other areas to estimate the spatial dependence of the residuals of the specified linear predictor . the spatial dependence is estimated with a maximum likelihood test , computing a spatial autocorrelation parameter , . the p value of the likelihood ratio test compares the model with no spatial autocorrelation ( = 0 ) to the one which allows for it . a binary matrix of spatial weights was used for this analysis ( see section 2.3.4 ) . model selection was started with following parameters : ger_p , age25_39 , age40_54 , ea55_74 , age_mean , ey8_11 , ey_mean , and edu_mean . in order to test if the spatial distribution of the major immigrant group ( main_img ) improves model prediction , the influence of the parameter main_imp on the selected model was tested . a set of models best fitting the data were selected according to the p values of the covariates ( p value < 0.05 ) . finally , the goodness of fit of the selected sar models was analyzed . in this step , the pixelwise divergence between predicted ( hm ) and observed values ( ho ) was quantified . interpolation surfaces were created based on the predicted values ( hm ) by each selected model . the pixelwise divergence in absolute values of these interpolation surfaces from the ho(ger ) landscape was used to compare prediction accuracy among the selected sar models . in order to facilitate comparison among models , the standardization was performed based on the maximal range of pixel values ( max_rgger ) measured in the ho(ger ) landscape . the parameter max_rgger was computed as follows:(3)max_rgger = zmax_intgerzmin_intger , where zmax_intger is the maximal value measured in the ho(ger ) landscape and zmin_intger the minimal value . for each sar model , a new elevation surface ( raster layer ) storing the respective pixelwise difference was created . following pixelwise computation was performed with the grass basic module r.mapcalc:(4)abshogerhmnmax_rgger100,where abs implies absolute value , ho(ger ) refers to the pixel values of the ho(ger ) landscape , h(mn ) refers to the pixel values of the interpolated surface created on the bases of the predicted h values of the mn sar model , and the parameter max_rgger = 1.64 ( zmin_int = 43.36 ; zmax_int = 45.00 ) . for each one of these elevation surfaces , spatial global statistics were computed . for this purpose , elevation surfaces were imported into the spatial r environment provided by the packages sp , rgdal , spdep , and spgrass6 ( r foundation for statistical computing , 2010 , http://www.r-project.org/foundation/ ) . mean , standard deviation ( sd ) , median , minimum ( min ) , and maximal values ( max ) of the elevation surfaces were computed with the r function summary ( ) . these statistics were applied as global quantitative measures of prediction goodness of each selected sar model and were used to select the model best fitting the data . the model with the lowest global difference between observed and predicted h values was selected as the one best fitting the data . based on this model , maps representing the spatial variation of predicted h values and the estimated divergence between observed and predicted h values were created . the former map represents the estimated variation in heterozygosity according to predictions obtained by the sar model best fitting the data . the latter maps allows a visual estimation of the agreement between observed and predicted heterozygosity in each land unit as well as the estimated spatial variation of divergence in the total study area . descriptive statistics of all measures , including mean , standard deviation , median , minimum value , and maximum value , are presented in table 2 . younger individuals ( 25 to 39 years old ) comprised more than 30 percent of the ger sample in the eastern sector and reached a proportion of 45 percent in pttmes ( figure 3(a ) ) . subjects corresponding to the intermediate age category ( 40 to 54 years old ) showed a lower proportion ( less than 30 percent ) in lus contiguous to augsburg city in the south and in the east ( figure 3(b ) ) . the upper age category ( 55 to 74 years old ) accounted for more than 50 percent in eight of the 13 lus , presenting the higher proportions ( > 70% ) southern from augsburg city ( figure 3(c ) ) . the lowest mean age values were recorded in pttmes , rehling , and neus ( figure 3(d ) ) . the percentage of individuals of the intermediate and the upper age category ( age40_54 , age55_74 ) showed a deviation of the expected moran 's i value with both tests ( moran.test , moran.mc ) on a significance level of = 0.05 . the percentage of individuals of the lower age category ( age25_39 ) showed a significant deviation of the expected moran 's i value with moran.test , but with the m - c permutation bootstrap test did not reach a significance at = 0.05 ( table 3 ) . the mean age ( age_mean ) did not show any indication of spatial dependence with either of both tests ( table 3 ) . with regard to the spatial distribution of education level in the study area , the largest values of education years , that is , lower values of ey8_11 , were observed in augsburg city and in neighboring lus in the east ( friedberg ) and in the west ( aystetten , neus ) , as well as in the southern lu of schwabmnchen ( figure 4(a ) ) . both mean variables , means of education years and education level ( ey_mean , edu_mean ) , showed the largest values in the center and in the south of the study area , while the peripheral lus aichach and altenmnster showed the lowest values ( figures 4(b ) and 4(c ) ) . whereas education level ( edu_mean ) showed a significant deviation from the expected moran 's i value with both tests ( moran.test , moran.mc ) at = 0.05 , the education years ( ey_mean ) presented just an indication of potential spatial dependency at this significance level ( table 3 ) . the percentage of natives ( ger_p ) showed a complex pattern ( figure 5(a ) ) . considering lus with an intermediate number of samples , the percentage of natives decreased with an increase of the absolute number of samples per land unit with the exception of altenmnster and neus ( table 1 ) . in augsburg city , with a quite larger number of samples in comparison with all other lus ( table 1 ) , the lowest percentages were measured around augsburg city , in knigsbrunn , followed by langweid . bobingen , contiguous to augsburg city on the south , and aystetten , relatively peripheral to augsburg city , presented the next lower frequencies of natives ( figure 5(a ) ) . samples included only natives in the eastern peripheral lus , pttmes and eurasburg ( figure 5(a ) ) , both accounting for the lowest sample counts as well ( table 1 ) . the distribution of the proportion of natives ( ger_p ) did not show any indication of a potential spatial dependency ( table 3 ) . the major group of immigrants ( main_img ) showed a higher ratio in the western lus ( figure 5(b ) ) . in augsburg city , where a considerably larger total number of immigrants were sampled , the major immigrant group composed 78 percent of the total immigrant data set ( table 1 ) . the results of both moran 's i tests of spatial autocorrelation ( moran.test , moran.mc ) pointed to a potential simple spatial dependency of the parameter main_imp , which however did not reach a significance level of = 0.05 ( table 3 ) . the search for indications of spatial patterns of genetic heterogeneity with well - established procedures did not provide any positive results . although a high number of different explorative runs with different parameters were performed , tests based either on eigenstrat | eigensoft , plink , geneland , or spagedi did not provide any indication of a potential geographic variation of genetic heterogeneity in the study area . a brief summary of a representative extract of these computations is presented in supplement 5 . geostatistical analysis based on the statistic observed heterozygosity ( ho ) provided indication of spatial patterning . table 3 presents results of the spatial autocorrelation analysis performed with test statistic moran 's i. on the one side , the variable ho(all ) showed an indication of association between genetic variation and geographic coordinates . on the other , in the native sample , tests of global spatial autocorrelation showed a significant deviation ( on a significance level of = 0.05 ) of a random spatial distribution of ho(ger ) values ( table 3 ) . significant results obtained in the native sample may indicate that the additional genetic variability contributed by the immigrant fraction of the sample could introduce noise , which diluted a subtle patterning of the genetic attributes of the native sample . h o(all ) landscape , which estimates the variation of the genetic heterogeneity of the extant german population in the study area , presented a marked depression in the east ( figure 6(a ) ) . intermediate ho values covered the central - northern sectors , including some areas of aichach - friedberg district , langweid , augsburg city , and schwabmnchen . the minimum values were found in neus and knigsbrunn . the ho(ger ) landscape , estimating the spatial variation of genetic heterogeneity of the native population , showed similar values to the ho(all ) landscape in the western and in the eastern periphery . in the central belt , running across the study area in north - south direction , this landscape showed higher values than the ho(all ) landscape ( figure 6(b ) ) . four spatial autoregressive models were selected according to the p value ( p value < 0.05 ) of the covariates ( table 4 ) . the four models included as covariates variables related to age and education ; two models ( m1 , m3 ) included as well the proportion of natives per lu ( ger_p ) , which is at the same time an indication of the proportion of immigrants per lu . the inclusion of the variable main_imp , which involves a differentiation of subgroups of immigrants , did not improve any of the selected models . out of these four selected models m2 showed a significant p value when the likelihood ratio of was tested , indicating left spatial correlation in the residuals ( p value = 0.018 ) . the h(m2 ) landscape ( figure 6(c ) ) showed as well the lowest mean and standard deviation of pixelwise difference to the ho(ger ) landscape ( table 4 ) , which we used as indicators of model goodness . the pixelwise difference between ho(ger ) and h(m2 ) surfaces showed a good agreement over a large area as well . areas where both surfaces showed very similar values are indicated in figure 7 with a white or light grey and correspond to a pixelwise h difference close to zero . the maximal differences were measured in augsburg city and in rehling , a small residential area in the country side . best fitting was obtained in the peripheral ring surrounding augsburg city ( figure 7 ) . fine - scale variation of genetic heterogeneity within a small region was detected and analyzed applying a geographic perspective . population genetics and geostatistics were combined with the open - source geographic information system grass . the capabilities of this approach were tested on a subset of the kora s4 survey [ 7 , 9 ] , collected in southern germany for prospective studies . tests on this data set with the well - known software structure previously reported by others did not provide evidence for population substructure . we assumed that within small urbanized areas of modern western countries , as is the case in germany , genetic composition may be strongly affected by migratory movements of the last half century , which may be still estimated by means of socio - demographic measures . genotypes ( 212 autosomal snps ) and socio - demographic information ( age , education , place of residence , and birth land ) of 728 healthy german citizens were analyzed . the estimated values of the observed heterozygosity showed to some extent a cline of decreasing values from east to west . in a first step to analyze spatial processes controlling the observed patterns , moran 's i tests for spatial autocorrelation were performed for all available parameters . indications of global spatial dependencies were observed in socio - demographic variables related to age categories and education level . while significant deviation of the expected moran 's i was obtained with the observed heterozygosity of the native subset ( ger ) , the observed heterozygosity of the total data set showed just an indication of a potential deviation of the expected moran 's i. in other words , these results could be interpreted as an indication that heterozygosity values of this small area may be regulated by a global spatial process , but such subtle process could only be observed when the subset of native germans is considered . this is consistent with the elemental assumption that because immigrants may differ to some extent in their genetic background , and this would be reflected in their observed heterozygosity , they would introduce additional variability which hinders detection of spatial variability pattern of the most frequent group , the german natives . spatial dependencies of socio - demographic variables at this local level could be axiomatically interpreted as a result of neighborhood preferences of recent local and regional migratory processes . it is to expect that social preferences affecting recent migration , such as regional and international migratory movements of the last half century , may be reflected by socio - demographic parameters . individuals would not choose new residence randomly and this may be reflected in similar socio - demographic attributes of contiguous neighborhoods . this expectation , which goes along with common sense , would also be in agreement with the fact that the level of admixture would associate to some extension with socio - demographic attributes of the location . this would be the result of new and recurrent resettlement of some areas or that inhabitants of areas presenting certain socio - demographic features would show higher predisposition to admixture . indeed , the fact that patterns of socio - demographic structure and genetic admixture account for spatial autocorrelation could indicate the occurrence of further unobserved phenomena influencing both , as it could be social preferences or official urban planning . nevertheless , it went beyond the scope of this work to search for such unobserved processes . spatial autoregressive models ( sar ) fitting the data were selected by a forward search . the four best - fit sar models contained as covariates socio - demographic measures related to age , school - attendance years , and education level . two of the four selected models included as well the percentage of natives per unit . this parameter directly accounts for degree of demographic admixture in sense of proportion of immigrants as measured by the parameter land of birth . the prediction strength of the selected model was estimated with quantitative comparisons between the genetic landscapes created on basis of observed and predicted measures of heterozygosity . we found a good agreement between the predicted and the observed patterns , which supports the assumption of a certain relationship between genetic admixture degree and socio - demographic structure . predicted and observed values showed the highest agreement in the surrounding belt of augsburg city the largest deviations were measured in augsburg city itself and in two small residential areas in the eastern countryside . the differentiation of augsburg city to the countryside ( see supplement 3 ) may be interpreted as the expected divergence of middle - size industrial settlements and countryside . this effect could still be detected even after excluding immigrants , subjects born outside of germany . this expected differentiation between urban and countryside areas in relation to genetic heterozygosity may be considered as a confirmation that the observed patterns are not artifacts . the differentiated area in the countryside , rehling , corresponded to a land unit with relatively low number of samples . the lower predictive capability of the best - fit model could be attributed to sampling bias . . a further possibility could be that this settlement offers an attractive residential area for individuals working in any of the larger urban centers located eastern from augsburg , as it could be ingolstadt or munich . both urban centers offer highly profitable working alternatives , in sense of carrier opportunities and higher income , and act as an attraction pole for domestic and foreign migration . as well , both cities are also among the ones with the most expensive living costs . this may reinforce the idea that this location may be attractive for newly settled workers willing to commute between their working place and a less expensive residential area , relatively close to a middle - size urban center as augsburg . if this would be the case , the inclusion of distance and accessibility to attractive urban centers could considerably improve model prediction . remarkably , almost 80 percent of them corresponded to individuals born either in czech republic , romania , poland , or ukraine . some areas situated in the periphery of augsburg city showed a stronger component of immigrants , mostly or totally represented by this major immigrant group . although this group involved four birth lands , it may be speculated that the concentration of these individuals is not casual . it could be expected that , within each provenance group , individuals could be originals of nearby regions or belonging to large related families . in total , a concentration of small groups , each one showing a higher degree of homozygosity , would stand out over a much more admixed group , as it is expected for the native german population . our additional analysis with vastly cited multivariate methods , geneland , eigenstrat | eigensoft , plink , and spagedi , did not also submit indication of population stratification in the study area . these tools proved to be successful in studies of groups with considerably larger genetic differences , significantly more polymorphic loci , or much larger number of loci than in the present work . the kora study was carefully designed for prospective studies aiming to reduce any type of genetic structuring . following this objective , consequently , the genetic differentiation of this subset of the kora cohort is expected to be considerably lower than in humans studies conducted at broader geographical scales or in further studies acknowledging larger evolutionary histories and dimensions ( i.e. , nonhuman species or samples with large population differences ) . these loci were not specifically selected by their informativeness for distinguishing among major regional groups ( i.e. , [ 35 , 36 ] ) . for this reason , the number of available snps was probably too low for detecting fine - scale population differences with these standard tools . using full capabilities of tools such as spagedi or geneland , both offering individual - based analysis tools , was not possible either since the search of patterns of variation of genetic heterogeneity of the kora sample can not be carried out on an individualized geographical basis . official restrictions concerning personal anonymity forbid the use of data which could individualize a subject , such as postal address or any other precise geographical reference . therefore , for human studies , precision of geographic references must be kept low with the consequence that data might be spatially aggregated . in opposite to studies analyzing other species with a continuous geographical distribution , the identification of spatial structures of humans inhabiting small areas may not be carried out on an individual basis . such an approach would jeopardize personal anonymity and would go against most official restrictions of human studies . accordingly , it is not surprising that well - established tools to detect human population stratification on a broader level or those which make use of geographic references on an individual basis ( mostly developed for studies of other species ) may not detect fine - scale patterns of genetic variation in small areas . most genetic studies on modern human groups address recruitment of control samples on extant populations or make use of available control cohorts . these samples may present some degree of heterogeneity even if recruitment was restricted to small areas , by citizenship , or in combination with homogeneous place of birth . the degree of spatial heterogeneity of small geographic areas , frequently assumed to be neglectful in the context of genetic studies of modern human groups , should be evaluated on a case - by - case basis . based on our outcomes , it could be stated that genetic heterogeneity could not be automatically assumed to be negligible . these results support the elemental assumption that within multiethnic , urban , and suburban groups , as found in medium - sized german cities and surroundings , the socio - economic parameter birth land allows a first reduction of genetic heterogeneity . as it was presented in this study ( see supplement 2 ) , even after removing immigrants from the kora s4 data set , the degree of genetic differentiation in natives still overlapped with the spatial frequency distribution of immigrants . if future studies verify our exploratory results obtained with visual examination of genetic landscapes based on fst analogous reynolds ' r genetic distance , potentially , the proportion of immigrants may be used as subrogate of degree of natives ' admixture , which could actually reflect native 's behavior in regard to choice of area of residence and tolerance or predisposition to admix . our results about a differentiation among urban , suburban , and periurban population are a suggestion of a true effect in the sense of subtle population differentiation ( cf . ) . as mentioned above in this section , it is to assume that such differentiation would not result from isolation between neighboring areas in the sense of evolutionary processes , but from differences in regard to the degree of migration , residential preferences and willingness to intermix . however , it must be stressed that our analysis is preliminary and it is predominantly aimed at a methodological evaluation . in particular , the coverage of the area is patchy and far from complete . in order to make use of these results for further genetic studies , first , the postulated fine - scale variation of genetic heterogeneity should be confirmed with a larger data set . second , the magnitude of the detected bias for the corresponding analysis should be evaluated . knowledge of fine - scale patterns of genetic variation could provide information about areas where expected genetic heterogeneity could introduce undesired bias . areas with an observed higher genetic heterogeneity than tolerable could be avoided . in case that spatial heterogeneity would be assessed after recruitment , examining the spatial pattern of genetic heterogeneity could serve as a basis to decide about a stratified analysis ( e.g. , grouping samples according to residence or any other relevant spatial reference ) or to correct for population stratification ( cf . [ 26 , 27 ] ) . our vision is to further develop our approach in order to be capable of testing as well as detecting and correcting , if it is applicable , for spatial patterns of genetic heterogeneity within the study sample ( cf . [ 26 , 27 ] ) . in contrast to the method implemented in eigensoft | eigenstrat [ 26 , 27 ] , which infers strata based on genetic data alone , such approach would make use of information on subject area membership to define the strata . this usage of additional a priori information potentially leads to improve strata definition ( cf . ) . taking these findings of the kora s4 sample altogether we can state that fine - scale spatial genetic variation may be assumed in the study area . our results indicate that patterns of genetic heterogeneity can be present in small regions within germany . in conclusion , it may be stated that the presented genetic geostatistical approach has the potential of being a powerful tool for detecting , modeling , and analyzing spatial patterns of genetic heterogeneity even within populations inhabiting small regions .

aiming to investigate fine - scale patterns of genetic heterogeneity in modern humans from a geographic perspective , a genetic geostatistical approach framed within a geographic information system is presented . a sample collected for prospective studies in a small area of southern germany was analyzed . none indication of genetic heterogeneity was detected in previous analysis . socio - demographic and genotypic data of german citizens were analyzed ( 212 snps ; n = 728 ) . genetic heterogeneity was evaluated with observed heterozygosity ( ho ) . best - fitting spatial autoregressive models were identified , using socio - demographic variables as covariates . spatial analysis included surface interpolation and geostatistics of observed and predicted patterns . prediction accuracy was quantified . spatial autocorrelation was detected for both socio - demographic and genetic variables . augsburg city and eastern suburban areas showed higher ho values . the selected model gave best predictions in suburban areas . fine - scale patterns of genetic heterogeneity were observed . in accordance to literature , more urbanized areas showed higher levels of admixture . this approach showed efficacy for detecting and analyzing subtle patterns of genetic heterogeneity within small areas . it is scalable in number of loci , even up to whole - genome analysis . it may be suggested that this approach may be applicable to investigate the underlying genetic history that is , at least partially , embedded in geographic data .

1. Introduction 2. Material and Methods 3. Results 4. Discussion and Conclusions

accurate assessment of genetic heterogeneity is relevant to manifold fields , ranging from clinical research , pharmacogenetics , and statistical genetics , over forensic sciences up to evolution ( for a review , cf . ) in planning genetic epidemiological studies or the collection of control cohorts for prospective studies it is crucial to prevent confounding effects due to undetected or disregarded population structure [ 2 , 3 ] . besides restricting recruitment to individuals of uniform ancestry , a common strategy applied to efficiently gain a representative sample of the inspected population and to control for potential unknown population substructure is to collect samples in smaller geographical areas , usually in medium to large urban centers ( e.g. it is to expect that genetic evolutionary forces , such as selection , mutation , drift , or barriers to gene flow , would play a relatively insignificant role in modeling fine - scale variation of genetic heterogeneity . at this geographical scale , it is more likely that neighborhood preferences and modern mating behavior would have a central role in modeling recent admixture , consequently , having strong influence on the observed pattern of genetic variation of modern small areas ( i.e. in other words , within modern western circumscribed areas , socio - demographic factors would probably explain a large proportion of the observed pattern of genetic heterogeneity . with the aim of unveiling modest amounts of population substructure in a small , admixed area we ( a ) searched for subtle patters of genetic heterogeneity and ( b ) explored potential predictors of the observed patterns . to this end , we combined statistical genetics with spatial statistics ( geostatistics ) within the framework of a geographic information system ( gis ) . a gis provides a computational environment designed for spatial analysis of geographic data , therefore the most suitable framework to detect , to model , and to analyze the geographic variation of genetic diversity . we analyzed a well - characterized cohort collected for prospective studies in a small area of southern germany . , the kora s4 sample shows a minimal but measurable increase of the inbreeding factor ( 8.4e 5% heterozygotes deficit ) measured in terms of fis values , that is , within - group deviation from expected heterozygosity but no indication of population substructure . regardless of intensive computations under several models , structure results did not provide any indication of a potential pattern of genetic heterogeneity in the kora s4 survey . the kora survey is an ongoing study , which takes place in a circumscribed region of southern germany : augsburg city and the two neighboring districts . the kora cohort was recruited for prospective studies . in 4 surveys ( s1s4 ) , a total of 18,000 participants were randomly selected from the adult population of resident german citizens ( 2574 years ) [ 7 , 9 ] . the study area comprised three administrative regions : the municipality of augsburg city and its two neighboring districts , aichach - friedberg district and augsburg district ( figure 1 ) . the mean population density is 212 inhabitants / km , a figure that is comparable with the german average . the aichach - friedberg and augsburg districts include a suburban area neighboring augsburg city and a periurban area , a patchy pattern of smaller cities and villages widespread across a rural landscape . genetic landscapes , in this work referred to matricial representations of genetic variation in the geographic space , were created with geostatistic methods of surface interpolation ( see section 2.7.1 ) . a finer geographical reference of subjects , that is , postal address , was not available and it would not be in agreement with local official restrictions in respect to personal anonymity . we considered the subdivision of the study area into postal areas adequate to identify and to analyze fine - scale patterns of genetic variation . the sampled area covered about 20% of the total study area , that is , approximately 600 km ( figure 1 ) . it included augsburg city and 15 settlements located in aichach - friedberg district and augsburg district . augsburg city samples were pooled together for frequency computations , since no information about postal area of residence was available for residents in this city . letting aside augsburg city , sample size per lu ranged between n = 9 samples ( eurasburg ) and n = 60 samples ( neus ) . socio - demographic information collected during recruitment included age , education years , degree of professional training and education , and place of birth . in the concrete case of the kora s4 it could be verified that the presence of immigrants introduced a small but significant effect on the total amount of genetic variation ( see supplement 4 ) . ( ii ) in order to inspect the effect of immigrant representation on total population from a model building perspective , variables related to birth land were included ; these variables were computed for the all data set . ( iii ) on the same line and for purpose of ascertaining a potential effect related to the presence of major fraction of immigrants incoming from a reduced number of countries , which could be acting as a differentiated population within the migrant group , a further variable was included : main_imp : percentage of the major group of immigrants ( subjects born in czech republic , romania , poland , or ukraine ) over all immigrants ( german citizens born outside of germany ) . we attempted to achieve a reduction in form of genetic landscapes of the complex georeferenced data available for this cohort ( genotype per snp and sample and geocoordinates of lus ) . these genetic landscapes should allow visualization of the estimated distribution of genetic diversity across geographic space and further assessment of associations between spatial patterns of genetic diversity and other georeferenced data , for example , average socio - demographic characteristics per lu . for this step it was necessary to select a genetic measure of diversity referred to each single lu , in opposition to relative measures such as wright 's fst or alike , which would characterize variation of genetic diversity in terms of pair of lus . we chose this simple measure of genetic diversity , the observed heterozygosity ( ho ) , to summarize sample genetic attributes of each lu . ho , the observed frequency of heterozygotes averaged over loci , was estimated using(1)ho=jlhjl , where l is the number of loci and hj indicates the proportion of heterozygote individuals per locus jth . in an attempt to frame the challenge embedded in this sample , first exploratory analysis with geostatistical methods indicated a potential differentiation of the periurban areas from augsburg city and its periphery ( see supplement 2 ) . potentially , the fine - scale patterns of genetic diversity observed in these first exploratory evaluations could be explained by various simple models of spatial variation . this is so because both geographic extension of the study area and evolutionary times of the study population ( here it refers to the number of generations necessary to fix the effects of any gene - flow process [ 23 , 24 ] ) are jointly , most probably , not large enough to have generated a local process of fixation of gradual variation of genetic features . , spatial autocorrelation ) and mantel tests would be the first methods of choice to detect spatial correlations of genetic distance with geographic distance . for instance , it is improbable that a modern western population inhabiting such a small area would be composed of several groups with reduced exchange ( low migration rates among the subareas and low predisposition to mate with individuals of other groups ) . this situation is even less probable if considered that spatial patterns of genetic variation were even detected within the group of natives . despite the large number of runs with varying models and parameters there was no indication of any population substructure . as a simplification , the usual denomination for this type of spatial object : interpolation based on splines proved to be a better choice for phenomena which combine a random component as well as processes which minimize energy , as it could be considered socio - demographic processes . with this step , we smoothed spatial interpolation results in the area of augsburg city and surroundings , while avoiding interpolation artifacts . in order to be able to adequately execute the v.surf.rst procedure the ho values computed using the raw data ( ho raw ) were transformed into percentage as follows:(2)ho raw100=ho , where ho raw , ho . interestingly , since the kora s4 genotypes conform a control population pool for genetic studies [ 7 , 9 ] , ho(all ) landscape may be examined as a representative estimation of the spatial variation of genetic diversity of the extant population and ho(ger ) landscape of the native fraction in the region of augsburg . spatial autocorrelation of each of the genetic and socio - demographic variables defined in this study was tested with the r package spdep . both implementations , moran.test and moran.mc , were used to test for spatial autocorrelation in measures of genetic variation : ( ho(all ) , ho(ger ) ) , as well as on the socio - demographic variables : ger_p , main_imp , age25_39 , age40_54 , ea55_74 , age_mean , ey8_11 , ey_mean , and edu_mean . socio - demographic measures were inspected as predictors of the observed pattern of ho(ger ) under the assumption that socio - demography would provide useful indication of spatial arrangement of recent migration processes , specially regional and national migration , which we assumed that it must have had a strong influence on fine - scale genetic variation . the contribution of socio - demographic factors to explain the observed spatial pattern was analyzed under the assumption of spatial dependence . best - fit spatial autoregressive models ( sar ) predicting heterozygosity ( hm ) were selected . in order to test if the spatial distribution of the major immigrant group ( main_img ) improves model prediction , the influence of the parameter main_imp on the selected model was tested . finally , the goodness of fit of the selected sar models was analyzed . in this step , the pixelwise divergence between predicted ( hm ) and observed values ( ho ) was quantified . the pixelwise divergence in absolute values of these interpolation surfaces from the ho(ger ) landscape was used to compare prediction accuracy among the selected sar models . the model with the lowest global difference between observed and predicted h values was selected as the one best fitting the data . based on this model , maps representing the spatial variation of predicted h values and the estimated divergence between observed and predicted h values were created . the latter maps allows a visual estimation of the agreement between observed and predicted heterozygosity in each land unit as well as the estimated spatial variation of divergence in the total study area . with regard to the spatial distribution of education level in the study area , the largest values of education years , that is , lower values of ey8_11 , were observed in augsburg city and in neighboring lus in the east ( friedberg ) and in the west ( aystetten , neus ) , as well as in the southern lu of schwabmnchen ( figure 4(a ) ) . in augsburg city , with a quite larger number of samples in comparison with all other lus ( table 1 ) , the lowest percentages were measured around augsburg city , in knigsbrunn , followed by langweid . in augsburg city , where a considerably larger total number of immigrants were sampled , the major immigrant group composed 78 percent of the total immigrant data set ( table 1 ) . the search for indications of spatial patterns of genetic heterogeneity with well - established procedures did not provide any positive results . although a high number of different explorative runs with different parameters were performed , tests based either on eigenstrat | eigensoft , plink , geneland , or spagedi did not provide any indication of a potential geographic variation of genetic heterogeneity in the study area . geostatistical analysis based on the statistic observed heterozygosity ( ho ) provided indication of spatial patterning . table 3 presents results of the spatial autocorrelation analysis performed with test statistic moran 's i. on the one side , the variable ho(all ) showed an indication of association between genetic variation and geographic coordinates . h o(all ) landscape , which estimates the variation of the genetic heterogeneity of the extant german population in the study area , presented a marked depression in the east ( figure 6(a ) ) . intermediate ho values covered the central - northern sectors , including some areas of aichach - friedberg district , langweid , augsburg city , and schwabmnchen . the ho(ger ) landscape , estimating the spatial variation of genetic heterogeneity of the native population , showed similar values to the ho(all ) landscape in the western and in the eastern periphery . four spatial autoregressive models were selected according to the p value ( p value < 0.05 ) of the covariates ( table 4 ) . the four models included as covariates variables related to age and education ; two models ( m1 , m3 ) included as well the proportion of natives per lu ( ger_p ) , which is at the same time an indication of the proportion of immigrants per lu . the maximal differences were measured in augsburg city and in rehling , a small residential area in the country side . fine - scale variation of genetic heterogeneity within a small region was detected and analyzed applying a geographic perspective . population genetics and geostatistics were combined with the open - source geographic information system grass . the capabilities of this approach were tested on a subset of the kora s4 survey [ 7 , 9 ] , collected in southern germany for prospective studies . we assumed that within small urbanized areas of modern western countries , as is the case in germany , genetic composition may be strongly affected by migratory movements of the last half century , which may be still estimated by means of socio - demographic measures . genotypes ( 212 autosomal snps ) and socio - demographic information ( age , education , place of residence , and birth land ) of 728 healthy german citizens were analyzed . in a first step to analyze spatial processes controlling the observed patterns , moran 's i tests for spatial autocorrelation were performed for all available parameters . indications of global spatial dependencies were observed in socio - demographic variables related to age categories and education level . while significant deviation of the expected moran 's i was obtained with the observed heterozygosity of the native subset ( ger ) , the observed heterozygosity of the total data set showed just an indication of a potential deviation of the expected moran 's i. in other words , these results could be interpreted as an indication that heterozygosity values of this small area may be regulated by a global spatial process , but such subtle process could only be observed when the subset of native germans is considered . spatial dependencies of socio - demographic variables at this local level could be axiomatically interpreted as a result of neighborhood preferences of recent local and regional migratory processes . it is to expect that social preferences affecting recent migration , such as regional and international migratory movements of the last half century , may be reflected by socio - demographic parameters . individuals would not choose new residence randomly and this may be reflected in similar socio - demographic attributes of contiguous neighborhoods . this expectation , which goes along with common sense , would also be in agreement with the fact that the level of admixture would associate to some extension with socio - demographic attributes of the location . this would be the result of new and recurrent resettlement of some areas or that inhabitants of areas presenting certain socio - demographic features would show higher predisposition to admixture . indeed , the fact that patterns of socio - demographic structure and genetic admixture account for spatial autocorrelation could indicate the occurrence of further unobserved phenomena influencing both , as it could be social preferences or official urban planning . spatial autoregressive models ( sar ) fitting the data were selected by a forward search . the four best - fit sar models contained as covariates socio - demographic measures related to age , school - attendance years , and education level . the prediction strength of the selected model was estimated with quantitative comparisons between the genetic landscapes created on basis of observed and predicted measures of heterozygosity . we found a good agreement between the predicted and the observed patterns , which supports the assumption of a certain relationship between genetic admixture degree and socio - demographic structure . the differentiation of augsburg city to the countryside ( see supplement 3 ) may be interpreted as the expected divergence of middle - size industrial settlements and countryside . this may reinforce the idea that this location may be attractive for newly settled workers willing to commute between their working place and a less expensive residential area , relatively close to a middle - size urban center as augsburg . although this group involved four birth lands , it may be speculated that the concentration of these individuals is not casual . in total , a concentration of small groups , each one showing a higher degree of homozygosity , would stand out over a much more admixed group , as it is expected for the native german population . these tools proved to be successful in studies of groups with considerably larger genetic differences , significantly more polymorphic loci , or much larger number of loci than in the present work . the kora study was carefully designed for prospective studies aiming to reduce any type of genetic structuring . for this reason , the number of available snps was probably too low for detecting fine - scale population differences with these standard tools . using full capabilities of tools such as spagedi or geneland , both offering individual - based analysis tools , was not possible either since the search of patterns of variation of genetic heterogeneity of the kora sample can not be carried out on an individualized geographical basis . accordingly , it is not surprising that well - established tools to detect human population stratification on a broader level or those which make use of geographic references on an individual basis ( mostly developed for studies of other species ) may not detect fine - scale patterns of genetic variation in small areas . these results support the elemental assumption that within multiethnic , urban , and suburban groups , as found in medium - sized german cities and surroundings , the socio - economic parameter birth land allows a first reduction of genetic heterogeneity . as it was presented in this study ( see supplement 2 ) , even after removing immigrants from the kora s4 data set , the degree of genetic differentiation in natives still overlapped with the spatial frequency distribution of immigrants . if future studies verify our exploratory results obtained with visual examination of genetic landscapes based on fst analogous reynolds ' r genetic distance , potentially , the proportion of immigrants may be used as subrogate of degree of natives ' admixture , which could actually reflect native 's behavior in regard to choice of area of residence and tolerance or predisposition to admix . in order to make use of these results for further genetic studies , first , the postulated fine - scale variation of genetic heterogeneity should be confirmed with a larger data set . knowledge of fine - scale patterns of genetic variation could provide information about areas where expected genetic heterogeneity could introduce undesired bias . in case that spatial heterogeneity would be assessed after recruitment , examining the spatial pattern of genetic heterogeneity could serve as a basis to decide about a stratified analysis ( e.g. our vision is to further develop our approach in order to be capable of testing as well as detecting and correcting , if it is applicable , for spatial patterns of genetic heterogeneity within the study sample ( cf . taking these findings of the kora s4 sample altogether we can state that fine - scale spatial genetic variation may be assumed in the study area . our results indicate that patterns of genetic heterogeneity can be present in small regions within germany . in conclusion , it may be stated that the presented genetic geostatistical approach has the potential of being a powerful tool for detecting , modeling , and analyzing spatial patterns of genetic heterogeneity even within populations inhabiting small regions .

glutamine is one of the 20 common amino acids , and thus it is a very important organic compound . although endogenous glutamine production is adequate in normal healthy people , glutamine depletion is associated with certain critical illnesses . it is said to become conditionally essential ' as the body 's requirements for glutamine can exceed the individual 's ability to produce it , and thus endogenous stores of glutamine are depleted rapidly . this dangerous scenario is demonstrated by studies showing that low plasma glutamine levels in patients admitted to intensive care units ( icus ) are an independent predictor of premature death . typically , the body 's glutamine levels will be most compromised for patients in hypercatabolic and/or hypermetabolic states ( e.g. , acute pancreatitis , severe burns , sepsis or following major surgical procedures ) , who thus may benefit most from an exogenous source of glutamine . practical problems with the use of free glutamine ( e.g. , instability and poor solubility ) have prevented its widespread use in the routine clinical setting . however , in recent years these problems have been overcome by the use of glutamine dipeptides ( e.g. , alanyl - glutamine ; dipeptiven ) . alanyl - glutamine is highly soluble , and it is stable in solution , allowing storage at room temperature and heat sterilisation . after its administration , the dipeptide is rapidly split into glutamine and alanine , which then flow as nutrients into their respective body pools and are metabolised according to the body 's needs . glutamine supplementation of standard parenteral nutrition ( pn ) for critically ill patients have included reductions in mortality rates , fewer infectious complications and reduced length of icu / hospital stay . glutamine supplementation at an adequate dose in patients most likely to benefit has prompted pharmacoeconomic cost - effectiveness studies . it combines different types of data ( e.g. , clinical and financial ) within a logical framework that represents the underlying data , thus allowing the cost - effectiveness of an intervention to be determined under conditions of uncertainty . glutamine supplementation has previously been investigated , and the results were highly favourable : net cost savings were achieved plus the costs of parenteral glutamine supplementation were offset completely . net - saving treatments ( i.e. , better quality of health care for less cost ) are exceptional among pharmacoeconomic evaluations . the model requires updating , as substantial new efficacy data have been published in the form a meta - analysis . this meta - analysis included a clinically realistic subgroup analysis based on adequate doses of parenteral glutamine ( > 0.2 g / kg body weight per day ) , as recommended by the guidelines . these doses were associated with reduced mortality rates , infection rates and shorter length of hospital stay for icu patients , and they form the basis of efficacy data used for the current model . in this article , we will combine the most up - to - date and clinically relevant efficacy data , the most recent cost data and the latest epidemiology data from a large italian icu database to determine whether i.v . glutamine supplementation remains a highly favourable cost - effective option in total pn therapy for intensive care patients . the model combines ( i ) baseline outcomes from the italian icu patient population , ( ii ) partially re - elaborated efficacy data from a meta - analysis of published randomised clinical trials and ( iii ) national ( italian ) cost data . the following text describes the model structure , baseline event rates and underlying assumptions , efficacy data sources and unit costs included in the modelling . treeage pro 2009 software ( treeage software inc . , williamstown , ma , usa ) was used to design a discrete event simulation , which is a type of decision model ( other types are decision trees and markov models ) . decision models are used to help decide whether treatment a or treatment b is preferable for a particular type of patient on the basis of two dimensions : costs and health effects . in a discrete event simulation , the experience of individuals is modelled over time in terms of the events that occur and the consequences of those events . a discrete event simulation is more practical than markov models when timing and chronology of events is important . moreover , discrete event simulation models are analysed as patient - level simulations , and this can add accuracy to the analysis . in brief , this model was used to compare the assignment of icu patients with standard pn or pn supplemented with i.v . every patient started in the icu , where they may ( or may not ) develop a new nosocomial infection . in either case , the patient admitted to the icu faced three alternative possibilities : ( i ) death in the icu , ( ii ) recovery and transfer to a general ward or ( iii ) recovery and discharge home . for those transferred to a general ward , there were two possibilities : ( i ) recovery and discharge or ( ii ) death ( figure 1 ) . consistent with this modelling technique , time was not discretised in cycles , but it was handled as time - to - event ; the time that patients spent in each state was sampled from distributions fitted to data reported in the progetto margherita ( table 1 ) . the progetto margherita report was used as the icu population data source for estimating baseline outcome rates ( death rates , infection rates and length of hospital / icu stay ) and transition probabilities ( probability of changing from one health state to another health state within a given period ) . elaborated data are reported in table 1 , which also details the choice of distributions allocated to each parameter in the model . the progetto margherita report used covers data from 2010 , which is an update on the 2007 data used in the previous model . it is an annual publication on behalf of the gruppo italiano per la valutazione degli interventi in terapia intensiva ( giviti ) , and it includes data collected in 230 italian icus , from a total of over 77 000 patients , covering over half of all icus operating in italy . this well - established project has been used as a data resource for observational investigations , including pharmacoeconomic studies . the duration of pn assumed that ( i ) all patients in the treatment arm received at least one full day of pn ; ( ii ) that pn never lasted as long as the hospital stay ( ends at least 2 days before the discharge from the hospital to allow for patient stabilisation , excluding the pre - icu length of stay ) ; and ( iii ) that for patients who die the duration of pn did not exceed the length of hospital stay . comparative efficacy data from clinical trials for standard pn vs supplementation of pn with i.v . glutamine for icu patients were taken from a subgroup in the meta - analysis of bollhalder et al . , and showed clear improvements in relative risk of death , infection and the length of hospital stay ( table 2 ) . this subgroup provided data for a clinically realistic and adequate dose of parenteral glutamine ( > 0.2 g / kg body weight per day ) , as recommended by the guidelines , and it represented the majority of the patients included in the meta - analysis ( 5463% , depending on the parameter analysed ) . the absolute length of stay reduction reported in the meta analysis was converted to a relative duration ( relative risk ( rr)=0.91 ; 95% confidence interval ( ci ) : 0.860.96 ) by using the mean hospital length of stay reported by patients given standard pn calculated from bollhalder et al . this additional processing is needed , as the absolute reduction estimated by bollhalder et al . can be applied only to total length of hospital stay , whereas in our model this is assessed in two stages ( length of stay in the icu and then in the general ward ) . the relative reduction can thus be applied to both the icu and the general ward for patients given parenteral glutamine . moreover , reductions in the length of hospital stay owing to glutamine supplementation are related to the time over which patients are given pn , and thus the efficacy of treatment results are better modelled by relative risk . the study perspective is that of the health - care provider ( i.e. , hospitals ) , and thus only direct costs ( e.g. , cost of stays in the icu or ward ) rather than indirect costs ( e.g. , working days lost ) were considered . thus , the price of infection , icu and ward costs were included , with these costs derived from official sources and published literature updated to 2012 values . adjustment to 2012 thus , the most up - to - date and publicly available figures for italian health care were used for the daily cost of icu stay , hospital ward stay and cost of new infections , as follows . the daily cost of icu stay ( 1581.76 ) was estimated using the results of an italian 2-year prospective case control study , subtracting the cost of surgical procedures and cost of extra antibiotics in sepsis patients ( which were considered separately for patients developing nosocomial infections in the simulation ) . the cost for an average day in an italian hospital ward ( 793.30 ) was taken from the report of the national agency for regional health services ( assr ) . the cost of new infections was included in the cost input for the model based on average prices reported for the treatment of icu - emergent bloodstream infections . these costs were limited to those for extra anti - infective treatments needed ( i.e. , 1114.87 ) , rather than effects of new infections on length of hospital stay or mortality rates , as length of stay and mortality rates were already included in the model as clinical outcomes . the cost of i.v . glutamine supplementation was calculated per simulated patient on the basis of their body weight and pn duration , assuming a dose of alanyl - glutamine of 0.5 g / kg per day , and a price of 2.15/g , which was derived from 50% of the wholesale price ( average price to italian hospitals ) of 85.97 for a 100- ml vial of alanyl - glutamine ( dipeptiven ; fresenius kabi deutschland gmbh , bad homburg , germany ) . supplementation cost was calculated for each simulated patient on the basis of ( i ) the duration of pn ( mean 6.13 days ; s.d . 4.77 ) sampled from a weibull distribution fitted on margherita data for icu patients , and ( ii ) individual patients ' weights in the model ( mean 70.55 kg ; s.d . 13.39 ) sampled from a gamma distribution reflecting the populations enrolled in the clinical trials used to determine treatment effectiveness estimates . the model accounts for variability among individuals ( as it is a patient - level simulation ) , and also the uncertainty of key parameters ( by performing a probabilistic sensitivity analysis ) . it was performed using a two - level monte carlo simulation : the inner loop ( 10 000 iterations ) was the patient - level simulation , which was averaged and repeated 1000 times ( outer loop ) each with a unique set of key model parameters drawn randomly from distributions representing the range of plausible values . a standard deviation of 20% of mean values was supposed for parameters without 95% cis ( i.e. , unit costs , infection probability and glutamine dose ) . a deterministic ( one - way ) sensitivity analysis was also used , which tested the sensitivity of estimated money savings per patient to extreme variations of base - case estimates . the 2.5th and 97.5th percentiles of the distributions used in the outer loop of the simulation were used as minimum and maximum values , whereas patient - level variables varied by shifting distributions to the left and right by 20% of the mean values . treeage pro 2009 software ( treeage software inc . , williamstown , ma , usa ) was used to design a discrete event simulation , which is a type of decision model ( other types are decision trees and markov models ) . decision models are used to help decide whether treatment a or treatment b is preferable for a particular type of patient on the basis of two dimensions : costs and health effects . in a discrete event simulation , the experience of individuals is modelled over time in terms of the events that occur and the consequences of those events . a discrete event simulation is more practical than markov models when timing and chronology of events is important . moreover , discrete event simulation models are analysed as patient - level simulations , and this can add accuracy to the analysis . in brief , this model was used to compare the assignment of icu patients with standard pn or pn supplemented with i.v . every patient started in the icu , where they may ( or may not ) develop a new nosocomial infection . in either case , the patient admitted to the icu faced three alternative possibilities : ( i ) death in the icu , ( ii ) recovery and transfer to a general ward or ( iii ) recovery and discharge home . for those transferred to a general ward , there were two possibilities : ( i ) recovery and discharge or ( ii ) death ( figure 1 ) . consistent with this modelling technique , time was not discretised in cycles , but it was handled as time - to - event ; the time that patients spent in each state was sampled from distributions fitted to data reported in the progetto margherita ( table 1 ) . the progetto margherita report was used as the icu population data source for estimating baseline outcome rates ( death rates , infection rates and length of hospital / icu stay ) and transition probabilities ( probability of changing from one health state to another health state within a given period ) . elaborated data are reported in table 1 , which also details the choice of distributions allocated to each parameter in the model . the progetto margherita report used covers data from 2010 , which is an update on the 2007 data used in the previous model . it is an annual publication on behalf of the gruppo italiano per la valutazione degli interventi in terapia intensiva ( giviti ) , and it includes data collected in 230 italian icus , from a total of over 77 000 patients , covering over half of all icus operating in italy . this well - established project has been used as a data resource for observational investigations , including pharmacoeconomic studies . the duration of pn assumed that ( i ) all patients in the treatment arm received at least one full day of pn ; ( ii ) that pn never lasted as long as the hospital stay ( ends at least 2 days before the discharge from the hospital to allow for patient stabilisation , excluding the pre - icu length of stay ) ; and ( iii ) that for patients who die the duration of pn did not exceed the length of hospital stay . comparative efficacy data from clinical trials for standard pn vs supplementation of pn with i.v . glutamine for icu patients were taken from a subgroup in the meta - analysis of bollhalder et al . , and showed clear improvements in relative risk of death , infection and the length of hospital stay ( table 2 ) . this subgroup provided data for a clinically realistic and adequate dose of parenteral glutamine ( > 0.2 g / kg body weight per day ) , as recommended by the guidelines , and it represented the majority of the patients included in the meta - analysis ( 5463% , depending on the parameter analysed ) . the absolute length of stay reduction reported in the meta analysis was converted to a relative duration ( relative risk ( rr)=0.91 ; 95% confidence interval ( ci ) : 0.860.96 ) by using the mean hospital length of stay reported by patients given standard pn calculated from bollhalder et al . this additional processing is needed , as the absolute reduction estimated by bollhalder et al . can be applied only to total length of hospital stay , whereas in our model this is assessed in two stages ( length of stay in the icu and then in the general ward ) . the relative reduction can thus be applied to both the icu and the general ward for patients given parenteral glutamine . moreover , reductions in the length of hospital stay owing to glutamine supplementation are related to the time over which patients are given pn , and thus the efficacy of treatment results are better modelled by relative risk . the study perspective is that of the health - care provider ( i.e. , hospitals ) , and thus only direct costs ( e.g. , cost of stays in the icu or ward ) rather than indirect costs ( e.g. , working days lost ) were considered . thus , the price of infection , icu and ward costs were included , with these costs derived from official sources and published literature updated to 2012 values . adjustment to 2012 values used figures for the italian consumer price index available from eurostat . thus , the most up - to - date and publicly available figures for italian health care were used for the daily cost of icu stay , hospital ward stay and cost of new infections , as follows . the daily cost of icu stay ( 1581.76 ) was estimated using the results of an italian 2-year prospective case control study , subtracting the cost of surgical procedures and cost of extra antibiotics in sepsis patients ( which were considered separately for patients developing nosocomial infections in the simulation ) . the cost for an average day in an italian hospital ward ( 793.30 ) was taken from the report of the national agency for regional health services ( assr ) . the cost of new infections was included in the cost input for the model based on average prices reported for the treatment of icu - emergent bloodstream infections . these costs were limited to those for extra anti - infective treatments needed ( i.e. , 1114.87 ) , rather than effects of new infections on length of hospital stay or mortality rates , as length of stay and mortality rates were already included in the model as clinical outcomes . glutamine supplementation was calculated per simulated patient on the basis of their body weight and pn duration , assuming a dose of alanyl - glutamine of 0.5 g / kg per day , and a price of 2.15/g , which was derived from 50% of the wholesale price ( average price to italian hospitals ) of 85.97 for a 100- ml vial of alanyl - glutamine ( dipeptiven ; fresenius kabi deutschland gmbh , bad homburg , germany ) . supplementation cost was calculated for each simulated patient on the basis of ( i ) the duration of pn ( mean 6.13 days ; s.d . 4.77 ) sampled from a weibull distribution fitted on margherita data for icu patients , and ( ii ) individual patients ' weights in the model ( mean 70.55 kg ; s.d . 13.39 ) sampled from a gamma distribution reflecting the populations enrolled in the clinical trials used to determine treatment effectiveness estimates . the model accounts for variability among individuals ( as it is a patient - level simulation ) , and also the uncertainty of key parameters ( by performing a probabilistic sensitivity analysis ) . it was performed using a two - level monte carlo simulation : the inner loop ( 10 000 iterations ) was the patient - level simulation , which was averaged and repeated 1000 times ( outer loop ) each with a unique set of key model parameters drawn randomly from distributions representing the range of plausible values . a standard deviation of 20% of mean values was supposed for parameters without 95% cis ( i.e. , unit costs , infection probability and glutamine dose ) . a deterministic ( one - way ) sensitivity analysis was also used , which tested the sensitivity of estimated money savings per patient to extreme variations of base - case estimates . the 2.5th and 97.5th percentiles of the distributions used in the outer loop of the simulation were used as minimum and maximum values , whereas patient - level variables varied by shifting distributions to the left and right by 20% of the mean values . pn regimens supplemented with glutamine offer the following advantages over standard pn : they prevent 29.0% of deaths ( 753 of 2598 ) and 21.2% of infections ( 247 of 1165 ) , and they reduce the mean overall length of hospital stay by 1.07 days per patient ( table 3 ) . alanyl - glutamine supplementation , is also expected to reduce overall costs compared with standard pn , with a mean net cost saving of 1047 per patient ( table 3 ) . these results indicate that alanyl - glutamine costs ( 369 ) are more than offset by savings in hospital stay costs ( 1388 ) , and also by reduced antibiotic costs for the treatment of icu - emergent infections ( 28 ) . consequently , alanyl - glutamine is expected to be a more cost - effective option than standard pn , as it is associated with a lower mean cost per patient discharged alive ( table 3 ) . indeed , on average , it is expected to dominate standard pn , as it is associated with better clinical and economic outcomes . the probabilistic sensitivity analysis confirmed the robustness of the model results : a strategy of parenteral glutamine supplementation was dominant in 91.2% of cases ( figure 2 ) . results of the deterministic ( one - way ) sensitivity analysis are displayed as a tornado diagram showing the influence of extreme variations in each key parameter on cost savings per patient ( figure 3 ) . this shows that the most influential parameters were the average daily cost of the icu , cost of parenteral glutamine supplementation , length of stay in the icu and icu mortality . none of the variations tested resulted in parenteral glutamine supplementation losing its dominance over standard pn regimens , finding the study to be robust and reliable that on average pn regimens supplemented with glutamine are cost saving compared with standard pn regimens . pn regimens supplemented with glutamine offer the following advantages over standard pn : they prevent 29.0% of deaths ( 753 of 2598 ) and 21.2% of infections ( 247 of 1165 ) , and they reduce the mean overall length of hospital stay by 1.07 days per patient ( table 3 ) . alanyl - glutamine supplementation , is also expected to reduce overall costs compared with standard pn , with a mean net cost saving of 1047 per patient ( table 3 ) . these results indicate that alanyl - glutamine costs ( 369 ) are more than offset by savings in hospital stay costs ( 1388 ) , and also by reduced antibiotic costs for the treatment of icu - emergent infections ( 28 ) . consequently , alanyl - glutamine is expected to be a more cost - effective option than standard pn , as it is associated with a lower mean cost per patient discharged alive ( table 3 ) . indeed , on average , it is expected to dominate standard pn , as it is associated with better clinical and economic outcomes . the probabilistic sensitivity analysis confirmed the robustness of the model results : a strategy of parenteral glutamine supplementation was dominant in 91.2% of cases ( figure 2 ) . results of the deterministic ( one - way ) sensitivity analysis are displayed as a tornado diagram showing the influence of extreme variations in each key parameter on cost savings per patient ( figure 3 ) . this shows that the most influential parameters were the average daily cost of the icu , cost of parenteral glutamine supplementation , length of stay in the icu and icu mortality . none of the variations tested resulted in parenteral glutamine supplementation losing its dominance over standard pn regimens , finding the study to be robust and reliable that on average pn regimens supplemented with glutamine are cost saving compared with standard pn regimens . the results of the analyses presented here suggest that parenteral glutamine supplementation has the potential to significantly improve outcomes for icu patients in italy , as our model shows that the additional cost of supplemented treatment is more than completely offset by cost savings in hospital care . thus , parenteral glutamine supplementation was more cost - effective ( cer=35 165 per patient discharged alive ) than standard , non - supplemented pn ( cer=40 156 per patient discharged alive ) , and it resulted in mean cost savings of 4991 per patient discharged alive or 1047 per patient admitted to the hospital . as supplementation costs were more than completely offset by cost savings to the hospital , glutamine supplementation is said to dominate ' standard , non - supplemented pn , as it provides better quality of health care for less cost . the conclusions for the current study are similar to those published previously , but the availability of more specific meta - analysis results have allowed improvements to the clinical sources used in the model to greater reflect best clinical practice in administering pn . this update also relies on a more up - to - date and wider group of icu patients than our previous study ( i.e. , not just patients admitted to the icu for intensive care therapies ' as in the previous study , but also including less critically ill patients such as those admitted for monitoring and weaning ' , which represent about one - third of all icu patients ) . thus , the current analysis may reflect recent italian patient populations in icus more closely . the current updated pharmacoeconomic analysis uses a longer length of hospital stay , resulting in somewhat higher costs per patient for both standard and glutamine - supplemented pn than in the previous study . moreover , there are slight differences between the two studies in reduction of infectious complications and mortality rates . taken together , these differences translate into variations in the economic analysis ( total mean net savings per patient of 752 vs 1047 , for the 2012 study and in this update , respectively ) . the stability of the model results has also been demonstrated by both types of sensitivity analysis performed , suggesting that these findings are likely to be applicable to other health - care settings and systems outside italy provided that there are no major differences in the icu patient population , and/or the organisation and costs of the icu or more generally in tertiary care . thus , the addition of alanyl - glutamine to standard pn regimens for icu patients is likely to be very cost - effective ( if not dominant ) in most european countries . nevertheless , increased acquisition costs still form a barrier to more widespread use of alanyl - glutamine supplementation of pn . several clinical studies have shown that addition of alanyl - glutamine to nutrition regimens can minimise resource health - care use . a randomised double - blind study comparing the use of supplemental alanyl - glutamine with standard pn regimens in patients following major abdominal surgery showed that those given alanyl - glutamine had a significantly reduced hospital stay , and thus this strategy allowed potential cost savings . another randomised double - blind study in patients with burns found that supplemental alanyl - glutamine significantly reduced wound healing times and hospital costs compared with standard pn . it is important , however , to understand that we do not suggest that supplementation of all patients ' standard pn regimens with alanyl - glutamine will be a cost - effective strategy . it is unlikely to be effective if it is used in an inappropriate clinical scenario : clinicians ' discernment and clinical judgment are necessary for the selection of patients in whom supplementation will be beneficial . alanyl - glutamine is indicated as part of a clinical nutrition regimen in patients in hypercatabolic and/or hypermetabolic states , and it should be given together with parenteral or enteral nutrition or a combination of both . although glutamine is normally a non - essential amino acid in healthy individuals , it can become conditionally essential under hypercatabolic and hypermetabolic conditions . glutamine supplementation include critically ill / icu patients , those with acute pancreatitis , severe burns , sepsis or following major surgical procedures ( e.g. , abdominal surgery ) . there are a number of well - known limitations to the modelling of cost - effectiveness : these are intrinsic to modelling itself , and they centre on the need for combining various data sources into a single logical construct . however , the use of modelling in economic evaluations is regarded widely as an unavoidable fact of life ' . models are often used in health economic evaluations , as relevant clinical trials have not been performed or they did not report economic data . decision analytic models are often used as a tool for health - care decision - makers , allowing the synthesis of the best available data rather than waiting for the perfect set of clinical and economic data to become available . glutamine ( in the form of alanyl - glutamine ) to standard pn is a clinically and economically attractive strategy for icu patients in italy . moreover , the robustness of these results mean that they may be applicable to selected icu patient populations in other countries .

background / objectives : intravenous ( i.v . ) glutamine supplementation of parenteral nutrition ( pn ) can improve clinical outcomes , reduce mortality and infection rates and shorten the length of hospital and/or intensive care unit ( icu ) stays compared with standard pn . this study is a pharmacoeconomic analysis to determine whether i.v . glutamine supplementation of pn remains both a highly favourable and cost - effective option for italian icu patients.subjects/methods:a previously published discrete event simulation model was updated by incorporating the most up - to - date and clinically relevant efficacy data ( a clinically realistic subgroup analysis from a published meta - analysis ) , recent cost data from the italian health - care system and the latest epidemiology data from a large italian icu database ( covering 230 italian icus and more than 77 000 patients ) . sensitivity analyses were performed to test the robustness of the results.results:parenteral glutamine supplementation can significantly improve icu efficiency in italy , as the additional cost of supplemented treatment is more than completely offset by cost savings in hospital care . supplementation was more cost - effective ( cost - effectiveness ratio ( cer)=35 165 per patient discharged alive ) than standard , non - supplemented pn ( cer=40 156 per patient discharged alive ) , and it resulted in mean cost savings of 4991 per patient discharged alive or 1047 per patient admitted to the hospital . sensitivity analyses confirmed the robustness of these results.conclusions:alanyl-glutamine supplementation of pn is a clinically and economically attractive strategy for icu patients in italy and may be applicable to selected icu patient populations in other countries .

Introduction Materials and methods Model structure Patient population: data sources Clinical efficacy: data sources Cost assessment: data sources Simulation and sensitivity analyses Results Main results Sensitivity analyses results Discussion Conclusions

glutamine is one of the 20 common amino acids , and thus it is a very important organic compound . glutamine supplementation of standard parenteral nutrition ( pn ) for critically ill patients have included reductions in mortality rates , fewer infectious complications and reduced length of icu / hospital stay . glutamine supplementation at an adequate dose in patients most likely to benefit has prompted pharmacoeconomic cost - effectiveness studies . glutamine supplementation has previously been investigated , and the results were highly favourable : net cost savings were achieved plus the costs of parenteral glutamine supplementation were offset completely . the model requires updating , as substantial new efficacy data have been published in the form a meta - analysis . this meta - analysis included a clinically realistic subgroup analysis based on adequate doses of parenteral glutamine ( > 0.2 g / kg body weight per day ) , as recommended by the guidelines . these doses were associated with reduced mortality rates , infection rates and shorter length of hospital stay for icu patients , and they form the basis of efficacy data used for the current model . in this article , we will combine the most up - to - date and clinically relevant efficacy data , the most recent cost data and the latest epidemiology data from a large italian icu database to determine whether i.v . glutamine supplementation remains a highly favourable cost - effective option in total pn therapy for intensive care patients . the model combines ( i ) baseline outcomes from the italian icu patient population , ( ii ) partially re - elaborated efficacy data from a meta - analysis of published randomised clinical trials and ( iii ) national ( italian ) cost data . , williamstown , ma , usa ) was used to design a discrete event simulation , which is a type of decision model ( other types are decision trees and markov models ) . in a discrete event simulation , the experience of individuals is modelled over time in terms of the events that occur and the consequences of those events . a discrete event simulation is more practical than markov models when timing and chronology of events is important . moreover , discrete event simulation models are analysed as patient - level simulations , and this can add accuracy to the analysis . in brief , this model was used to compare the assignment of icu patients with standard pn or pn supplemented with i.v . in either case , the patient admitted to the icu faced three alternative possibilities : ( i ) death in the icu , ( ii ) recovery and transfer to a general ward or ( iii ) recovery and discharge home . the progetto margherita report was used as the icu population data source for estimating baseline outcome rates ( death rates , infection rates and length of hospital / icu stay ) and transition probabilities ( probability of changing from one health state to another health state within a given period ) . it is an annual publication on behalf of the gruppo italiano per la valutazione degli interventi in terapia intensiva ( giviti ) , and it includes data collected in 230 italian icus , from a total of over 77 000 patients , covering over half of all icus operating in italy . the duration of pn assumed that ( i ) all patients in the treatment arm received at least one full day of pn ; ( ii ) that pn never lasted as long as the hospital stay ( ends at least 2 days before the discharge from the hospital to allow for patient stabilisation , excluding the pre - icu length of stay ) ; and ( iii ) that for patients who die the duration of pn did not exceed the length of hospital stay . comparative efficacy data from clinical trials for standard pn vs supplementation of pn with i.v . glutamine for icu patients were taken from a subgroup in the meta - analysis of bollhalder et al . , and showed clear improvements in relative risk of death , infection and the length of hospital stay ( table 2 ) . this subgroup provided data for a clinically realistic and adequate dose of parenteral glutamine ( > 0.2 g / kg body weight per day ) , as recommended by the guidelines , and it represented the majority of the patients included in the meta - analysis ( 5463% , depending on the parameter analysed ) . the absolute length of stay reduction reported in the meta analysis was converted to a relative duration ( relative risk ( rr)=0.91 ; 95% confidence interval ( ci ) : 0.860.96 ) by using the mean hospital length of stay reported by patients given standard pn calculated from bollhalder et al . can be applied only to total length of hospital stay , whereas in our model this is assessed in two stages ( length of stay in the icu and then in the general ward ) . moreover , reductions in the length of hospital stay owing to glutamine supplementation are related to the time over which patients are given pn , and thus the efficacy of treatment results are better modelled by relative risk . the study perspective is that of the health - care provider ( i.e. adjustment to 2012 thus , the most up - to - date and publicly available figures for italian health care were used for the daily cost of icu stay , hospital ward stay and cost of new infections , as follows . the daily cost of icu stay ( 1581.76 ) was estimated using the results of an italian 2-year prospective case control study , subtracting the cost of surgical procedures and cost of extra antibiotics in sepsis patients ( which were considered separately for patients developing nosocomial infections in the simulation ) . , 1114.87 ) , rather than effects of new infections on length of hospital stay or mortality rates , as length of stay and mortality rates were already included in the model as clinical outcomes . glutamine supplementation was calculated per simulated patient on the basis of their body weight and pn duration , assuming a dose of alanyl - glutamine of 0.5 g / kg per day , and a price of 2.15/g , which was derived from 50% of the wholesale price ( average price to italian hospitals ) of 85.97 for a 100- ml vial of alanyl - glutamine ( dipeptiven ; fresenius kabi deutschland gmbh , bad homburg , germany ) . supplementation cost was calculated for each simulated patient on the basis of ( i ) the duration of pn ( mean 6.13 days ; s.d . 4.77 ) sampled from a weibull distribution fitted on margherita data for icu patients , and ( ii ) individual patients ' weights in the model ( mean 70.55 kg ; s.d . 13.39 ) sampled from a gamma distribution reflecting the populations enrolled in the clinical trials used to determine treatment effectiveness estimates . the model accounts for variability among individuals ( as it is a patient - level simulation ) , and also the uncertainty of key parameters ( by performing a probabilistic sensitivity analysis ) . , williamstown , ma , usa ) was used to design a discrete event simulation , which is a type of decision model ( other types are decision trees and markov models ) . in a discrete event simulation , the experience of individuals is modelled over time in terms of the events that occur and the consequences of those events . a discrete event simulation is more practical than markov models when timing and chronology of events is important . moreover , discrete event simulation models are analysed as patient - level simulations , and this can add accuracy to the analysis . in brief , this model was used to compare the assignment of icu patients with standard pn or pn supplemented with i.v . in either case , the patient admitted to the icu faced three alternative possibilities : ( i ) death in the icu , ( ii ) recovery and transfer to a general ward or ( iii ) recovery and discharge home . consistent with this modelling technique , time was not discretised in cycles , but it was handled as time - to - event ; the time that patients spent in each state was sampled from distributions fitted to data reported in the progetto margherita ( table 1 ) . the progetto margherita report was used as the icu population data source for estimating baseline outcome rates ( death rates , infection rates and length of hospital / icu stay ) and transition probabilities ( probability of changing from one health state to another health state within a given period ) . it is an annual publication on behalf of the gruppo italiano per la valutazione degli interventi in terapia intensiva ( giviti ) , and it includes data collected in 230 italian icus , from a total of over 77 000 patients , covering over half of all icus operating in italy . the duration of pn assumed that ( i ) all patients in the treatment arm received at least one full day of pn ; ( ii ) that pn never lasted as long as the hospital stay ( ends at least 2 days before the discharge from the hospital to allow for patient stabilisation , excluding the pre - icu length of stay ) ; and ( iii ) that for patients who die the duration of pn did not exceed the length of hospital stay . comparative efficacy data from clinical trials for standard pn vs supplementation of pn with i.v . glutamine for icu patients were taken from a subgroup in the meta - analysis of bollhalder et al . , and showed clear improvements in relative risk of death , infection and the length of hospital stay ( table 2 ) . this subgroup provided data for a clinically realistic and adequate dose of parenteral glutamine ( > 0.2 g / kg body weight per day ) , as recommended by the guidelines , and it represented the majority of the patients included in the meta - analysis ( 5463% , depending on the parameter analysed ) . the absolute length of stay reduction reported in the meta analysis was converted to a relative duration ( relative risk ( rr)=0.91 ; 95% confidence interval ( ci ) : 0.860.96 ) by using the mean hospital length of stay reported by patients given standard pn calculated from bollhalder et al . moreover , reductions in the length of hospital stay owing to glutamine supplementation are related to the time over which patients are given pn , and thus the efficacy of treatment results are better modelled by relative risk . the study perspective is that of the health - care provider ( i.e. , hospitals ) , and thus only direct costs ( e.g. thus , the most up - to - date and publicly available figures for italian health care were used for the daily cost of icu stay , hospital ward stay and cost of new infections , as follows . the daily cost of icu stay ( 1581.76 ) was estimated using the results of an italian 2-year prospective case control study , subtracting the cost of surgical procedures and cost of extra antibiotics in sepsis patients ( which were considered separately for patients developing nosocomial infections in the simulation ) . , 1114.87 ) , rather than effects of new infections on length of hospital stay or mortality rates , as length of stay and mortality rates were already included in the model as clinical outcomes . glutamine supplementation was calculated per simulated patient on the basis of their body weight and pn duration , assuming a dose of alanyl - glutamine of 0.5 g / kg per day , and a price of 2.15/g , which was derived from 50% of the wholesale price ( average price to italian hospitals ) of 85.97 for a 100- ml vial of alanyl - glutamine ( dipeptiven ; fresenius kabi deutschland gmbh , bad homburg , germany ) . supplementation cost was calculated for each simulated patient on the basis of ( i ) the duration of pn ( mean 6.13 days ; s.d . 4.77 ) sampled from a weibull distribution fitted on margherita data for icu patients , and ( ii ) individual patients ' weights in the model ( mean 70.55 kg ; s.d . the model accounts for variability among individuals ( as it is a patient - level simulation ) , and also the uncertainty of key parameters ( by performing a probabilistic sensitivity analysis ) . pn regimens supplemented with glutamine offer the following advantages over standard pn : they prevent 29.0% of deaths ( 753 of 2598 ) and 21.2% of infections ( 247 of 1165 ) , and they reduce the mean overall length of hospital stay by 1.07 days per patient ( table 3 ) . alanyl - glutamine supplementation , is also expected to reduce overall costs compared with standard pn , with a mean net cost saving of 1047 per patient ( table 3 ) . these results indicate that alanyl - glutamine costs ( 369 ) are more than offset by savings in hospital stay costs ( 1388 ) , and also by reduced antibiotic costs for the treatment of icu - emergent infections ( 28 ) . consequently , alanyl - glutamine is expected to be a more cost - effective option than standard pn , as it is associated with a lower mean cost per patient discharged alive ( table 3 ) . the probabilistic sensitivity analysis confirmed the robustness of the model results : a strategy of parenteral glutamine supplementation was dominant in 91.2% of cases ( figure 2 ) . results of the deterministic ( one - way ) sensitivity analysis are displayed as a tornado diagram showing the influence of extreme variations in each key parameter on cost savings per patient ( figure 3 ) . this shows that the most influential parameters were the average daily cost of the icu , cost of parenteral glutamine supplementation , length of stay in the icu and icu mortality . none of the variations tested resulted in parenteral glutamine supplementation losing its dominance over standard pn regimens , finding the study to be robust and reliable that on average pn regimens supplemented with glutamine are cost saving compared with standard pn regimens . pn regimens supplemented with glutamine offer the following advantages over standard pn : they prevent 29.0% of deaths ( 753 of 2598 ) and 21.2% of infections ( 247 of 1165 ) , and they reduce the mean overall length of hospital stay by 1.07 days per patient ( table 3 ) . alanyl - glutamine supplementation , is also expected to reduce overall costs compared with standard pn , with a mean net cost saving of 1047 per patient ( table 3 ) . these results indicate that alanyl - glutamine costs ( 369 ) are more than offset by savings in hospital stay costs ( 1388 ) , and also by reduced antibiotic costs for the treatment of icu - emergent infections ( 28 ) . consequently , alanyl - glutamine is expected to be a more cost - effective option than standard pn , as it is associated with a lower mean cost per patient discharged alive ( table 3 ) . the probabilistic sensitivity analysis confirmed the robustness of the model results : a strategy of parenteral glutamine supplementation was dominant in 91.2% of cases ( figure 2 ) . results of the deterministic ( one - way ) sensitivity analysis are displayed as a tornado diagram showing the influence of extreme variations in each key parameter on cost savings per patient ( figure 3 ) . this shows that the most influential parameters were the average daily cost of the icu , cost of parenteral glutamine supplementation , length of stay in the icu and icu mortality . none of the variations tested resulted in parenteral glutamine supplementation losing its dominance over standard pn regimens , finding the study to be robust and reliable that on average pn regimens supplemented with glutamine are cost saving compared with standard pn regimens . the results of the analyses presented here suggest that parenteral glutamine supplementation has the potential to significantly improve outcomes for icu patients in italy , as our model shows that the additional cost of supplemented treatment is more than completely offset by cost savings in hospital care . thus , parenteral glutamine supplementation was more cost - effective ( cer=35 165 per patient discharged alive ) than standard , non - supplemented pn ( cer=40 156 per patient discharged alive ) , and it resulted in mean cost savings of 4991 per patient discharged alive or 1047 per patient admitted to the hospital . as supplementation costs were more than completely offset by cost savings to the hospital , glutamine supplementation is said to dominate ' standard , non - supplemented pn , as it provides better quality of health care for less cost . the conclusions for the current study are similar to those published previously , but the availability of more specific meta - analysis results have allowed improvements to the clinical sources used in the model to greater reflect best clinical practice in administering pn . this update also relies on a more up - to - date and wider group of icu patients than our previous study ( i.e. , not just patients admitted to the icu for intensive care therapies ' as in the previous study , but also including less critically ill patients such as those admitted for monitoring and weaning ' , which represent about one - third of all icu patients ) . thus , the current analysis may reflect recent italian patient populations in icus more closely . the current updated pharmacoeconomic analysis uses a longer length of hospital stay , resulting in somewhat higher costs per patient for both standard and glutamine - supplemented pn than in the previous study . the stability of the model results has also been demonstrated by both types of sensitivity analysis performed , suggesting that these findings are likely to be applicable to other health - care settings and systems outside italy provided that there are no major differences in the icu patient population , and/or the organisation and costs of the icu or more generally in tertiary care . thus , the addition of alanyl - glutamine to standard pn regimens for icu patients is likely to be very cost - effective ( if not dominant ) in most european countries . nevertheless , increased acquisition costs still form a barrier to more widespread use of alanyl - glutamine supplementation of pn . several clinical studies have shown that addition of alanyl - glutamine to nutrition regimens can minimise resource health - care use . a randomised double - blind study comparing the use of supplemental alanyl - glutamine with standard pn regimens in patients following major abdominal surgery showed that those given alanyl - glutamine had a significantly reduced hospital stay , and thus this strategy allowed potential cost savings . another randomised double - blind study in patients with burns found that supplemental alanyl - glutamine significantly reduced wound healing times and hospital costs compared with standard pn . it is important , however , to understand that we do not suggest that supplementation of all patients ' standard pn regimens with alanyl - glutamine will be a cost - effective strategy . alanyl - glutamine is indicated as part of a clinical nutrition regimen in patients in hypercatabolic and/or hypermetabolic states , and it should be given together with parenteral or enteral nutrition or a combination of both . there are a number of well - known limitations to the modelling of cost - effectiveness : these are intrinsic to modelling itself , and they centre on the need for combining various data sources into a single logical construct . decision analytic models are often used as a tool for health - care decision - makers , allowing the synthesis of the best available data rather than waiting for the perfect set of clinical and economic data to become available . glutamine ( in the form of alanyl - glutamine ) to standard pn is a clinically and economically attractive strategy for icu patients in italy . moreover , the robustness of these results mean that they may be applicable to selected icu patient populations in other countries .

the worldwide prevalence of diabetes mellitus ( dm ) has risen dramatically over the past two decades because of increasing obesity and reduced activity levels . india has the largest number of diabetic population in the world , and it is expected that there will be 69.9 million diabetic populations in india by 2025 . type 2 dm ( t2 dm ) and major depressive disorder ( mdd ) are both chronic diseases that may progress for years before diagnosis . worldwide estimates of the prevalence of depression and anxiety among diabetic patients appear to vary by nations ; though data is scarce from developing countries , studies from asia ( including india ) report prevalence rates of depression ranging from 17% to 44% and for anxiety it is from 4% to 58% . due to varying prevalence rate of diabetes as well as depression and anxiety from various parts of india therefore , the present study was carried out to find out the prevalence and risk factors of depression and anxiety , among patients of t2 dm in a tertiary care center of haryana . it was carried out from june 2010 to march 2011 in endocrine department of pt . the study protocol was approved by the ethics committee of the institute . after taking informed and written consent , a total of 820 participants patients above 18 years of agepatients of either sexpatients having t2 dm irrespective of their duration of illness or diabetic treatment . patients above 18 years of age patients of either sex patients having t2 dm irrespective of their duration of illness or diabetic treatment . patients with chronic medical or surgical illness other than dmpatients on long - term treatment for other medical illnesspatients who were terminally illpatients having renal , neurological , or cardiovascular dysfunction who require immediate hospitalization for serious illness andpatients who were on corticosteroids or any psychotropic drug . patients with chronic medical or surgical illness other than dm patients on long - term treatment for other medical illness patients who were terminally ill patients having renal , neurological , or cardiovascular dysfunction who require immediate hospitalization for serious illness and patients who were on corticosteroids or any psychotropic drug . a semi - structured proforma was used to obtain sociodemographic variables of patients and relevant past medical and psychiatric history and duration of illness , treatment taken for diabetes . anthropometric measurements including weight , waist circumference , body mass index ( bmi ) , were recorded . furthermore , lipid profile , serum creatinine , spot urine for microalbuminuria , fundoscopy was done to assess for any diabetes related complicationshamilton depression rating scale was administered to assess the presence of depression as well as severity . the score ranges from 0 to 54 where 07 indicate a normal person with regard to depression , 813 indicate mild depression , 1418 indicate moderate depression and 19 indicate severe depression hamilton anxiety rating scale was used to assess the presence of anxiety . scale consists of 14 items with a total score range of 056 , where 05 indicate no anxiety , 614 indicate mild anxiety , 1528 indicate moderate anxiety and 29 indicate severe anxiety . a semi - structured proforma was used to obtain sociodemographic variables of patients and relevant past medical and psychiatric history and duration of illness , treatment taken for diabetes . anthropometric measurements including weight , waist circumference , body mass index ( bmi ) , were recorded . furthermore , lipid profile , serum creatinine , spot urine for microalbuminuria , fundoscopy was done to assess for any diabetes related complications hamilton depression rating scale was administered to assess the presence of depression as well as severity . the score ranges from 0 to 54 where 07 indicate a normal person with regard to depression , 813 indicate mild depression , 1418 indicate moderate depression and 19 indicate severe depression hamilton anxiety rating scale was used to assess the presence of anxiety . scale consists of 14 items with a total score range of 056 , where 05 indicate no anxiety , 614 indicate mild anxiety , 1528 indicate moderate anxiety and 29 indicate severe anxiety . the data collected during the study was entered in the microsoft excel format and was analyzed using ibm spss statistics software version 17 . differences in characteristics between participants were tested with unpaired t - test for normally distributed variables and with the chi - square test for categorical variables . binary logistic regression model was used to examine the association between predictor variables and risk of depression . the p values were two - tailed , and probability level of significant difference was set at < 0.05 . patients above 18 years of agepatients of either sexpatients having t2 dm irrespective of their duration of illness or diabetic treatment . patients above 18 years of age patients of either sex patients having t2 dm irrespective of their duration of illness or diabetic treatment . patients with chronic medical or surgical illness other than dmpatients on long - term treatment for other medical illnesspatients who were terminally illpatients having renal , neurological , or cardiovascular dysfunction who require immediate hospitalization for serious illness andpatients who were on corticosteroids or any psychotropic drug . patients with chronic medical or surgical illness other than dm patients on long - term treatment for other medical illness patients who were terminally ill patients having renal , neurological , or cardiovascular dysfunction who require immediate hospitalization for serious illness and patients who were on corticosteroids or any psychotropic drug . a semi - structured proforma was used to obtain sociodemographic variables of patients and relevant past medical and psychiatric history and duration of illness , treatment taken for diabetes . anthropometric measurements including weight , waist circumference , body mass index ( bmi ) , were recorded . furthermore , lipid profile , serum creatinine , spot urine for microalbuminuria , fundoscopy was done to assess for any diabetes related complicationshamilton depression rating scale was administered to assess the presence of depression as well as severity . the score ranges from 0 to 54 where 07 indicate a normal person with regard to depression , 813 indicate mild depression , 1418 indicate moderate depression and 19 indicate severe depression hamilton anxiety rating scale was used to assess the presence of anxiety . scale consists of 14 items with a total score range of 056 , where 05 indicate no anxiety , 614 indicate mild anxiety , 1528 indicate moderate anxiety and 29 indicate severe anxiety . a semi - structured proforma was used to obtain sociodemographic variables of patients and relevant past medical and psychiatric history and duration of illness , treatment taken for diabetes . anthropometric measurements including weight , waist circumference , body mass index ( bmi ) , were recorded . furthermore , lipid profile , serum creatinine , spot urine for microalbuminuria , fundoscopy was done to assess for any diabetes related complications hamilton depression rating scale was administered to assess the presence of depression as well as severity . the score ranges from 0 to 54 where 07 indicate a normal person with regard to depression , 813 indicate mild depression , 1418 indicate moderate depression and 19 indicate severe depression hamilton anxiety rating scale was used to assess the presence of anxiety . scale consists of 14 items with a total score range of 056 , where 05 indicate no anxiety , 614 indicate mild anxiety , 1528 indicate moderate anxiety and 29 indicate severe anxiety . the data collected during the study was entered in the microsoft excel format and was analyzed using ibm spss statistics software version 17 . differences in characteristics between participants were tested with unpaired t - test for normally distributed variables and with the chi - square test for categorical variables . binary logistic regression model was used to examine the association between predictor variables and risk of depression . the p values were two - tailed , and probability level of significant difference was set at < 0.05 . four hundred ten patients had t2 dm and 410 participants were taken as healthy controls . there were 213 females ( 52.0% ) and 197 males ( 48.0% ) in both groups , respectively . most of the diabetic cases ( 63.4% ) and healthy controls ( 61.7% ) were in the 4160 years age group . sociodemographic and clinical profile of the participants the prevalence rate of depression , anxiety , and comorbid depression and anxiety among diabetic and healthy controls group are shown in table 2 . table 3 shows independent risk factors for depression and anxiety respectively among the patients of dm calculated by applying logistic regression analysis . for depression or was higher for retinopathy with the value of 5.24 , followed by 4.48 for marital status and 3.57 for nephropathy and insulin therapy . for anxiety , or was higher in retinopathy with the value of 4.95 , followed by 4.41 for nephropathy and insulin therapy . the presence of complications as independent risk factors for depression and anxiety in patients of dm has been depicted in table 4 . prevalence rate of depression , anxiety and comorbid depression and anxiety in diabetic and healthy control group predictors of depression and anxiety among patients of diabetes mellitus ( results of logistic regression analysis ) presence of complications as independent risk factors for depression and anxiety in patients of diabetes mellitus the findings of the present study suggested that depression and anxiety were 2 times more prevalent patients with t2 dm ( 26.3% vs. 11.2% ) than in healthy controls . a rising trend in prevalence of depression and anxiety in diabetic patients has been suggested by the studies done in various parts of the world as well as in india . in a population - based study in chennai , it was found that the prevalence of depression was 23.4% . . found a very high prevalence ( 41% ) of depression in 300 patients with type 2 diabetes in a tertiary care hospital in northern india . another study conducted at a tertiary care center found the prevalence of depression in t2 dm patients to be 16.9% . a multi - center study done in pakistan found prevalence as 43.5% . various other studies found that prevalence of depression in t2 dm ranging from 13.6% to 67.5% . however , studies from us and uk reported the prevalence of depression in patients with t2 dm varying from 30% to 83% . similarly , a meta - analysis identified the prevalence of depression in diabetes ranging from 8% to 61% . the varying rates of prevalence may be accounted for methodological differences such as self - reported depressive symptoms versus clinically diagnosed depression . in addition , the lack of documentation regarding relevant factors associated with the disease state ( e.g. number of diabetes complications , other medical comorbidity ) may confound results of clinical studies and skew prevalence rates . the prevalence rate of depression was higher in age between 41 and 60 years ( 18.8% vs. 7.6% , respectively ) in diabetic and healthy control group . this may be because diabetic patients require adherence to a complex set of treatment regimens including daily multiple insulin injections , monitoring blood glucose level , adherence to specific dietary guidelines , and attending regular medical check - ups . furthermore , a number of risk factors may increase the likelihood of depression including female gender , family dysfunction , and stressful experience in this age group . these results correlate with previous findings of higher prevalence rate of depression in age group of 3159 years . raval et al . also found a higher rate of depression in diabetic patients with age > 54 years . diabetic women had higher prevalence of depression ( 17.1% vs. 9.3% ) as compared to diabetic men . this could be attributable to gender - specific issues such as pregnancy , menstrual cycle changes , postpartum , and stresses such as responsibilities at work and home , caring for children and aging parents which could all lead to depression . this may be due to the high cost involved in the management of diabetes which adds to higher psychological stress . this finding is supported by katon et al . in their study . also , the prevalence rate was higher in married patients . this may be attributed to the greater responsibilities on them such as managing their career aspirations and family responsibilities together , upbringing their children and most importantly managing the chronic illness like diabetes which adds to their financial and emotional burden . this finding is in contrast with the previous study where it was found that the prevalence rate of depression is higher in single diabetic patients . they found that marriage integrates people into wider social networks , buffers life from strains and provide personal security , meaningfulness , and purpose . depression was found to be significantly associated with joint family system in patients of diabetes . this may be attributable to the fact that in a joint family system , number of individuals is more as compared to earning hands which adds to financial burden , lack of freedom in decision making which adds to emotional burden , greater interference and loss of individuality making them more prone to psychological problems . this is in contrast with an earlier study which found that depression was more common in nuclear family as middle aged person in a single family might find no help from elders in the face of chronic or severe stress and develop depressive symptoms . in western world , limited literature is available on association between joint family system and depression as nuclear families are more common in these countries . depression was found significantly associated with retinopathy 25.4% ( 0.001 ) , nephropathy 13.9% ( 0.001 ) and ischemic heart disease ( ihd ) 13.4% ( 0.001 ) in the present study . this could be attributable to difficulty in adhering to diet , exercise and poor compliance with prescribed medications by diabetic patients , which would lead to development and progression of both microvascular and macrovascular complications . the results of the present study are similar to earlier studies which showed that the prevalence of depression was significantly higher among diabetic subjects with retinopathy , neuropathy , nephropathy and macrovascular complications ( e.g. coronary artery disease , peripheral vascular disease [ pvd ] , etc . ) . poongothai et al . also observed that retinopathy , neuropathy , nephropathy , and pvd were associated with depression while raval et al . found a significant associated between depression and neuropathy , nephropathy , pvd and diabetic foot disease , but no significant association of depression with coronary artery disease was found in both studies . this finding suggests that the diabetic patients on insulin therapy face more daily stresses ( routine multiple injections , repeated investigations , and hospitalizations and fear of complications ) as compared to diabetic patients on oral therapy . previous literature also suggested that the severity of depressive symptoms was significantly higher ( < 0.001 ) in insulin dependent diabetic patients as compared to noninsulin dependents . the present study also found that the prevalence rate of anxiety was 27.6% which was almost similar to depression ( 26.3% ) and it was twice as high as compared to healthy controls ( 27.6% vs. 12.7% ) . in contrast , the prevalence rate of anxiety was found to be high in few previous studies as 35.357.9% and a low prevalence of about 3.9% in other studies as compared to the present study . the differences in prevalence rate may be attributed to various scales used and methods that were applied in calculating the prevalence rate of anxiety . in this study age , female sex , marital status , type of family , retinopathy , nephropathy , ihd , and insulin therapy were significant predictors of anxiety . diabetic women were found to have significant higher anxiety scores ( 17.6 vs. 10.0% ) as compared to diabetic men . similar findings were found in a previous study which showed that anxiety scores in women and men were 70.161.6% , respectively . the prevalence rate of anxiety in diabetic patients was also found to be significantly high in age group of 4160 years ( 20.5% ) . the microvascular ( retinopathy and nephropathy ) and macrovascular ( ihd ) complications were also significantly associated with anxiety . khuwaja et al . also showed a significant association between anxiety and macrovascular complications like hypertension and ihd . a few indian studies observed anxiety to be significantly associated with bmi , physical activity , hba1c level and postprandial blood glucose level , but no such associations were found in the present study . the prevalence rate of comorbid depression and anxiety in diabetic patients was ( 21.0% vs. 7.3% , p = 0.001 ) as compared to healthy controls . . found a higher prevalence rate of comorbid depression and anxiety in diabetic patients ( 36.0% ) as compared to the present study . in a study conducted in southern india , it was found that clinical depression was present in 12.1% , generalized anxiety disorder ( gad ) in 19.0% and as high as 87% patients of depression were also suffering from anxiety disorder . in the national comorbidity survey from us , 58% of mdd patients were found to have an anxiety disorder ; among these patients , the rate of comorbidity with gad was 17.2% and with panic disorder was 9.9% . patients with diagnosed anxiety disorder also had high rates of comorbid depression , including 22.4% of patients with social phobia , 9.4% with agoraphobia and 2.3% with panic disorder . for most of the patients , the symptoms of both depression and anxiety were not severe enough ( i.e. subsyndromal ) to justify a primary diagnosis of either mdd or an anxiety disorder indicating the need for appropriate management to alleviate symptoms and prevent the emergence of more serious disease . accumulating evidence indicates that patients with comorbid depression and anxiety tend to have greater illness severity and a lower treatment response than those with either disorder alone . there is limited data available which showed the determinants of comorbid depression and anxiety in patients of diabetes ; however thomas et al . found that the comorbid depression and anxiety was significantly associated with age ( 45.16 years ) and high education level . in the present study , comorbid depression and anxiety was significantly associated with age ( 4061 years ) , obesity ( bmi 25 kg / m ) , poor glycemic control , insulin therapy , nephropathy , neuropathy , and ihd but no significant association was found with education level . thus , it is suggested that depression and anxiety symptoms were two - fold higher in diabetic patients as compared to healthy controls , and major predictors of depression and anxiety among diabetic cases were increasing age , female sex , marital status ( married ) , joint family system , insulin therapy , retinopathy , nephropathy , and ihd . first , due to the cross - sectional study design , it is not possible to draw long - term conclusions . second , the study had a small sample size and carried out in a tertiary care center ; the results can not be generalized to general population setting . third , there are certain confounding variables such as smoking , alcohol use , and duration of diabetes , present in the study group . further , multi - central , longitudinal studies in different geographical areas need to be considered to establish causal relationship between depression and diabetes . the findings of the present study suggested that depression and anxiety were 2 times more prevalent patients with t2 dm ( 26.3% vs. 11.2% ) than in healthy controls . a rising trend in prevalence of depression and anxiety in diabetic patients has been suggested by the studies done in various parts of the world as well as in india . in a population - based study in chennai , it was found that the prevalence of depression was 23.4% . . found a very high prevalence ( 41% ) of depression in 300 patients with type 2 diabetes in a tertiary care hospital in northern india . another study conducted at a tertiary care center found the prevalence of depression in t2 dm patients to be 16.9% . a multi - center study done in pakistan found prevalence as 43.5% . various other studies found that prevalence of depression in t2 dm ranging from 13.6% to 67.5% . however , studies from us and uk reported the prevalence of depression in patients with t2 dm varying from 30% to 83% . similarly , a meta - analysis identified the prevalence of depression in diabetes ranging from 8% to 61% . the varying rates of prevalence may be accounted for methodological differences such as self - reported depressive symptoms versus clinically diagnosed depression . in addition , the lack of documentation regarding relevant factors associated with the disease state ( e.g. number of diabetes complications , other medical comorbidity ) may confound results of clinical studies and skew prevalence rates . the prevalence rate of depression was higher in age between 41 and 60 years ( 18.8% vs. 7.6% , respectively ) in diabetic and healthy control group . this may be because diabetic patients require adherence to a complex set of treatment regimens including daily multiple insulin injections , monitoring blood glucose level , adherence to specific dietary guidelines , and attending regular medical check - ups . furthermore , a number of risk factors may increase the likelihood of depression including female gender , family dysfunction , and stressful experience in this age group . these results correlate with previous findings of higher prevalence rate of depression in age group of 3159 years . raval et al . also found a higher rate of depression in diabetic patients with age > 54 years . diabetic women had higher prevalence of depression ( 17.1% vs. 9.3% ) as compared to diabetic men . this could be attributable to gender - specific issues such as pregnancy , menstrual cycle changes , postpartum , and stresses such as responsibilities at work and home , caring for children and aging parents which could all lead to depression . this may be due to the high cost involved in the management of diabetes which adds to higher psychological stress . this finding is supported by katon et al . in their study . also , the prevalence rate was higher in married patients . this may be attributed to the greater responsibilities on them such as managing their career aspirations and family responsibilities together , upbringing their children and most importantly managing the chronic illness like diabetes which adds to their financial and emotional burden . this finding is in contrast with the previous study where it was found that the prevalence rate of depression is higher in single diabetic patients . they found that marriage integrates people into wider social networks , buffers life from strains and provide personal security , meaningfulness , and purpose . depression was found to be significantly associated with joint family system in patients of diabetes . this may be attributable to the fact that in a joint family system , number of individuals is more as compared to earning hands which adds to financial burden , lack of freedom in decision making which adds to emotional burden , greater interference and loss of individuality making them more prone to psychological problems . this is in contrast with an earlier study which found that depression was more common in nuclear family as middle aged person in a single family might find no help from elders in the face of chronic or severe stress and develop depressive symptoms . in western world , limited literature is available on association between joint family system and depression as nuclear families are more common in these countries . depression was found significantly associated with retinopathy 25.4% ( 0.001 ) , nephropathy 13.9% ( 0.001 ) and ischemic heart disease ( ihd ) 13.4% ( 0.001 ) in the present study . this could be attributable to difficulty in adhering to diet , exercise and poor compliance with prescribed medications by diabetic patients , which would lead to development and progression of both microvascular and macrovascular complications . the results of the present study are similar to earlier studies which showed that the prevalence of depression was significantly higher among diabetic subjects with retinopathy , neuropathy , nephropathy and macrovascular complications ( e.g. coronary artery disease , peripheral vascular disease [ pvd ] , etc . ) . poongothai et al . also observed that retinopathy , neuropathy , nephropathy , and pvd were associated with depression while raval et al . found a significant associated between depression and neuropathy , nephropathy , pvd and diabetic foot disease , but no significant association of depression with coronary artery disease was found in both studies . this finding suggests that the diabetic patients on insulin therapy face more daily stresses ( routine multiple injections , repeated investigations , and hospitalizations and fear of complications ) as compared to diabetic patients on oral therapy . previous literature also suggested that the severity of depressive symptoms was significantly higher ( < 0.001 ) in insulin dependent diabetic patients as compared to noninsulin dependents . the present study also found that the prevalence rate of anxiety was 27.6% which was almost similar to depression ( 26.3% ) and it was twice as high as compared to healthy controls ( 27.6% vs. 12.7% ) . in contrast , the prevalence rate of anxiety was found to be high in few previous studies as 35.357.9% and a low prevalence of about 3.9% in other studies as compared to the present study . the differences in prevalence rate may be attributed to various scales used and methods that were applied in calculating the prevalence rate of anxiety . in this study age , female sex , marital status , type of family , retinopathy , nephropathy , ihd , and insulin therapy were significant predictors of anxiety . diabetic women were found to have significant higher anxiety scores ( 17.6 vs. 10.0% ) as compared to diabetic men . similar findings were found in a previous study which showed that anxiety scores in women and men were 70.161.6% , respectively . the prevalence rate of anxiety in diabetic patients was also found to be significantly high in age group of 4160 years ( 20.5% ) . the microvascular ( retinopathy and nephropathy ) and macrovascular ( ihd ) complications were also significantly associated with anxiety . khuwaja et al . also showed a significant association between anxiety and macrovascular complications like hypertension and ihd . a few indian studies observed anxiety to be significantly associated with bmi , physical activity , hba1c level and postprandial blood glucose level , but no such associations were found in the present study . the prevalence rate of comorbid depression and anxiety in diabetic patients was ( 21.0% vs. 7.3% , p = 0.001 ) as compared to healthy controls . . found a higher prevalence rate of comorbid depression and anxiety in diabetic patients ( 36.0% ) as compared to the present study . in a study conducted in southern india , it was found that clinical depression was present in 12.1% , generalized anxiety disorder ( gad ) in 19.0% and as high as 87% patients of depression were also suffering from anxiety disorder . in the national comorbidity survey from us , 58% of mdd patients were found to have an anxiety disorder ; among these patients , the rate of comorbidity with gad was 17.2% and with panic disorder was 9.9% . patients with diagnosed anxiety disorder also had high rates of comorbid depression , including 22.4% of patients with social phobia , 9.4% with agoraphobia and 2.3% with panic disorder . for most of the patients , the symptoms of both depression and anxiety were not severe enough ( i.e. subsyndromal ) to justify a primary diagnosis of either mdd or an anxiety disorder indicating the need for appropriate management to alleviate symptoms and prevent the emergence of more serious disease . accumulating evidence indicates that patients with comorbid depression and anxiety tend to have greater illness severity and a lower treatment response than those with either disorder alone . there is limited data available which showed the determinants of comorbid depression and anxiety in patients of diabetes ; however thomas et al . found that the comorbid depression and anxiety was significantly associated with age ( 45.16 years ) and high education level . in the present study , comorbid depression and anxiety was significantly associated with age ( 4061 years ) , obesity ( bmi 25 kg / m ) , poor glycemic control , insulin therapy , nephropathy , neuropathy , and ihd but no significant association was found with education level . thus , it is suggested that depression and anxiety symptoms were two - fold higher in diabetic patients as compared to healthy controls , and major predictors of depression and anxiety among diabetic cases were increasing age , female sex , marital status ( married ) , joint family system , insulin therapy , retinopathy , nephropathy , and ihd . first , due to the cross - sectional study design , it is not possible to draw long - term conclusions . second , the study had a small sample size and carried out in a tertiary care center ; the results can not be generalized to general population setting . third , there are certain confounding variables such as smoking , alcohol use , and duration of diabetes , present in the study group . further , multi - central , longitudinal studies in different geographical areas need to be considered to establish causal relationship between depression and diabetes . our study showed a high prevalence of depression and anxiety in patients with t2 dm . the presence of risk factors for depression among patients of diabetes predicts a causal relationship and deserves attention from clinicians . therefore , screening for depression and anxiety among diabetic patients needs to be done at each clinical contact , particularly in older adults , female sex , and those on insulin therapies and with complications .

background : diabetes is one of the most common chronic diseases and affects virtually every organ of the human system . depression and anxiety is common among patients with diabetes and associated with worse diabetes outcomes.aims and objective : to study the prevalence and predictors of depression and anxiety in patients of type 2 diabetes mellitus ( t2 dm ) in pt . b.d . sharma , pgims , rohtak , haryana , india , a tertiary care center in northern india.materials and methods : four hundred ten consecutive patients having t2 dm and 410 healthy controls matched for age and sex attending the endocrine out - patient department of a tertiary care center of northern india were included in the study . sociodemographic and relevant clinical variables were collected . they were evaluated for depression and anxiety using hamilton depression rating scale and hamilton anxiety rating scale respectively.results:it was found that a significantly larger proportion of diabetic patients had depression ( 26.3% vs. 11.2% , p = 0.001 ) , anxiety ( 27.6% vs. 12.7% , p = 0.001 ) and comorbid depression and anxiety ( 21.0% vs. 7.3% , p = 0.001 ) as compared to healthy controls . diabetic women had higher depression ( 17.1% vs. 9.3% ) and anxiety ( 17.6% vs. 10.0% ) than men . the major predictors for a severe form of depression and anxiety among t2 dm cases were age , female sex , insulin therapy , retinopathy , nephropathy , and ischemic heart disease.conclusion:the present findings reveal that diabetic cases had significantly higher depression and anxiety as compared to healthy controls . the risk factors for depression and anxiety were age , female sex , insulin therapy , and diabetic complications .

I M Inclusion criteria Exclusion criteria Measures Statistical analysis R D Prevalence and predictors of depression Prevalence and predictors of anxiety Comorbid depression and anxiety with diabetes mellitus C Financial support and sponsorship Conflicts of interest

the worldwide prevalence of diabetes mellitus ( dm ) has risen dramatically over the past two decades because of increasing obesity and reduced activity levels . india has the largest number of diabetic population in the world , and it is expected that there will be 69.9 million diabetic populations in india by 2025 . type 2 dm ( t2 dm ) and major depressive disorder ( mdd ) are both chronic diseases that may progress for years before diagnosis . worldwide estimates of the prevalence of depression and anxiety among diabetic patients appear to vary by nations ; though data is scarce from developing countries , studies from asia ( including india ) report prevalence rates of depression ranging from 17% to 44% and for anxiety it is from 4% to 58% . due to varying prevalence rate of diabetes as well as depression and anxiety from various parts of india therefore , the present study was carried out to find out the prevalence and risk factors of depression and anxiety , among patients of t2 dm in a tertiary care center of haryana . the study protocol was approved by the ethics committee of the institute . after taking informed and written consent , a total of 820 participants patients above 18 years of agepatients of either sexpatients having t2 dm irrespective of their duration of illness or diabetic treatment . patients above 18 years of age patients of either sex patients having t2 dm irrespective of their duration of illness or diabetic treatment . patients with chronic medical or surgical illness other than dm patients on long - term treatment for other medical illness patients who were terminally ill patients having renal , neurological , or cardiovascular dysfunction who require immediate hospitalization for serious illness and patients who were on corticosteroids or any psychotropic drug . furthermore , lipid profile , serum creatinine , spot urine for microalbuminuria , fundoscopy was done to assess for any diabetes related complicationshamilton depression rating scale was administered to assess the presence of depression as well as severity . the score ranges from 0 to 54 where 07 indicate a normal person with regard to depression , 813 indicate mild depression , 1418 indicate moderate depression and 19 indicate severe depression hamilton anxiety rating scale was used to assess the presence of anxiety . furthermore , lipid profile , serum creatinine , spot urine for microalbuminuria , fundoscopy was done to assess for any diabetes related complications hamilton depression rating scale was administered to assess the presence of depression as well as severity . the score ranges from 0 to 54 where 07 indicate a normal person with regard to depression , 813 indicate mild depression , 1418 indicate moderate depression and 19 indicate severe depression hamilton anxiety rating scale was used to assess the presence of anxiety . the data collected during the study was entered in the microsoft excel format and was analyzed using ibm spss statistics software version 17 . patients above 18 years of age patients of either sex patients having t2 dm irrespective of their duration of illness or diabetic treatment . patients with chronic medical or surgical illness other than dm patients on long - term treatment for other medical illness patients who were terminally ill patients having renal , neurological , or cardiovascular dysfunction who require immediate hospitalization for serious illness and patients who were on corticosteroids or any psychotropic drug . furthermore , lipid profile , serum creatinine , spot urine for microalbuminuria , fundoscopy was done to assess for any diabetes related complicationshamilton depression rating scale was administered to assess the presence of depression as well as severity . the score ranges from 0 to 54 where 07 indicate a normal person with regard to depression , 813 indicate mild depression , 1418 indicate moderate depression and 19 indicate severe depression hamilton anxiety rating scale was used to assess the presence of anxiety . furthermore , lipid profile , serum creatinine , spot urine for microalbuminuria , fundoscopy was done to assess for any diabetes related complications hamilton depression rating scale was administered to assess the presence of depression as well as severity . the score ranges from 0 to 54 where 07 indicate a normal person with regard to depression , 813 indicate mild depression , 1418 indicate moderate depression and 19 indicate severe depression hamilton anxiety rating scale was used to assess the presence of anxiety . the data collected during the study was entered in the microsoft excel format and was analyzed using ibm spss statistics software version 17 . four hundred ten patients had t2 dm and 410 participants were taken as healthy controls . there were 213 females ( 52.0% ) and 197 males ( 48.0% ) in both groups , respectively . most of the diabetic cases ( 63.4% ) and healthy controls ( 61.7% ) were in the 4160 years age group . sociodemographic and clinical profile of the participants the prevalence rate of depression , anxiety , and comorbid depression and anxiety among diabetic and healthy controls group are shown in table 2 . table 3 shows independent risk factors for depression and anxiety respectively among the patients of dm calculated by applying logistic regression analysis . for depression or was higher for retinopathy with the value of 5.24 , followed by 4.48 for marital status and 3.57 for nephropathy and insulin therapy . the presence of complications as independent risk factors for depression and anxiety in patients of dm has been depicted in table 4 . prevalence rate of depression , anxiety and comorbid depression and anxiety in diabetic and healthy control group predictors of depression and anxiety among patients of diabetes mellitus ( results of logistic regression analysis ) presence of complications as independent risk factors for depression and anxiety in patients of diabetes mellitus the findings of the present study suggested that depression and anxiety were 2 times more prevalent patients with t2 dm ( 26.3% vs. 11.2% ) than in healthy controls . a rising trend in prevalence of depression and anxiety in diabetic patients has been suggested by the studies done in various parts of the world as well as in india . in a population - based study in chennai , it was found that the prevalence of depression was 23.4% . found a very high prevalence ( 41% ) of depression in 300 patients with type 2 diabetes in a tertiary care hospital in northern india . another study conducted at a tertiary care center found the prevalence of depression in t2 dm patients to be 16.9% . various other studies found that prevalence of depression in t2 dm ranging from 13.6% to 67.5% . however , studies from us and uk reported the prevalence of depression in patients with t2 dm varying from 30% to 83% . similarly , a meta - analysis identified the prevalence of depression in diabetes ranging from 8% to 61% . the prevalence rate of depression was higher in age between 41 and 60 years ( 18.8% vs. 7.6% , respectively ) in diabetic and healthy control group . this may be because diabetic patients require adherence to a complex set of treatment regimens including daily multiple insulin injections , monitoring blood glucose level , adherence to specific dietary guidelines , and attending regular medical check - ups . furthermore , a number of risk factors may increase the likelihood of depression including female gender , family dysfunction , and stressful experience in this age group . also found a higher rate of depression in diabetic patients with age > 54 years . diabetic women had higher prevalence of depression ( 17.1% vs. 9.3% ) as compared to diabetic men . this finding is in contrast with the previous study where it was found that the prevalence rate of depression is higher in single diabetic patients . they found that marriage integrates people into wider social networks , buffers life from strains and provide personal security , meaningfulness , and purpose . depression was found to be significantly associated with joint family system in patients of diabetes . this may be attributable to the fact that in a joint family system , number of individuals is more as compared to earning hands which adds to financial burden , lack of freedom in decision making which adds to emotional burden , greater interference and loss of individuality making them more prone to psychological problems . depression was found significantly associated with retinopathy 25.4% ( 0.001 ) , nephropathy 13.9% ( 0.001 ) and ischemic heart disease ( ihd ) 13.4% ( 0.001 ) in the present study . the results of the present study are similar to earlier studies which showed that the prevalence of depression was significantly higher among diabetic subjects with retinopathy , neuropathy , nephropathy and macrovascular complications ( e.g. also observed that retinopathy , neuropathy , nephropathy , and pvd were associated with depression while raval et al . found a significant associated between depression and neuropathy , nephropathy , pvd and diabetic foot disease , but no significant association of depression with coronary artery disease was found in both studies . this finding suggests that the diabetic patients on insulin therapy face more daily stresses ( routine multiple injections , repeated investigations , and hospitalizations and fear of complications ) as compared to diabetic patients on oral therapy . previous literature also suggested that the severity of depressive symptoms was significantly higher ( < 0.001 ) in insulin dependent diabetic patients as compared to noninsulin dependents . the present study also found that the prevalence rate of anxiety was 27.6% which was almost similar to depression ( 26.3% ) and it was twice as high as compared to healthy controls ( 27.6% vs. 12.7% ) . in contrast , the prevalence rate of anxiety was found to be high in few previous studies as 35.357.9% and a low prevalence of about 3.9% in other studies as compared to the present study . the differences in prevalence rate may be attributed to various scales used and methods that were applied in calculating the prevalence rate of anxiety . in this study age , female sex , marital status , type of family , retinopathy , nephropathy , ihd , and insulin therapy were significant predictors of anxiety . diabetic women were found to have significant higher anxiety scores ( 17.6 vs. 10.0% ) as compared to diabetic men . the prevalence rate of anxiety in diabetic patients was also found to be significantly high in age group of 4160 years ( 20.5% ) . the microvascular ( retinopathy and nephropathy ) and macrovascular ( ihd ) complications were also significantly associated with anxiety . a few indian studies observed anxiety to be significantly associated with bmi , physical activity , hba1c level and postprandial blood glucose level , but no such associations were found in the present study . the prevalence rate of comorbid depression and anxiety in diabetic patients was ( 21.0% vs. 7.3% , p = 0.001 ) as compared to healthy controls . found a higher prevalence rate of comorbid depression and anxiety in diabetic patients ( 36.0% ) as compared to the present study . in a study conducted in southern india , it was found that clinical depression was present in 12.1% , generalized anxiety disorder ( gad ) in 19.0% and as high as 87% patients of depression were also suffering from anxiety disorder . patients with diagnosed anxiety disorder also had high rates of comorbid depression , including 22.4% of patients with social phobia , 9.4% with agoraphobia and 2.3% with panic disorder . for most of the patients , the symptoms of both depression and anxiety were not severe enough ( i.e. accumulating evidence indicates that patients with comorbid depression and anxiety tend to have greater illness severity and a lower treatment response than those with either disorder alone . there is limited data available which showed the determinants of comorbid depression and anxiety in patients of diabetes ; however thomas et al . found that the comorbid depression and anxiety was significantly associated with age ( 45.16 years ) and high education level . in the present study , comorbid depression and anxiety was significantly associated with age ( 4061 years ) , obesity ( bmi 25 kg / m ) , poor glycemic control , insulin therapy , nephropathy , neuropathy , and ihd but no significant association was found with education level . thus , it is suggested that depression and anxiety symptoms were two - fold higher in diabetic patients as compared to healthy controls , and major predictors of depression and anxiety among diabetic cases were increasing age , female sex , marital status ( married ) , joint family system , insulin therapy , retinopathy , nephropathy , and ihd . second , the study had a small sample size and carried out in a tertiary care center ; the results can not be generalized to general population setting . third , there are certain confounding variables such as smoking , alcohol use , and duration of diabetes , present in the study group . the findings of the present study suggested that depression and anxiety were 2 times more prevalent patients with t2 dm ( 26.3% vs. 11.2% ) than in healthy controls . a rising trend in prevalence of depression and anxiety in diabetic patients has been suggested by the studies done in various parts of the world as well as in india . in a population - based study in chennai , it was found that the prevalence of depression was 23.4% . found a very high prevalence ( 41% ) of depression in 300 patients with type 2 diabetes in a tertiary care hospital in northern india . another study conducted at a tertiary care center found the prevalence of depression in t2 dm patients to be 16.9% . various other studies found that prevalence of depression in t2 dm ranging from 13.6% to 67.5% . however , studies from us and uk reported the prevalence of depression in patients with t2 dm varying from 30% to 83% . similarly , a meta - analysis identified the prevalence of depression in diabetes ranging from 8% to 61% . the prevalence rate of depression was higher in age between 41 and 60 years ( 18.8% vs. 7.6% , respectively ) in diabetic and healthy control group . this may be because diabetic patients require adherence to a complex set of treatment regimens including daily multiple insulin injections , monitoring blood glucose level , adherence to specific dietary guidelines , and attending regular medical check - ups . furthermore , a number of risk factors may increase the likelihood of depression including female gender , family dysfunction , and stressful experience in this age group . also found a higher rate of depression in diabetic patients with age > 54 years . diabetic women had higher prevalence of depression ( 17.1% vs. 9.3% ) as compared to diabetic men . this finding is in contrast with the previous study where it was found that the prevalence rate of depression is higher in single diabetic patients . they found that marriage integrates people into wider social networks , buffers life from strains and provide personal security , meaningfulness , and purpose . depression was found to be significantly associated with joint family system in patients of diabetes . this may be attributable to the fact that in a joint family system , number of individuals is more as compared to earning hands which adds to financial burden , lack of freedom in decision making which adds to emotional burden , greater interference and loss of individuality making them more prone to psychological problems . depression was found significantly associated with retinopathy 25.4% ( 0.001 ) , nephropathy 13.9% ( 0.001 ) and ischemic heart disease ( ihd ) 13.4% ( 0.001 ) in the present study . the results of the present study are similar to earlier studies which showed that the prevalence of depression was significantly higher among diabetic subjects with retinopathy , neuropathy , nephropathy and macrovascular complications ( e.g. also observed that retinopathy , neuropathy , nephropathy , and pvd were associated with depression while raval et al . found a significant associated between depression and neuropathy , nephropathy , pvd and diabetic foot disease , but no significant association of depression with coronary artery disease was found in both studies . this finding suggests that the diabetic patients on insulin therapy face more daily stresses ( routine multiple injections , repeated investigations , and hospitalizations and fear of complications ) as compared to diabetic patients on oral therapy . previous literature also suggested that the severity of depressive symptoms was significantly higher ( < 0.001 ) in insulin dependent diabetic patients as compared to noninsulin dependents . the present study also found that the prevalence rate of anxiety was 27.6% which was almost similar to depression ( 26.3% ) and it was twice as high as compared to healthy controls ( 27.6% vs. 12.7% ) . in contrast , the prevalence rate of anxiety was found to be high in few previous studies as 35.357.9% and a low prevalence of about 3.9% in other studies as compared to the present study . the differences in prevalence rate may be attributed to various scales used and methods that were applied in calculating the prevalence rate of anxiety . in this study age , female sex , marital status , type of family , retinopathy , nephropathy , ihd , and insulin therapy were significant predictors of anxiety . diabetic women were found to have significant higher anxiety scores ( 17.6 vs. 10.0% ) as compared to diabetic men . the prevalence rate of anxiety in diabetic patients was also found to be significantly high in age group of 4160 years ( 20.5% ) . the microvascular ( retinopathy and nephropathy ) and macrovascular ( ihd ) complications were also significantly associated with anxiety . the prevalence rate of comorbid depression and anxiety in diabetic patients was ( 21.0% vs. 7.3% , p = 0.001 ) as compared to healthy controls . found a higher prevalence rate of comorbid depression and anxiety in diabetic patients ( 36.0% ) as compared to the present study . in a study conducted in southern india , it was found that clinical depression was present in 12.1% , generalized anxiety disorder ( gad ) in 19.0% and as high as 87% patients of depression were also suffering from anxiety disorder . for most of the patients , the symptoms of both depression and anxiety were not severe enough ( i.e. accumulating evidence indicates that patients with comorbid depression and anxiety tend to have greater illness severity and a lower treatment response than those with either disorder alone . there is limited data available which showed the determinants of comorbid depression and anxiety in patients of diabetes ; however thomas et al . found that the comorbid depression and anxiety was significantly associated with age ( 45.16 years ) and high education level . in the present study , comorbid depression and anxiety was significantly associated with age ( 4061 years ) , obesity ( bmi 25 kg / m ) , poor glycemic control , insulin therapy , nephropathy , neuropathy , and ihd but no significant association was found with education level . thus , it is suggested that depression and anxiety symptoms were two - fold higher in diabetic patients as compared to healthy controls , and major predictors of depression and anxiety among diabetic cases were increasing age , female sex , marital status ( married ) , joint family system , insulin therapy , retinopathy , nephropathy , and ihd . second , the study had a small sample size and carried out in a tertiary care center ; the results can not be generalized to general population setting . third , there are certain confounding variables such as smoking , alcohol use , and duration of diabetes , present in the study group . our study showed a high prevalence of depression and anxiety in patients with t2 dm . the presence of risk factors for depression among patients of diabetes predicts a causal relationship and deserves attention from clinicians . therefore , screening for depression and anxiety among diabetic patients needs to be done at each clinical contact , particularly in older adults , female sex , and those on insulin therapies and with complications .

regular audits in the literature attempt to track trends in availability of radiation therapy ( rt ) technology such as intensitymodulated radiotherapy ( imrt)1 from a range of countries.2 , 3 , 4 there is a big difference , however , between the availability of resources and actual usage with the resourcehungry nature of commissioning5 in particular affecting implementation and usage of the available technology and equipment.6 mayles ' 2010 uk study7 remains one of the few to investigate the link between availability and use of technology and determined that lack of personnel and funding was a common inhibitory factor rather than availability of technology . in the absence of formal national data collection procedures such as the uk rt dataset,8 there is a lack of data describing australian rt patterns . this national study was built on the findings of a 2012 statewide audit9 of practice in queensland that used a fast and simple method to collect data concerning rt indications and routine technology usage . the primary aim of the study was to ascertain patterns of rt practice and technology in use across australia . these data would then be used to inform curriculum development of academic programs , thereby ensuring that training is matched to workforce patterns of practice . the secondary aim of the study was to compare the findings with those of the previous audit and additional published data to determine trends in practice . the study design was a census method as previously validated.9 all 59 australian rt centres in operation at the time of the study were invited to provide quantitative summary data relating to patient case mix and technology use on a randomly selected but common date ( thursday 5th december 2013 ) . anonymous demographicfree data were harvested using a microsoft excel spreadsheet proforma adapted and validated from the previous study9 and was analysed using descriptive statistics . modifications to the original format included separate consideration of left and right breast , radical and palliative classification and slightly amended tumour categories as informed by the previous iteration . clinical centres were approached by local clinical and academic rt educators who sought permission of centre managers and data collection was coordinated by state educator representatives . as with the previous study , ethical exemption was provided by the coordinating university research ethics committee due to the anonymous nature of the data collection and the use of already existing datasets . the results were combined to provide summary data relating to a number of key themes including tumour case mix and usage of imrt , image fusion and ontreatment imaging modalities . tumour case mix analysis indicated the relative incidence of the patients on the chosen day along with the range of techniques used . indication data identified the tumour sites that were most likely to use the technology . results from this audit were directly compared to the previous results from the queenslandwide study and published data to determine trends . overall data were provided from centres in all six australian states by 29 centres of a possible 59 , yielding a response rate of 49% . a total of 2743 patients were treated on the chosen date across these centres and table 1 presents a summary of the national and statewide responses . radical treatments comprised 81.6% of the total workload and over 91% of treatments used megavoltage equipment with only 2.5% of treatments using kilovoltage therapy . a total of 198 patients ( 7.2% ) received electron treatment either standalone or concurrent with megavoltage ( table 1 ) . summary of statewide data nsw , new south wales ; qld , queensland ; sa , south australia ; tas , tasmania ; vic , victoria ; wa , western australia ; mv , megavoltage radiotherapy ; e , electrons ; kv , kilovoltage radiotherapy ; imrt , intensitymodulated radiation therapy . table 2 illustrates the 10 most common tumour sites treated radically as both an absolute number and as a percentage of all radial treatments . the table also compares this list with results from the previous queenslandbased audit9 as well as barton 's 201310 summary of the most common sites indicated for radiotherapy nationally in australia . the most common sites receiving rt were as expected and largely as indicated in the previous study.9 rt of the breast , prostate and head and neck together comprised 62% of the total radical fractions as seen in table 2 . the five most common tumour sites according to the adopted categories for this study were breast , prostate , head and neck , skin and breast with nodes ( including additional axillary or supraclavicular fields ) . the exception was breast which comprised almost 33% of radical treatments in this iteration but only 18.4% in the previous . top 10 indications for radical radiotherapy imrtrelated data are summarised in table 3 with the left hand section showing data for fixed angle imrt and the right hand showing data for volumetricmodulated arc therapy ( vmat ) , including tomotherapy . a total of 1007 ( 37% ) of the treated patients received imrt and 16% of these received vmat using dynamic linear accelerator or tomotherapy technology . the % of imrt column shows how imrt is distributed among the various tumour sites with prostate accounting for over 31% of usage . % use per site values indicate the percentage of each tumour site patients receiving imrt ; it can thus be seen that over 71% of prostate patients receive imrt . as in the previous study , the most common sites for imrt ( as a proportion of all imrt treatments ) were still prostate , breast and head and neck , although vmat was not used for breast treatments . it was interesting to note that imrt was more commonly delivered to the right breast ( 31% ) than the left ( 23.5% ) , despite the increased challenge of cardiac toxicity on the left . this is at odds with published data11 which reported more frequent use of imrt for the left breast . overall , arc therapies were used for 160 ( 6% ) of all patient cases , with tomotherapy delivering a little under onethird of these . most tomotherapy treatments were delivered to head and neck cancers with the oropharynx accounting for 41% . vmat delivered the remaining arc therapy treatments , with 62% of being used for prostate , and 17% for head and neck . intensity modulation was used in preference to other rt modalities for prostate and head and neck , with imrt or vmat used in 71% and 65% of these sites respectively . top 10 intensitymodulated radiotherapy ( imrt ) indications and usage data in table 4 relate to usage of magnetic resonance imaging ( mri ) and computed tomography ( ct ) fusion , where it can be seen that prostate and brain tumours made by far the most demands on the modality . the left hand section presents the percentage of total mri fusions for each tumour site while the right hand columns show the percentage of tumour site patients receiving mri fusion . it can be seen that over 68% of benign tumours benefited from mri fusion ; significantly more than any other tumour site . top 10 magnetic resonance imaging ( mri ) fusion indications and usage table 5 illustrates the main indications for positron emission tomography ( pet)ct fusion with lung and head and neck comprising over 50% of the indications . the data from this study suggested that 9% of patients were planned with petct . as indicated in table 5 , cancers of the lung , head and neck , oesophagus and rectum were the most common sites for this form of image fusion , with over 30% of lung cancer patients benefitting from the technology . top 10 pet fusion indications and usage data were also gathered to highlight usage of kilovoltage and megavoltage portal imaging as well as conebeam ct modalities . planar imaging modalities are the most common modalities , comprising over 80% of instances as seen in table 6 . overall 99% of patients received imaging ; some patients received multiple imaging modalities but the summary data will not permit further analysis . although this was below that reported in previous studies,1 , 9 it does provide a reasonable indication of national practice . some interesting themes were extracted from the data and further discussion and comparison with published findings follows . the 2012 study reported slightly higher rates of electron therapy ( 10.7% ) and lower rates of kilovoltage therapy ( 0.5% ) . interestingly , the queenslandonly data for this study reported a 0.9% kilovoltage rate , suggesting a reduction in this iteration compared to the previous queenslandbased study . in 2010 , the five most commonly diagnosed cancers in australia were prostate ( 19,821 cases ) , bowel ( 14,860 ) , breast ( 14,308 ) , melanoma ( 11,405 ) and lung ( 10,296).12 these figures map consistently with the data from this study as prostate and breast cancer are commonly treated with rt , although it must be acknowledged that some of the most common cancers are not demonstrated . an australian review of optimal rt utilisation rates based on evidencebased treatment guidelines was published in march 2013.10 this suggested that in australia , rt ( alone or with chemotherapy or brachytherapy ) is the treatment of choice for 48.3% of notifiable cancers . table 2 illustrates the new indicated rt utilisation rate for the 10 most common sitespecific cancers and the national proportion of new cases of cancer with an indication for radiotherapy , which compares well to our data . this study 's data are therefore well validated with the published findings in terms of the incidence of sitespecific cancers and those treated in the highest 10 categories . there was considerable variability between states in terms of imrt use ranging from over 99% of patients in western australia to 15.5% in tasmania . these extreme figures , however , are only drawn from singlecentre data so should be interpreted with caution . most states used imrt for between 21% and 26% of patients with new south wales ( nsw ) treating over half of all patients with imrt . it would be interesting to determine factors affecting this variation but the need for individual centre anonymity precluded this for this study . the previous iteration of this study reported a 19.6% use of imrt.9 when compared to the current results for the national ( 37% ) and queensland data ( 25.9% ) it is clear that imrt use in australia is increasing in prevalence . a comparison with published data also confirms this trend in other countries.3 , 11 , 13 it was noteworthy that of the 19 patients treated for benign conditions 10 received resourceintensive imrt . it is clear that imrt usage in australia is relatively high when compared to published data and is steadily increasing . jacobs14 raises the interesting issue of increasing imrt incidence linked to medicare funding , although anecdotal evidence suggests that the costworkload balance in australia makes it less likely to be a factor . this strong utilisation of arc therapy technologies for prostate and head and neck tumours is supported by the literature and associated with improved outcomes for these patients.15 while arc therapy was not delivered to any of the six paediatric cases treated , half the cases were treated with imrt despite the decreased monitor unit and treatment times offered by vmat . this may be due to concerns around dose baths and thereby potential increased risk of secondary malignancies and growth defects . it is also important to note that funding and reimbursement challenges within the private sector may have influenced vmat implementation . the superior softtissue visualisation offered by mrict fusion significantly enhances localisation of intracranial , head and neck16 and prostate tumours.17 this study 's data indicated an overall increase in mrict fusion use when compared to the previous study 's 6.9%.9 the mri data from this study again highlighted the resourceintensive nature of benign tumours with 13 of the 19 patient datasets using the technique . over the past decade , use of pet and ct image fusion ( petct ) has improved precision of tumour volume delineation . in particular , this allows for a reduction in margins and sparing of normal tissue with potentialdose escalation for certain tumours . the literature suggests that petct image fusion is of particular use in delineating lung cancers.18 the results from this study demonstrate high uptake of petct fusion for lung patients and correlates well with existing literature.19 interestingly , when compared to data from the previous study,9 where petct fusion was used in 11.3% of patients , it can be seen that there was an overall decrease in its use . this may be influenced by different state practices but the corresponding increase in the use of mrict fusion may go some way to explaining this . statewide variations indicated a slightly lower uptake of mri fusion for queensland and victoria and a comparatively low uptake of pet fusion for south australia and western australian despite average numbers of indicated patients being treated . to some extent , this may be influenced by cyclotron availability with facilities in place in victoria , nsw , western australia and queensland . . it will be interesting to see if future iterations of this study reveal how this evidence affects pet fusion data and whether image fusion becomes an established method of monitoring tumour regression during rt . the data demonstrated increasing use of kv conebeam equipment with 12% of patients nationally receiving a cone beam computed tomography ( cbct ) image compared to 8.5% last time ; this is consistent with the published data.20 when comparing the uptake in queensland directly between the two studies , there was a modest increase from 11.2% to 12% suggesting the findings may be skewed slightly by increased usage in other states . although too small to be significant with this method , any increase may reflect the reticence to add to the dose burden when compared to kv planar imaging . igrt for head and neck cancer was well established across queensland in 2012 and this is repeated again . examination of table 1 reveals that nsw used cbct for 17.3% of patients ; most other states were on a par at between 9% and 12% . of the 321 cbct images taken on the census date 36% were prostate patients , 16% head and neck and 13% lung . it will be interesting to see how increasing use of adaptive rt protocols influence use of cbct in future iterations . fiducial markers were utilised in 53% of prostate patients despite recommendations for their use in the evidence base.21 with cbct being indicated for patients without fiducial markers with only 36% of prostate patients receiving cbct , it is unclear how some patients ' motion was accounted for most planar mv images were used for verification of the breast , with bone , brain and lung treatments also ahead of the imagereliant sites such as the prostate and head and neck . the benefits of softtissue definition associated with kv imaging are obviously not as clear for these tumour sites . it was interesting to see that of the 19 benign tumours treated there were 16 imaging incidences reported . it is not known how many of these represented multiple imaging modalities for the same patient , but reinforces the finding of benign tumours being resource intensive . as with the previous study , there are some limitations associated with the method . although sufficient data existed to determine trends on a national scale , the participation rate was lower than expected . some centres did not employ a rt educator to collate the data , while others had concerns about data sensitivity . measures to ameliorate the latter concern were taken but future data collection may be enhanced by provision of a research assistant to those centres struggling with staffing levels . this would also allow random audit of data to check robustness of data collection . while the design of the study allows useful information to be captured about the range and mix of patients and treatment approaches , it provides limited insight as to why individual centres adopt particular technologies or modalities . a more widespread data collection technique such as the national rt dataset in the uk8 would help determine factors influencing local decisions about routine treatment approaches . recruitment issues aside , the study method also allows for possible limitations with the random date possibly not being representative . responses could have been influenced by timing in relation to regular clinics , oncologist availability and resource availability . such random fluctuations are expected to decrease as sample size increases and future wider participation may reveal the extent of this . by far , the greatest limitation of the study relates to data entry error with a large number of individuals responsible for transcribing large quantities of data on top of a busy workload . reported singleentry errors are in the region of 36 per 10,000 entries22 ; this can potentially be reduced by using double data entry . although guidelines were developed and validated in a previous study , there was a definite discrepancy with interpretation of imaging frequency reporting . these data were discarded during the analysis phase and future iterations will need more specific guidance . as with the previous study , the proforma utilised a simplified system of data classification for ease of interpretation , speed of entry and reporting purposes . while this is valuable for broad trend spotting , there is a loss of accuracy with tumour pathologies and stages and inability to perform correlation analysis for individual patients . the results of the study in general confirm the validity of the research method when compared with those from the previous study . the method is a relatively convenient and powerful means of monitoring radiotherapy treatment and technology uptake trends . the overall trend demonstrated by the two studies is that of increasing use of technology in rt ; in particular there is increasing use of imaging technology as part of the standard workload in line with recommendations for igrt use from the usa23 and the uk.24 from an educational perspective these findings support increased emphasis on igrt at both pre and postregistration provision . the authors are particularly interested in using the data to inform curriculum development of academic programs in australia and to ensure training is matched to current workforce patterns of practice.25 with the implementation of new technologies in radiotherapy comes the challenge to keep up to date with teaching content . what is common practice in one state may not be reflective of national trends and best practice . a regular audit such as this study provides a broader national perspective of practice and techniques . the results allow mapping of content to reflect current practice and respond to trends in new techniques for future review . examples from this are the broad increased use of igrt nationally and the specific increase in imrt for prostate cases in particular ; these results have led to earlier and more intensive teaching in imrt . in considering the future formation of a national competency development framework for rt students , the results of this audit form a valuable component . an interesting finding from this data analysis has been the apparent resourceintensive nature of benign tumour radiotherapy . for a traditionally small component of the radiotherapy workload , there is a high uptake of imrt , fusion and igrt ; some of these technologies contribute an increased dose to these patients . it may be cogent to further examine the extent to which benign tumours utilise resources compared with malignancies . another point to consider is whether differences in uptake of technology between states are indicative of different policy implementation and resource availability or a byproduct of the random audit method used . there has been much discussion related to the postcode lottery of cancer treatment26 , 27 with variability between and across different countries ; these data could suggest that this also applies to more widespread differences . certainly within australia , newer technology is established in some states more than others with both geographical and political factors impacting on their availability . the 2012 study reported slightly higher rates of electron therapy ( 10.7% ) and lower rates of kilovoltage therapy ( 0.5% ) . interestingly , the queenslandonly data for this study reported a 0.9% kilovoltage rate , suggesting a reduction in this iteration compared to the previous queenslandbased study . in 2010 , the five most commonly diagnosed cancers in australia were prostate ( 19,821 cases ) , bowel ( 14,860 ) , breast ( 14,308 ) , melanoma ( 11,405 ) and lung ( 10,296).12 these figures map consistently with the data from this study as prostate and breast cancer are commonly treated with rt , although it must be acknowledged that some of the most common cancers are not demonstrated . an australian review of optimal rt utilisation rates based on evidencebased treatment guidelines was published in march 2013.10 this suggested that in australia , rt ( alone or with chemotherapy or brachytherapy ) is the treatment of choice for 48.3% of notifiable cancers . table 2 illustrates the new indicated rt utilisation rate for the 10 most common sitespecific cancers and the national proportion of new cases of cancer with an indication for radiotherapy , which compares well to our data . this study 's data are therefore well validated with the published findings in terms of the incidence of sitespecific cancers and those treated in the highest 10 categories . there was considerable variability between states in terms of imrt use ranging from over 99% of patients in western australia to 15.5% in tasmania . these extreme figures , however , are only drawn from singlecentre data so should be interpreted with caution . most states used imrt for between 21% and 26% of patients with new south wales ( nsw ) treating over half of all patients with imrt . it would be interesting to determine factors affecting this variation but the need for individual centre anonymity precluded this for this study . the previous iteration of this study reported a 19.6% use of imrt.9 when compared to the current results for the national ( 37% ) and queensland data ( 25.9% ) it is clear that imrt use in australia is increasing in prevalence . a comparison with published data also confirms this trend in other countries.3 , 11 , 13 it was noteworthy that of the 19 patients treated for benign conditions 10 received resourceintensive imrt . it is clear that imrt usage in australia is relatively high when compared to published data and is steadily increasing . jacobs14 raises the interesting issue of increasing imrt incidence linked to medicare funding , although anecdotal evidence suggests that the costworkload balance in australia makes it less likely to be a factor . this strong utilisation of arc therapy technologies for prostate and head and neck tumours is supported by the literature and associated with improved outcomes for these patients.15 while arc therapy was not delivered to any of the six paediatric cases treated , half the cases were treated with imrt despite the decreased monitor unit and treatment times offered by vmat . this may be due to concerns around dose baths and thereby potential increased risk of secondary malignancies and growth defects . it is also important to note that funding and reimbursement challenges within the private sector may have influenced vmat implementation . the superior softtissue visualisation offered by mrict fusion significantly enhances localisation of intracranial , head and neck16 and prostate tumours.17 this study 's data indicated an overall increase in mrict fusion use when compared to the previous study 's 6.9%.9 the mri data from this study again highlighted the resourceintensive nature of benign tumours with 13 of the 19 patient datasets using the technique . over the past decade , use of pet and ct image fusion ( petct ) has improved precision of tumour volume delineation . in particular , this allows for a reduction in margins and sparing of normal tissue with potentialdose escalation for certain tumours . the literature suggests that petct image fusion is of particular use in delineating lung cancers.18 the results from this study demonstrate high uptake of petct fusion for lung patients and correlates well with existing literature.19 interestingly , when compared to data from the previous study,9 where petct fusion was used in 11.3% of patients , it can be seen that there was an overall decrease in its use . this may be influenced by different state practices but the corresponding increase in the use of mrict fusion may go some way to explaining this . statewide variations indicated a slightly lower uptake of mri fusion for queensland and victoria and a comparatively low uptake of pet fusion for south australia and western australian despite average numbers of indicated patients being treated . to some extent , this may be influenced by cyclotron availability with facilities in place in victoria , nsw , western australia and queensland . . it will be interesting to see if future iterations of this study reveal how this evidence affects pet fusion data and whether image fusion becomes an established method of monitoring tumour regression during rt . the data demonstrated increasing use of kv conebeam equipment with 12% of patients nationally receiving a cone beam computed tomography ( cbct ) image compared to 8.5% last time ; this is consistent with the published data.20 when comparing the uptake in queensland directly between the two studies , there was a modest increase from 11.2% to 12% suggesting the findings may be skewed slightly by increased usage in other states . although too small to be significant with this method , any increase may reflect the reticence to add to the dose burden when compared to kv planar imaging . igrt for head and neck cancer was well established across queensland in 2012 and this is repeated again . examination of table 1 reveals that nsw used cbct for 17.3% of patients ; most other states were on a par at between 9% and 12% . of the 321 cbct images taken on the census date 36% were prostate patients , 16% head and neck and 13% lung . it will be interesting to see how increasing use of adaptive rt protocols influence use of cbct in future iterations . fiducial markers were utilised in 53% of prostate patients despite recommendations for their use in the evidence base.21 with cbct being indicated for patients without fiducial markers with only 36% of prostate patients receiving cbct , it is unclear how some patients ' motion was accounted for . further study in this area most planar mv images were used for verification of the breast , with bone , brain and lung treatments also ahead of the imagereliant sites such as the prostate and head and neck . the benefits of softtissue definition associated with kv imaging are obviously not as clear for these tumour sites . it was interesting to see that of the 19 benign tumours treated there were 16 imaging incidences reported . it is not known how many of these represented multiple imaging modalities for the same patient , but reinforces the finding of benign tumours being resource intensive . as with the previous study , there are some limitations associated with the method . although sufficient data existed to determine trends on a national scale , the participation rate was lower than expected . some centres did not employ a rt educator to collate the data , while others had concerns about data sensitivity . measures to ameliorate the latter concern were taken but future data collection may be enhanced by provision of a research assistant to those centres struggling with staffing levels . this would also allow random audit of data to check robustness of data collection . while the design of the study allows useful information to be captured about the range and mix of patients and treatment approaches , it provides limited insight as to why individual centres adopt particular technologies or modalities . a more widespread data collection technique such as the national rt dataset in the uk8 would help determine factors influencing local decisions about routine treatment approaches . recruitment issues aside , the study method also allows for possible limitations with the random date possibly not being representative . responses could have been influenced by timing in relation to regular clinics , oncologist availability and resource availability . such random fluctuations are expected to decrease as sample size increases and future wider participation may reveal the extent of this . by far , the greatest limitation of the study relates to data entry error with a large number of individuals responsible for transcribing large quantities of data on top of a busy workload . reported singleentry errors are in the region of 36 per 10,000 entries22 ; this can potentially be reduced by using double data entry . although guidelines were developed and validated in a previous study , there was a definite discrepancy with interpretation of imaging frequency reporting . these data were discarded during the analysis phase and future iterations will need more specific guidance . as with the previous study , the proforma utilised a simplified system of data classification for ease of interpretation , speed of entry and reporting purposes . while this is valuable for broad trend spotting , there is a loss of accuracy with tumour pathologies and stages and inability to perform correlation analysis for individual patients . the results of the study in general confirm the validity of the research method when compared with those from the previous study . the method is a relatively convenient and powerful means of monitoring radiotherapy treatment and technology uptake trends . the overall trend demonstrated by the two studies is that of increasing use of technology in rt ; in particular there is increasing use of imaging technology as part of the standard workload in line with recommendations for igrt use from the usa23 and the uk.24 from an educational perspective these findings support increased emphasis on igrt at both pre and postregistration provision . the authors are particularly interested in using the data to inform curriculum development of academic programs in australia and to ensure training is matched to current workforce patterns of practice.25 with the implementation of new technologies in radiotherapy comes the challenge to keep up to date with teaching content . what is common practice in one state may not be reflective of national trends and best practice . a regular audit such as this study provides a broader national perspective of practice and techniques . the results allow mapping of content to reflect current practice and respond to trends in new techniques for future review . examples from this are the broad increased use of igrt nationally and the specific increase in imrt for prostate cases in particular ; these results have led to earlier and more intensive teaching in imrt . in considering the future formation of a national competency development framework for rt students , the results of this audit form a valuable component . an interesting finding from this data analysis has been the apparent resourceintensive nature of benign tumour radiotherapy . for a traditionally small component of the radiotherapy workload , there is a high uptake of imrt , fusion and igrt ; some of these technologies contribute an increased dose to these patients . it may be cogent to further examine the extent to which benign tumours utilise resources compared with malignancies . another point to consider is whether differences in uptake of technology between states are indicative of different policy implementation and resource availability or a byproduct of the random audit method used . there has been much discussion related to the postcode lottery of cancer treatment26 , 27 with variability between and across different countries ; these data could suggest that this also applies to more widespread differences . certainly within australia , newer technology is established in some states more than others with both geographical and political factors impacting on their availability . findings from this study indicate the increasing use of emerging imrt , image fusion and igrt technology in australian rt planning and delivery phases . this must be reflected in national rt curriculum development with increased provision of theoretical teaching and practical experience with these technologies . the results also highlight the resourceintensive nature of planning and treating benign tumours ; frequently requiring additional radiation dose . the singleday census method offers a relatively convenient means of measuring and tracking rt resources . in the absence of more formal national data collection procedures , wider use of this tool has the potential to not only track trends in technology implementation but also inform evidencebased guidelines for referral and resource planning.27 , 28

abstractintroductionthis article presents the results of a singleday census of radiation therapy ( rt ) treatment and technology use in australia . the primary aim of the study was to ascertain patterns of rt practice and technology in use across australia . these data were primarily collated to inform curriculum development of academic programs , thereby ensuring that training is matched to workforce patterns of practice.methodsthe study design was a census method with all 59 rt centres in australia being invited to provide quantitative summary data relating to patient case mix and technology use on a randomly selected but common date . anonymous and demographicfree data were analysed using descriptive statistics.resultsoverall data were provided across all six australian states by 29 centres of a possible 59 , yielding a response rate of 49% and representing a total of 2743 patients . findings from this study indicate the increasing use of emerging intensitymodulated radiotherapy ( imrt ) , image fusion and imageguided radiation therapy ( igrt ) technology in australian rt planning and delivery phases . imrt in particular was used for 37% of patients , indicating a high uptake of the technology in australia when compared to other published data . the results also highlight the resourceintensive nature of benign tumour radiotherapy.conclusionsin the absence of routine national data collection , the singleday census method offers a relatively convenient means of measuring and tracking rt resource utilisation . wider use of this tool has the potential to not only track trends in technology implementation but also inform evidencebased guidelines for referral and resource planning .

Introduction Methods Results Discussion Indications for RT Patterns of IMRT use Patterns of image fusion use Imageguided radiotherapy Limitations of the study Implications of the study Conclusions Conflict of Interest

regular audits in the literature attempt to track trends in availability of radiation therapy ( rt ) technology such as intensitymodulated radiotherapy ( imrt)1 from a range of countries.2 , 3 , 4 there is a big difference , however , between the availability of resources and actual usage with the resourcehungry nature of commissioning5 in particular affecting implementation and usage of the available technology and equipment.6 mayles ' 2010 uk study7 remains one of the few to investigate the link between availability and use of technology and determined that lack of personnel and funding was a common inhibitory factor rather than availability of technology . in the absence of formal national data collection procedures such as the uk rt dataset,8 there is a lack of data describing australian rt patterns . this national study was built on the findings of a 2012 statewide audit9 of practice in queensland that used a fast and simple method to collect data concerning rt indications and routine technology usage . the primary aim of the study was to ascertain patterns of rt practice and technology in use across australia . these data would then be used to inform curriculum development of academic programs , thereby ensuring that training is matched to workforce patterns of practice . the secondary aim of the study was to compare the findings with those of the previous audit and additional published data to determine trends in practice . the study design was a census method as previously validated.9 all 59 australian rt centres in operation at the time of the study were invited to provide quantitative summary data relating to patient case mix and technology use on a randomly selected but common date ( thursday 5th december 2013 ) . anonymous demographicfree data were harvested using a microsoft excel spreadsheet proforma adapted and validated from the previous study9 and was analysed using descriptive statistics . as with the previous study , ethical exemption was provided by the coordinating university research ethics committee due to the anonymous nature of the data collection and the use of already existing datasets . the results were combined to provide summary data relating to a number of key themes including tumour case mix and usage of imrt , image fusion and ontreatment imaging modalities . tumour case mix analysis indicated the relative incidence of the patients on the chosen day along with the range of techniques used . results from this audit were directly compared to the previous results from the queenslandwide study and published data to determine trends . overall data were provided from centres in all six australian states by 29 centres of a possible 59 , yielding a response rate of 49% . a total of 2743 patients were treated on the chosen date across these centres and table 1 presents a summary of the national and statewide responses . radical treatments comprised 81.6% of the total workload and over 91% of treatments used megavoltage equipment with only 2.5% of treatments using kilovoltage therapy . a total of 198 patients ( 7.2% ) received electron treatment either standalone or concurrent with megavoltage ( table 1 ) . the table also compares this list with results from the previous queenslandbased audit9 as well as barton 's 201310 summary of the most common sites indicated for radiotherapy nationally in australia . top 10 indications for radical radiotherapy imrtrelated data are summarised in table 3 with the left hand section showing data for fixed angle imrt and the right hand showing data for volumetricmodulated arc therapy ( vmat ) , including tomotherapy . a total of 1007 ( 37% ) of the treated patients received imrt and 16% of these received vmat using dynamic linear accelerator or tomotherapy technology . % use per site values indicate the percentage of each tumour site patients receiving imrt ; it can thus be seen that over 71% of prostate patients receive imrt . as in the previous study , the most common sites for imrt ( as a proportion of all imrt treatments ) were still prostate , breast and head and neck , although vmat was not used for breast treatments . overall , arc therapies were used for 160 ( 6% ) of all patient cases , with tomotherapy delivering a little under onethird of these . vmat delivered the remaining arc therapy treatments , with 62% of being used for prostate , and 17% for head and neck . intensity modulation was used in preference to other rt modalities for prostate and head and neck , with imrt or vmat used in 71% and 65% of these sites respectively . top 10 intensitymodulated radiotherapy ( imrt ) indications and usage data in table 4 relate to usage of magnetic resonance imaging ( mri ) and computed tomography ( ct ) fusion , where it can be seen that prostate and brain tumours made by far the most demands on the modality . top 10 magnetic resonance imaging ( mri ) fusion indications and usage table 5 illustrates the main indications for positron emission tomography ( pet)ct fusion with lung and head and neck comprising over 50% of the indications . the data from this study suggested that 9% of patients were planned with petct . as indicated in table 5 , cancers of the lung , head and neck , oesophagus and rectum were the most common sites for this form of image fusion , with over 30% of lung cancer patients benefitting from the technology . top 10 pet fusion indications and usage data were also gathered to highlight usage of kilovoltage and megavoltage portal imaging as well as conebeam ct modalities . overall 99% of patients received imaging ; some patients received multiple imaging modalities but the summary data will not permit further analysis . interestingly , the queenslandonly data for this study reported a 0.9% kilovoltage rate , suggesting a reduction in this iteration compared to the previous queenslandbased study . in 2010 , the five most commonly diagnosed cancers in australia were prostate ( 19,821 cases ) , bowel ( 14,860 ) , breast ( 14,308 ) , melanoma ( 11,405 ) and lung ( 10,296).12 these figures map consistently with the data from this study as prostate and breast cancer are commonly treated with rt , although it must be acknowledged that some of the most common cancers are not demonstrated . this study 's data are therefore well validated with the published findings in terms of the incidence of sitespecific cancers and those treated in the highest 10 categories . most states used imrt for between 21% and 26% of patients with new south wales ( nsw ) treating over half of all patients with imrt . it would be interesting to determine factors affecting this variation but the need for individual centre anonymity precluded this for this study . the previous iteration of this study reported a 19.6% use of imrt.9 when compared to the current results for the national ( 37% ) and queensland data ( 25.9% ) it is clear that imrt use in australia is increasing in prevalence . a comparison with published data also confirms this trend in other countries.3 , 11 , 13 it was noteworthy that of the 19 patients treated for benign conditions 10 received resourceintensive imrt . it is clear that imrt usage in australia is relatively high when compared to published data and is steadily increasing . this strong utilisation of arc therapy technologies for prostate and head and neck tumours is supported by the literature and associated with improved outcomes for these patients.15 while arc therapy was not delivered to any of the six paediatric cases treated , half the cases were treated with imrt despite the decreased monitor unit and treatment times offered by vmat . the superior softtissue visualisation offered by mrict fusion significantly enhances localisation of intracranial , head and neck16 and prostate tumours.17 this study 's data indicated an overall increase in mrict fusion use when compared to the previous study 's 6.9%.9 the mri data from this study again highlighted the resourceintensive nature of benign tumours with 13 of the 19 patient datasets using the technique . over the past decade , use of pet and ct image fusion ( petct ) has improved precision of tumour volume delineation . the literature suggests that petct image fusion is of particular use in delineating lung cancers.18 the results from this study demonstrate high uptake of petct fusion for lung patients and correlates well with existing literature.19 interestingly , when compared to data from the previous study,9 where petct fusion was used in 11.3% of patients , it can be seen that there was an overall decrease in its use . it will be interesting to see if future iterations of this study reveal how this evidence affects pet fusion data and whether image fusion becomes an established method of monitoring tumour regression during rt . the data demonstrated increasing use of kv conebeam equipment with 12% of patients nationally receiving a cone beam computed tomography ( cbct ) image compared to 8.5% last time ; this is consistent with the published data.20 when comparing the uptake in queensland directly between the two studies , there was a modest increase from 11.2% to 12% suggesting the findings may be skewed slightly by increased usage in other states . although too small to be significant with this method , any increase may reflect the reticence to add to the dose burden when compared to kv planar imaging . examination of table 1 reveals that nsw used cbct for 17.3% of patients ; most other states were on a par at between 9% and 12% . of the 321 cbct images taken on the census date 36% were prostate patients , 16% head and neck and 13% lung . it will be interesting to see how increasing use of adaptive rt protocols influence use of cbct in future iterations . fiducial markers were utilised in 53% of prostate patients despite recommendations for their use in the evidence base.21 with cbct being indicated for patients without fiducial markers with only 36% of prostate patients receiving cbct , it is unclear how some patients ' motion was accounted for most planar mv images were used for verification of the breast , with bone , brain and lung treatments also ahead of the imagereliant sites such as the prostate and head and neck . although sufficient data existed to determine trends on a national scale , the participation rate was lower than expected . measures to ameliorate the latter concern were taken but future data collection may be enhanced by provision of a research assistant to those centres struggling with staffing levels . while the design of the study allows useful information to be captured about the range and mix of patients and treatment approaches , it provides limited insight as to why individual centres adopt particular technologies or modalities . recruitment issues aside , the study method also allows for possible limitations with the random date possibly not being representative . by far , the greatest limitation of the study relates to data entry error with a large number of individuals responsible for transcribing large quantities of data on top of a busy workload . these data were discarded during the analysis phase and future iterations will need more specific guidance . the results of the study in general confirm the validity of the research method when compared with those from the previous study . the method is a relatively convenient and powerful means of monitoring radiotherapy treatment and technology uptake trends . the overall trend demonstrated by the two studies is that of increasing use of technology in rt ; in particular there is increasing use of imaging technology as part of the standard workload in line with recommendations for igrt use from the usa23 and the uk.24 from an educational perspective these findings support increased emphasis on igrt at both pre and postregistration provision . the authors are particularly interested in using the data to inform curriculum development of academic programs in australia and to ensure training is matched to current workforce patterns of practice.25 with the implementation of new technologies in radiotherapy comes the challenge to keep up to date with teaching content . a regular audit such as this study provides a broader national perspective of practice and techniques . the results allow mapping of content to reflect current practice and respond to trends in new techniques for future review . examples from this are the broad increased use of igrt nationally and the specific increase in imrt for prostate cases in particular ; these results have led to earlier and more intensive teaching in imrt . in considering the future formation of a national competency development framework for rt students , the results of this audit form a valuable component . an interesting finding from this data analysis has been the apparent resourceintensive nature of benign tumour radiotherapy . for a traditionally small component of the radiotherapy workload , there is a high uptake of imrt , fusion and igrt ; some of these technologies contribute an increased dose to these patients . another point to consider is whether differences in uptake of technology between states are indicative of different policy implementation and resource availability or a byproduct of the random audit method used . interestingly , the queenslandonly data for this study reported a 0.9% kilovoltage rate , suggesting a reduction in this iteration compared to the previous queenslandbased study . in 2010 , the five most commonly diagnosed cancers in australia were prostate ( 19,821 cases ) , bowel ( 14,860 ) , breast ( 14,308 ) , melanoma ( 11,405 ) and lung ( 10,296).12 these figures map consistently with the data from this study as prostate and breast cancer are commonly treated with rt , although it must be acknowledged that some of the most common cancers are not demonstrated . this study 's data are therefore well validated with the published findings in terms of the incidence of sitespecific cancers and those treated in the highest 10 categories . it would be interesting to determine factors affecting this variation but the need for individual centre anonymity precluded this for this study . the previous iteration of this study reported a 19.6% use of imrt.9 when compared to the current results for the national ( 37% ) and queensland data ( 25.9% ) it is clear that imrt use in australia is increasing in prevalence . a comparison with published data also confirms this trend in other countries.3 , 11 , 13 it was noteworthy that of the 19 patients treated for benign conditions 10 received resourceintensive imrt . it is clear that imrt usage in australia is relatively high when compared to published data and is steadily increasing . the superior softtissue visualisation offered by mrict fusion significantly enhances localisation of intracranial , head and neck16 and prostate tumours.17 this study 's data indicated an overall increase in mrict fusion use when compared to the previous study 's 6.9%.9 the mri data from this study again highlighted the resourceintensive nature of benign tumours with 13 of the 19 patient datasets using the technique . over the past decade , use of pet and ct image fusion ( petct ) has improved precision of tumour volume delineation . the literature suggests that petct image fusion is of particular use in delineating lung cancers.18 the results from this study demonstrate high uptake of petct fusion for lung patients and correlates well with existing literature.19 interestingly , when compared to data from the previous study,9 where petct fusion was used in 11.3% of patients , it can be seen that there was an overall decrease in its use . it will be interesting to see if future iterations of this study reveal how this evidence affects pet fusion data and whether image fusion becomes an established method of monitoring tumour regression during rt . the data demonstrated increasing use of kv conebeam equipment with 12% of patients nationally receiving a cone beam computed tomography ( cbct ) image compared to 8.5% last time ; this is consistent with the published data.20 when comparing the uptake in queensland directly between the two studies , there was a modest increase from 11.2% to 12% suggesting the findings may be skewed slightly by increased usage in other states . although too small to be significant with this method , any increase may reflect the reticence to add to the dose burden when compared to kv planar imaging . examination of table 1 reveals that nsw used cbct for 17.3% of patients ; most other states were on a par at between 9% and 12% . of the 321 cbct images taken on the census date 36% were prostate patients , 16% head and neck and 13% lung . it will be interesting to see how increasing use of adaptive rt protocols influence use of cbct in future iterations . further study in this area most planar mv images were used for verification of the breast , with bone , brain and lung treatments also ahead of the imagereliant sites such as the prostate and head and neck . although sufficient data existed to determine trends on a national scale , the participation rate was lower than expected . measures to ameliorate the latter concern were taken but future data collection may be enhanced by provision of a research assistant to those centres struggling with staffing levels . this would also allow random audit of data to check robustness of data collection . while the design of the study allows useful information to be captured about the range and mix of patients and treatment approaches , it provides limited insight as to why individual centres adopt particular technologies or modalities . recruitment issues aside , the study method also allows for possible limitations with the random date possibly not being representative . such random fluctuations are expected to decrease as sample size increases and future wider participation may reveal the extent of this . by far , the greatest limitation of the study relates to data entry error with a large number of individuals responsible for transcribing large quantities of data on top of a busy workload . these data were discarded during the analysis phase and future iterations will need more specific guidance . the results of the study in general confirm the validity of the research method when compared with those from the previous study . the method is a relatively convenient and powerful means of monitoring radiotherapy treatment and technology uptake trends . the overall trend demonstrated by the two studies is that of increasing use of technology in rt ; in particular there is increasing use of imaging technology as part of the standard workload in line with recommendations for igrt use from the usa23 and the uk.24 from an educational perspective these findings support increased emphasis on igrt at both pre and postregistration provision . the authors are particularly interested in using the data to inform curriculum development of academic programs in australia and to ensure training is matched to current workforce patterns of practice.25 with the implementation of new technologies in radiotherapy comes the challenge to keep up to date with teaching content . a regular audit such as this study provides a broader national perspective of practice and techniques . the results allow mapping of content to reflect current practice and respond to trends in new techniques for future review . examples from this are the broad increased use of igrt nationally and the specific increase in imrt for prostate cases in particular ; these results have led to earlier and more intensive teaching in imrt . in considering the future formation of a national competency development framework for rt students , the results of this audit form a valuable component . an interesting finding from this data analysis has been the apparent resourceintensive nature of benign tumour radiotherapy . for a traditionally small component of the radiotherapy workload , there is a high uptake of imrt , fusion and igrt ; some of these technologies contribute an increased dose to these patients . another point to consider is whether differences in uptake of technology between states are indicative of different policy implementation and resource availability or a byproduct of the random audit method used . findings from this study indicate the increasing use of emerging imrt , image fusion and igrt technology in australian rt planning and delivery phases . the results also highlight the resourceintensive nature of planning and treating benign tumours ; frequently requiring additional radiation dose . the singleday census method offers a relatively convenient means of measuring and tracking rt resources . in the absence of more formal national data collection procedures , wider use of this tool has the potential to not only track trends in technology implementation but also inform evidencebased guidelines for referral and resource planning.27 , 28

while the number of protein sequences deposited in public databases continues to expand exponentially , determining the function of the encoded proteins remains slow . except where sequence identity to a protein of known function is high , the activity of a newly sequenced protein must be interrogated with candidate ligands or substrates . this can be done empirically , by screening for binding or substrate turnover or by a mixture of computational prediction , for instance by docking molecular libraries and subsequent experimental testing . both approaches rely on screening libraries of small molecules , such as metabolites . if the right metabolite , or a close analogue , is present in the library , it may be detected as substrate , whereas if it is not , either no activity will be assigned or it may be mis - assigned . in the latter case , however , the multiple chemotypes present in biological small molecules , and their exponential scaling when combined into more complex biological compounds , make full coverage of biorelevant chemical space difficult to ensure . in drug discovery , the combinatorial explosion of chemotypes with molecular size has been addressed by screening libraries of fragment molecules . because fragments are smaller than druglike molecules ( typically less than 17 non - hydrogen atoms ) , fragment chemical space is about 50 orders - of - magnitude smaller than druglike chemical space , enabling fragment libraries to cover chemical space better than libraries of more complex molecules . individual fragment inhibitors usually present simple chemotypes that are only expanded out to fully elaborated molecules after initial hits are discovered ; this has been a remarkably successful approach . a fragment - based strategy could be an attractive alternative to the full enumeration of metabolite space for substrate discovery . not only would it cover potential substrate space far more efficiently , but it would also increase the number of representative molecules that can be physically sourced ; currently , many known metabolites and biogenic molecules are simply unavailable for testing this is far less of a problem for fragments , where molecules containing core reactant groups are readily available ; for instance , over 700,000 accessible fragments are cataloged in the zinc database . a key question is whether a substrate , stripped to the core reactive group on which the catalytic machinery of an enzyme acts , retains enough recognition elements to be an effective , or at least a detectable , enzyme substrate . it could be that enzyme catalysis is so demanding that most of the atoms of the substrate must be engaged with the enzyme before catalysis will occur . several lines of evidence support this view , including studies showing that fragmentation of cytidine into component fragments lowered the activity for cytidine deaminase by 49 orders - of - magnitude and that fragmentation of a transition - state analogue of calf adenosine deaminase led to losses of up to 6 orders - of - magnitude in affinity . also , as shown by jencks , there is no reason why the binding energies of component fragments should sum up to the binding or catalytic activity of a full substrate , owing to the nonadditive , nonequilibrium effects of chemical connectivity . conversely , other studies suggest that fragments can be built up additively for affinity and catalytic recognition . for instance , the well - studied enzyme chymotrypsin hydrolyzes a variety of substrates , including p - nitrophenyl acetate and p - nitroanilides . these compounds only share a very reduced fragment at the reaction center ( two common heavy atoms : c(=o ) ) , which would argue in favor of a fragment - based approach . it has also been possible to deconstruct at least some substrates and transition - state analogues into component fragments . for example , the transition - state analogue immucillin - h , which inhibits purine nucleoside phosphorylase with a ki of 28 pm , was deconstructed into purine and iminoribitol with a loss of only 30-fold in the multiplied affinities of the component fragments . crystallographic studies confirmed that the geometry of these fragments , when bound to the enzyme , recapitulates that of the entire immucillin - h . fragments of the thymidylate synthase substrate deoxyuridine monophosphate , 2-deoxyuridine , and 2,5-dideoxyuridine , also conserve their binding mode when crystallized in the presence of phosphate , although smaller fragments are either poorly ordered in the active site or bind nonspecifically . more recently , the substrate specificity of the mis - annotated enzyme atu3266 from agrobacterium tumefaciens c58 was determined , starting from a very weak fragment hit ( kcat / km = 4 m s ) and resulting in a potent substrate ( kcat / km = 2.8 10 m s ) . similarly , for triosephosphate isomerase , the difference in activation barrier for the isomerization of whole substrate and substrate in pieces is large ( 6.6 kcal / mol ) but product formation is still detectable . large increases in proteolytic activity also have been observed when long - chain substrates are hydrolyzed by pepsin and elastase . lastly , in addition to the successes in stepwise optimization of fragment inhibitors for drug discovery , a fragment - based approach has been used to develop enzyme inhibitors , based on initial substrate turnover . in short , there is evidence to both support and undermine the use of fragments for substrate discovery . the potential benefit of fragment screens against genomic targets , which may dramatically expand our ability to probe chemotype space , spurred us to explore this question further . if it is true that fragments can be used as probe substrates , fragments of extant substrates should retain substantial turnover , assuming that they keep the reactive center intact . here we explore this idea systematically on the substrates of six enzymes from three different enzyme superfamilies : the amidohydrolase superfamily members adenosine deaminase ( ada ) , 5-methylthioadenosine / s - adenosyl - homocysteine deaminase ( sahd ) , and isoaspartyl dipeptidase ( iad ) ; the haloacid dehalogenase superfamily members phosphoserine phosphatase ( psp ) and flavin mononucleotide phosphatase ( fmp ) ; and ampc -lactamase ( ampc ) . substrates for each enzyme were deconstructed into fragments ( figure 1 ) , which contained either the catalytic core of the full - length substrate , or side - chains of the substrate not directly involved in catalysis . the fragments were tested for enzyme turnover or enzyme inhibition , typically one at a time . in almost every case , the resulting fragments several variants of which were tried for each enzyme showed a drop of 10 or more in kcat / km relative to the canonical substrate , and for most compounds binding could not be detected . these results have implications for our ability to extend the fragment approach , so successful for inhibitor discovery , to substrate discovery , and may illuminate the nature of the small - molecule environment against which enzymes have evolved . multiple ways of fragmenting the substrates of adenosine deaminase ( ada ) ; mta / sah deaminase ( sahd ) ; phosphoserine phosphatase ( psp ) ; flavin mononucleotide phosphatase ( fmp ) ; and ampc -lactamase ( ampc ) . only one fragment was typically tested at a time . for isoaspartyl dipeptidase ( iad ) , recognition of the component amino - acid monomers was investigated . the canonical substrate of ada , adenosine ( 1 ) , was fragmented in stages , beginning with the removal of the hydroxyl groups at c2 , c3 , and c5 of the molecule ( 2 , 3 , 4 ) , and with the dideoxy derivative , 2,5-dideoxy adenosine ( 5 ) . subsequently , the entire ribose was removed to give 9-hydroxymethyladenine ( 6 ) , 9-methyladenine ( 7 ) , and adenine ( 8) ; all retained the core reactive purine amine on which the deamination reaction occurs . value for adenosine is 7.6 10 m s. while the 2-deoxy derivative 2 retained most of that activity ( kcat / km 4.1 10 m s ) , the 3-deoxy 3 lost over 1 order of magnitude of activity ( kcat /km 2 10 m s ) , and in the 5-deoxy and dideoxy analogues 4 and 5 no activity could be detected . for the adenine derivatives 6 , 7 , and 8 , kcat / km values were reduced by 5 orders of magnitude ( table 1 ) . it may be that , while purines themselves are poor substrates , adding a purine and a ribose fragment simultaneously to the enzyme would enhance activity . to investigate this , adenine ( 8) was added to the enzyme which had been preincubated with 1-deoxy - d - ribose ( 9 ) ( 5 mm ) , but correspondingly , deamination of adenosine ( 1 ) was not inhibited by addition of either 9 or ribosylamine ( 10 ) at concentrations up to 6.5 mm , indicating that these two fragments do not bind detectably to the active site of the enzyme . to explore the specificity of ada , cytosine ( 11 ) and guanine ( 12 ) were tested as substrates ; neither showed detectable activity . sahd deaminates 5-methyl - thioadenosine ( 13 ) , s - adenosyl - homocysteine ( 14 ) and adenosine ( 1 ) with kcat /km values of 5 10 m s , 5.8 10 m s , and 9.2 10 m s , respectively ( table 2 ) . this enzyme is mechanistically related to ada , but their sequence identity is low ( 22% ) ; these two enzymes fall into two different clades of the amidohydrolase superfamily and are from different clusters of orthologous groups . purine fragments that retain the reactive core of the larger substrates were tested for activity . no deamination could be detected for 9-hydroxymethyladenine ( 6 ) , 9-methyladenine ( 7 ) , and adenine ( 8) after incubating these compounds ( 0.1 mm ) for 12 h with 0.001 mm enzyme ( kcat / km < 2.3 m s ) ( table 2 ) . to further probe binding , the ribose fragments 1-deoxy - d - ribose ( 9 ) and ribosyl amine ( 10 ) , and methionine ( 15 ) were tested as inhibitors of sahd using 13 as a substrate . at concentrations of 5.0 mm , 9 , 10 , and 15 did not inhibit the enzyme detectably ( ki > 5 mm ) . we note that the crystal structure of sahd , determined in the presence of a high concentration of methionine ( 15 ) as an antioxidant , demonstrates that this fragment can bind to the protein in the absence of other substrates . to investigate a possible synergistic role of 15 , the enzyme was preincubated with this fragment ( up to 10 mm ) before addition of 8 , but still no deamination was observed . thus , for mta - sah deaminase , purine fragments of the canonical substrate , even though they retain the core adenine reactive center , lose over 5 orders of magnitude of activity relative to the natural substrate of the enzyme . iad hydrolyzes several -aspartyl peptides , of which -aspartyl - leucine ( 16 ) is the best recognized , with a kcat / km of 10 m s. we were interested in determining if the enzyme would recognize any of the component amino acid monomers , even as inhibitors . accordingly , iad was assayed with 0.5 mm of the substrate 16 in the presence and absence of 2 mm concentrations of the following l - amino acids : leucine ( 17 ) , alanine ( 18 ) , glutamate ( 19 ) , phenylalanine ( 20 ) , glycine ( 21 ) , histidine ( 22 ) , isoleucine ( 23 ) , lysine ( 24 ) , methionine ( 15 ) , asparagine ( 25 ) , proline ( 26 ) , glutamine ( 27 ) , arginine ( 28 ) , serine ( 29 ) , threonine ( 30 ) , valine ( 31 ) , tryptophan ( 32 ) , and tyrosine ( 33 ) ( table s1 supporting information [ si ] ) . no measurable inhibition was observed for any of these amino acids ( ki < 4 mm ) . psp dephosphorylates phosphoserine ( 34 ) with a kcat / km of 3.6 10 m s and phosphothreonine ( 35 ) with a kcat /km of 3 10 m s ( table 3 ) . here too , the substrates are small to begin with , but a reasonable fragmentation was tested using the decarboxy analogue of phosphoserine , ethanolamine phosphate ( 36 ) . this perturbation was sufficient to reduce activity on this molecule , relative to phosphoserine , by 5 orders of magnitude , to a kcat / km of 4.2 m s. meanwhile , serine ( 29 ) itself is a weak competitive inhibitor with a ki of 0.5 mm . fmp catalyzes the dephosphorylation of flavin mononucleotide ( 37 ) with a kcat / km of 2.4 10 m s ( table 4 ) . fragments where the flavin ring was removed , leaving sugar phosphate analogues , were tested as substrates . mannose-1-phosphate ( 38 ) , glucose-1-phosphate ( 39 ) and ribose-5-phosphate ( 40 ) , though far less active as substrates than flavin mononucleotide , do retain substantial activity , with kcat / km of 2 10 m s , 2.6 10 m s , and 2.1 10 m s , respectively . the fragments lumichrome ( 41 ) , mannose ( 42 ) , and glucose ( 43 ) showed no measurable inhibition of fmp - catalyzed dephosphorylation of 38 ( ki values > 0.5 , 20 , and 20 mm , respectively ) . unfortunately , investigating a possible synergistic effect of 41 on the activity of 38 , 39 , and 40 was not possible due to technical issues ( fluorescence and background phosphate interference , see methods in si ) . ampc catalyzes the hydrolysis of cephalothin ( 44 ) with a kcat / km of 7 10 m s ( table 5 ) . we began with fragments that retained the -lactam ring , on which hydrolysis takes place , but lacked most of the side chains characteristic of -lactam antibiotics . starting with the minimal bicyclic cephalosporin ring system ( 45 ) and building up through 46 to cephalosporanic acid ( 47 ) itself , no measurable activity was observed . only for 7-n - formyl - cephalosporanic acid ( 48 ) , which almost entirely recapitulates cephalothin , is measurable activity achieved , and then it is almost entirely restored with a kcat /km of 2.9 10 m s ( figure s1 in the si ) . thus , the addition of a single formyl moiety , distal to the catalytic center , restores 5 log - orders of activity to the cephalosporin family . correspondingly , even binding for the smaller fragments 46 and 47 was difficult to detect , with ki values of about 2 and 5 mm , respectively . though the amidation of the n7 was sufficient to confer activity , the full thiophene acetamide fragment 49 did not detectably inhibit the enzyme ( ki > 20 mm ) , nor did the methyl acetate fragment 50 , representing the right - hand side of cephalothin ( ki > 20 mm ) . the structures of three cephalothin fragments , 45 , 46 , and 48 in complex with ampc were determined by x - ray crystallography , with resolutions ranging from 1.37 to 1.71 . fc difference electron density maps generated early in refinement ( figure s2 and table s2 in the si ) , allowing reliable model building of the enzyme electron density for 48 in the ampc active site is unambiguous in both monomers , with a clear covalent bond between ser64 and the fragment substrate , as expected ( figure 2a and figure s2 in the si ) . the hydrolysis of the beta - lactam ring results in a carbonyl oxygen that interacts with s64 and a318 backbone nitrogens in the oxyanion hole of the enzyme . this structure thus captures the stable acyl - enzyme intermediate step between the transition - state acylation and deacylation complexes . ( a ) the structure of ampc in complex with 48 shows a covalent bond between ser64 and the fragment substrate , ( see also figure s2 in the si ) and captures the stable acyl - enzyme intermediate step between the transition state acylation and deacylation complexes . ( b ) the structure of ampc in complex with 45 shows the fragment in its product form , bound in two orientations to a distal subsite of the large overall binding site , interacting with ser212 , tyr221 , and gly320 . although we could detect neither turnover of 45 nor binding to the enzyme , we were able to observe this compound in the hydrolyzed form in both monomers ( figure 2b and figure s2 in the si ) , via x - ray crystallography after a 10 min soak at 100 mm . the hydrolyzed form of 45 does not bind to the catalytic site , but rather to a distal subsite of the large binding site , interacting with ser212 , tyr221 , and gly320 . two orientations of the ligand are observed , but in both the carboxylates hydrogen bond with the backbone nitrogens of ser212 and gly320 , as previously observed for anionic ligands targeting this site . it may be that this orientation represents an intermediate step in the exit of the product in ampc the contacting residues are well - conserved , even though ser212 and gly320 rarely interact with the better cephalosporin substrates of the enzyme . while 46 was also inert to catalysis by ampc , this compound did weakly inhibit the enzyme . in the crystal structure of the ampc/46 complex , 46 is bound at the surface of the protein , away from the active site , in its hydrolyzed form ( figure s2 in the si ) . intriguingly , the substrate cephalotin was previously observed to bind to the same site , also in the hydrolyzed form . neither the complex with 46 nor 45 supports a catalytically competent recognition of fragment substrates by -lactamase , though both contain the core cephalosporin ring . the appearance of this hydrolysis product in the structure may reflect slow turnover by the enzyme , over the course of the crystallization experiment , but we can not exclude the possibility that the hydrolysis product reflects a background reaction in solvent , to which -lactams are prone . a key observation from this study is that substrate fragments for five of the six enzymes lost essentially all measurable activity or binding , with a reduction of at least 56 orders of magnitude relative to that of the canonical substrate . detectable activity was not regained until most of the substrate had been recapitulated , typically by adding back recognition elements far from the reactive center ( figure 3 ) . clipping only two or three heavy atoms , well removed from the reactive site , was sufficient to almost completely oblate enzymatic activity . for instance , removing a single hydroxyl from adenosine ( 1 ) to form 2- or 3-deoxyadenosine ( 2 and 3 ) reduces the activity of adenosine deaminase by 10-fold ( table 1 ) , as does eliminating the thiophene ring from cephalothin for -lactamase ( table 5 ) . removing one additional hydroxyl moiety from 2 to form 2,5-dideoxy adenosine ( 5 ) completely abolishes the adenosine deaminase activity , while removing the formyl group from 48 eliminates detectable ampc activity . molecules representing the core of the substrate , containing the key reactive group , and representing over 70% of its atoms and functionality , typically had little or no measurable activity against their respective enzymes ( figure 3 ) . moreover , most showed no detectable enzyme inhibition . whereas fragment - based approaches are sometimes suitable for substrate discovery , as observed earlier for purine nucleoside phosphorylase and atu3266 , they seem unreliable as a general strategy . plot of % compound activity ( as compared to the entire substrate ) as a function of size . the high sensitivity of substrate reactivity to small chemical insults seems to contrast with successful efforts to use fragment substrates as inhibitor leads and with efforts to additively improve the affinity of inhibitory fragments . peptide- and phosphate - bearing fragments have been used as substrate leads for proteases and phosphatases with great success . even in that work , however , substrate recognition was often decreased by 5 orders of magnitude compared to that of canonical substrates . this activity loss can be accepted when one knows the fundamental activity for which one is probing ; it is harder to tolerate when seeking to identify the core enzyme activity from a screen of a diverse library of potential substrates . it is challenging to reconcile the large effects on substrate recognition with those of inhibitors , where it is uncommon to find that removal of a single atom or small moiety reduces affinity by 79 kcal / mol , as was observed here . not only did small changes in the substrate have pronounced effects , the residual pieces often did not measurably bind to the enzymes . at least partly , this may reflect the limits of detection over background , and more importantly the compounding effects of reduced substrate recognition on catalytic competence . because substrate binding is usually weak , a loss of 3 kcal / mol in substrate affinity will not only reduce turnover by 100-fold ( in the kcat / km regime ) but may also ramify through poor substrate alignment to a lower kcat . however , it also seems that , for many enzymes , there is a substantial nonadditivity to substrate recognition in catalysis . thus , functional groups that contribute substantially to recognition when presented as part of the full substrate , are not recognized in substrate fragments . for inhibitors , conversely , recognition is often more additive . the idea of integrated , nonadditive recognition of substrates has precedence in experimental and theoretical enzymology returning to jencks idea that groups distal to the reactive center may provide crucial binding energy for catalytic recognition and these results may have implications for promiscuity on the substrate side of enzyme substrate interactions , providing a counterpoint to the promiscuity of enzyme recognition and evolution . enzyme promiscuity and moonlighting activities seem central to the evolution of new activities , and noncanonical substrates and ligands may be specifically recognized . what this study suggests is that enzyme recognition of optimal substrates for any given reaction is mostly punctate , tolerating little diminution in substrate functional groups . physically , this may reflect the integrated chemistry of small molecules , where each atom contributes to the electronic properties of the entire substrate . the sharp dependence on substrate structure may also reflect specificity constraints faced by enzymes confronted with multiple related metabolites , upon whose product multiple downstream enzymes depend . in such a system , it is possible to duplicate and subsequently mutate an enzyme without detrimental effect on other dependencies , exploiting intrinsic enzyme promiscuity . conversely , changes in the substrate or the product could have cascading effects on the larger cellular system . thus , while enzymes remain plastic to evolution overall , and may be promiscuous with unrelated substrates , they may be selected for high fidelity around the structure of their cognate substrate , in which little change is tolerated ; this idea has also been suggested for signaling molecules and their receptors . that was observed here for flavin nucleotide phosphatase , and has been observed previously for purine nucleoside phosphorylase . also , one can always imagine larger substrates than the core molecules we have investigated . for instance , there are -lactamase substrates that are substantially larger than cephalothin , and for these the removal of functional groups to yield a substrate just below the size of cephalothin only marginally affects their reactivity to -lactamase . however , the larger side chains of these molecules do not make strong interactions with -lactamase , and these may be seen as extraneous groups that are not germane to substrate recognition by the enzyme . the tolerance to diversity of substrate sizes may be more common for a detoxification enzyme like -lactamase , which operates by itself and not in a pathway . whereas we suspect that in most cases substrates do not tolerate fragmenting , and fragments will not be a pragmatic general strategy for function discovery , there will be cases where such a strategy may well be fruitful . for instance , phosphoserine phosphatase only recognizes phosphoserine and phosphothreonine , whereas other phosphatases such as alkaline phosphatase are able to hydrolyze a large range of monophosphate substrates . such broad - specificity enzymes are expected to be less sensitive to small chemical changes in their substrates and to lend themselves more easily to substrate fragmentation . finally , using substrate fragments as probes for new inhibitors and fragments as leads for inhibitor discovery is unaffected by this study ; these approaches to inhibitor discovery continue to hold great promise . most substrate functional groups may be essential for recognition and turnover by a cognate enzyme , and removal of even a small portion of the substrate , distal to the reactive center , can dramatically reduce the activity . once deconstructed into smaller parts , the resulting fragments show little to no activity , and often do not measurably bind to the enzyme active site . enzymes typically did not evolve for inhibitor binding as they did for substrates , and fidelity requirements appear much higher for the latter . this may reflect a need to engage a full substrate to drive reactivity , as distal functional groups can contribute critically to stabilization of a transition state . the idea that the stringent specificity constraints on substrates , as opposed to inhibitors , reflects downstream affects of products on the cell is more speculative . even here , one can imagine testable implications , as designed enzymes , not evolved against pathway constraints , may exhibit broader promiscuity against related substrates and fragments than do natural enzymes . pragmatically , this study supports programs to synthesize and collect a relatively full repertoire of the core metabolites recognized by biology with which to interrogate protein function . this space , though far larger than one would prefer , is not , in the end , unbounded . isoaspartyl dipeptidase ( iad ) and ampc -lactamase ( ampc ) were expressed and purified as previously described . for adenosine deaminase ( ada ) , mta - sah deaminase ( sahd ) , phosphoserine phosphatase ( psp ) and flavin mononucleotide phosphatase ( fmp ) expression and purification , see methods in the si . the hydrolysis rates and inhibition constants for adenosine deaminase and mta - sah deaminase activities were monitored spectrophotometrically in a direct assay . inhibition of isoaspartyl dipeptidase was measured in a spectrophotometric assay coupling the formation of aspartate to the oxidation of nadh . the substrate hydrolysis rates and inhibition constants for phosphoserine phosphatase were measured spectrophotometrically using the enzchek kit ( invitrogen ) . the hydrolysis rates and inhibition constants for flavin mononucleotide phosphatase were monitored using either the enzchek kit or biomol green ( enzo life sciences ) . the hydrolysis of ampc -lactamase substrates and enzyme inhibition were monitored spectrophotometrically ( methods in the si ) . ampc -lactamase structures in complex with 45 , 46 , and 48 were obtained by soaking the crystals in the respective ligand solution . structures were determined between 1.37 and 1.71 resolution , and the phases for all structures were determined by difference fourier methods ( methods in the si ) .

predicting substrates for enzymes of unknown function is a major postgenomic challenge . substrate discovery , like inhibitor discovery , is constrained by our ability to explore chemotypes ; it would be expanded by orders of magnitude if reactive sites could be probed with fragments rather than fully elaborated substrates , as is done for inhibitor discovery . to explore the feasibility of this approach , substrates of six enzymes from three different superfamilies were deconstructed into 41 overlapping fragments that were tested for activity or binding . surprisingly , even those fragments containing the key reactive group had little activity , and most fragments did not bind measurably , until they captured most of the substrate features . removing a single atom from a recognized substrate could often reduce catalytic recognition by 6 log - orders . to explore recognition at atomic resolution , the structures of three fragment complexes of the -lactamase substrate cephalothin were determined by x - ray crystallography . substrate discovery may be difficult to reduce to the fragment level , with implications for function discovery and for the tolerance of enzymes to metabolite promiscuity . pragmatically , this study supports the development of libraries of fully elaborated metabolites as probes for enzyme function , which currently do not exist .

Introduction Results Discussion Methods

except where sequence identity to a protein of known function is high , the activity of a newly sequenced protein must be interrogated with candidate ligands or substrates . if the right metabolite , or a close analogue , is present in the library , it may be detected as substrate , whereas if it is not , either no activity will be assigned or it may be mis - assigned . in the latter case , however , the multiple chemotypes present in biological small molecules , and their exponential scaling when combined into more complex biological compounds , make full coverage of biorelevant chemical space difficult to ensure . in drug discovery , the combinatorial explosion of chemotypes with molecular size has been addressed by screening libraries of fragment molecules . a fragment - based strategy could be an attractive alternative to the full enumeration of metabolite space for substrate discovery . a key question is whether a substrate , stripped to the core reactive group on which the catalytic machinery of an enzyme acts , retains enough recognition elements to be an effective , or at least a detectable , enzyme substrate . it could be that enzyme catalysis is so demanding that most of the atoms of the substrate must be engaged with the enzyme before catalysis will occur . also , as shown by jencks , there is no reason why the binding energies of component fragments should sum up to the binding or catalytic activity of a full substrate , owing to the nonadditive , nonequilibrium effects of chemical connectivity . for instance , the well - studied enzyme chymotrypsin hydrolyzes a variety of substrates , including p - nitrophenyl acetate and p - nitroanilides . for example , the transition - state analogue immucillin - h , which inhibits purine nucleoside phosphorylase with a ki of 28 pm , was deconstructed into purine and iminoribitol with a loss of only 30-fold in the multiplied affinities of the component fragments . crystallographic studies confirmed that the geometry of these fragments , when bound to the enzyme , recapitulates that of the entire immucillin - h . fragments of the thymidylate synthase substrate deoxyuridine monophosphate , 2-deoxyuridine , and 2,5-dideoxyuridine , also conserve their binding mode when crystallized in the presence of phosphate , although smaller fragments are either poorly ordered in the active site or bind nonspecifically . more recently , the substrate specificity of the mis - annotated enzyme atu3266 from agrobacterium tumefaciens c58 was determined , starting from a very weak fragment hit ( kcat / km = 4 m s ) and resulting in a potent substrate ( kcat / km = 2.8 10 m s ) . similarly , for triosephosphate isomerase , the difference in activation barrier for the isomerization of whole substrate and substrate in pieces is large ( 6.6 kcal / mol ) but product formation is still detectable . lastly , in addition to the successes in stepwise optimization of fragment inhibitors for drug discovery , a fragment - based approach has been used to develop enzyme inhibitors , based on initial substrate turnover . the potential benefit of fragment screens against genomic targets , which may dramatically expand our ability to probe chemotype space , spurred us to explore this question further . here we explore this idea systematically on the substrates of six enzymes from three different enzyme superfamilies : the amidohydrolase superfamily members adenosine deaminase ( ada ) , 5-methylthioadenosine / s - adenosyl - homocysteine deaminase ( sahd ) , and isoaspartyl dipeptidase ( iad ) ; the haloacid dehalogenase superfamily members phosphoserine phosphatase ( psp ) and flavin mononucleotide phosphatase ( fmp ) ; and ampc -lactamase ( ampc ) . substrates for each enzyme were deconstructed into fragments ( figure 1 ) , which contained either the catalytic core of the full - length substrate , or side - chains of the substrate not directly involved in catalysis . the fragments were tested for enzyme turnover or enzyme inhibition , typically one at a time . in almost every case , the resulting fragments several variants of which were tried for each enzyme showed a drop of 10 or more in kcat / km relative to the canonical substrate , and for most compounds binding could not be detected . these results have implications for our ability to extend the fragment approach , so successful for inhibitor discovery , to substrate discovery , and may illuminate the nature of the small - molecule environment against which enzymes have evolved . the canonical substrate of ada , adenosine ( 1 ) , was fragmented in stages , beginning with the removal of the hydroxyl groups at c2 , c3 , and c5 of the molecule ( 2 , 3 , 4 ) , and with the dideoxy derivative , 2,5-dideoxy adenosine ( 5 ) . subsequently , the entire ribose was removed to give 9-hydroxymethyladenine ( 6 ) , 9-methyladenine ( 7 ) , and adenine ( 8) ; all retained the core reactive purine amine on which the deamination reaction occurs . value for adenosine is 7.6 10 m s. while the 2-deoxy derivative 2 retained most of that activity ( kcat / km 4.1 10 m s ) , the 3-deoxy 3 lost over 1 order of magnitude of activity ( kcat /km 2 10 m s ) , and in the 5-deoxy and dideoxy analogues 4 and 5 no activity could be detected . for the adenine derivatives 6 , 7 , and 8 , kcat / km values were reduced by 5 orders of magnitude ( table 1 ) . it may be that , while purines themselves are poor substrates , adding a purine and a ribose fragment simultaneously to the enzyme would enhance activity . to investigate this , adenine ( 8) was added to the enzyme which had been preincubated with 1-deoxy - d - ribose ( 9 ) ( 5 mm ) , but correspondingly , deamination of adenosine ( 1 ) was not inhibited by addition of either 9 or ribosylamine ( 10 ) at concentrations up to 6.5 mm , indicating that these two fragments do not bind detectably to the active site of the enzyme . to explore the specificity of ada , cytosine ( 11 ) and guanine ( 12 ) were tested as substrates ; neither showed detectable activity . purine fragments that retain the reactive core of the larger substrates were tested for activity . no deamination could be detected for 9-hydroxymethyladenine ( 6 ) , 9-methyladenine ( 7 ) , and adenine ( 8) after incubating these compounds ( 0.1 mm ) for 12 h with 0.001 mm enzyme ( kcat / km < 2.3 m s ) ( table 2 ) . to further probe binding , the ribose fragments 1-deoxy - d - ribose ( 9 ) and ribosyl amine ( 10 ) , and methionine ( 15 ) were tested as inhibitors of sahd using 13 as a substrate . at concentrations of 5.0 mm , 9 , 10 , and 15 did not inhibit the enzyme detectably ( ki > 5 mm ) . thus , for mta - sah deaminase , purine fragments of the canonical substrate , even though they retain the core adenine reactive center , lose over 5 orders of magnitude of activity relative to the natural substrate of the enzyme . iad hydrolyzes several -aspartyl peptides , of which -aspartyl - leucine ( 16 ) is the best recognized , with a kcat / km of 10 m s. we were interested in determining if the enzyme would recognize any of the component amino acid monomers , even as inhibitors . accordingly , iad was assayed with 0.5 mm of the substrate 16 in the presence and absence of 2 mm concentrations of the following l - amino acids : leucine ( 17 ) , alanine ( 18 ) , glutamate ( 19 ) , phenylalanine ( 20 ) , glycine ( 21 ) , histidine ( 22 ) , isoleucine ( 23 ) , lysine ( 24 ) , methionine ( 15 ) , asparagine ( 25 ) , proline ( 26 ) , glutamine ( 27 ) , arginine ( 28 ) , serine ( 29 ) , threonine ( 30 ) , valine ( 31 ) , tryptophan ( 32 ) , and tyrosine ( 33 ) ( table s1 supporting information [ si ] ) . this perturbation was sufficient to reduce activity on this molecule , relative to phosphoserine , by 5 orders of magnitude , to a kcat / km of 4.2 m s. meanwhile , serine ( 29 ) itself is a weak competitive inhibitor with a ki of 0.5 mm . mannose-1-phosphate ( 38 ) , glucose-1-phosphate ( 39 ) and ribose-5-phosphate ( 40 ) , though far less active as substrates than flavin mononucleotide , do retain substantial activity , with kcat / km of 2 10 m s , 2.6 10 m s , and 2.1 10 m s , respectively . we began with fragments that retained the -lactam ring , on which hydrolysis takes place , but lacked most of the side chains characteristic of -lactam antibiotics . only for 7-n - formyl - cephalosporanic acid ( 48 ) , which almost entirely recapitulates cephalothin , is measurable activity achieved , and then it is almost entirely restored with a kcat /km of 2.9 10 m s ( figure s1 in the si ) . thus , the addition of a single formyl moiety , distal to the catalytic center , restores 5 log - orders of activity to the cephalosporin family . correspondingly , even binding for the smaller fragments 46 and 47 was difficult to detect , with ki values of about 2 and 5 mm , respectively . though the amidation of the n7 was sufficient to confer activity , the full thiophene acetamide fragment 49 did not detectably inhibit the enzyme ( ki > 20 mm ) , nor did the methyl acetate fragment 50 , representing the right - hand side of cephalothin ( ki > 20 mm ) . the structures of three cephalothin fragments , 45 , 46 , and 48 in complex with ampc were determined by x - ray crystallography , with resolutions ranging from 1.37 to 1.71 . fc difference electron density maps generated early in refinement ( figure s2 and table s2 in the si ) , allowing reliable model building of the enzyme electron density for 48 in the ampc active site is unambiguous in both monomers , with a clear covalent bond between ser64 and the fragment substrate , as expected ( figure 2a and figure s2 in the si ) . ( b ) the structure of ampc in complex with 45 shows the fragment in its product form , bound in two orientations to a distal subsite of the large overall binding site , interacting with ser212 , tyr221 , and gly320 . although we could detect neither turnover of 45 nor binding to the enzyme , we were able to observe this compound in the hydrolyzed form in both monomers ( figure 2b and figure s2 in the si ) , via x - ray crystallography after a 10 min soak at 100 mm . the hydrolyzed form of 45 does not bind to the catalytic site , but rather to a distal subsite of the large binding site , interacting with ser212 , tyr221 , and gly320 . two orientations of the ligand are observed , but in both the carboxylates hydrogen bond with the backbone nitrogens of ser212 and gly320 , as previously observed for anionic ligands targeting this site . it may be that this orientation represents an intermediate step in the exit of the product in ampc the contacting residues are well - conserved , even though ser212 and gly320 rarely interact with the better cephalosporin substrates of the enzyme . intriguingly , the substrate cephalotin was previously observed to bind to the same site , also in the hydrolyzed form . the appearance of this hydrolysis product in the structure may reflect slow turnover by the enzyme , over the course of the crystallization experiment , but we can not exclude the possibility that the hydrolysis product reflects a background reaction in solvent , to which -lactams are prone . a key observation from this study is that substrate fragments for five of the six enzymes lost essentially all measurable activity or binding , with a reduction of at least 56 orders of magnitude relative to that of the canonical substrate . detectable activity was not regained until most of the substrate had been recapitulated , typically by adding back recognition elements far from the reactive center ( figure 3 ) . for instance , removing a single hydroxyl from adenosine ( 1 ) to form 2- or 3-deoxyadenosine ( 2 and 3 ) reduces the activity of adenosine deaminase by 10-fold ( table 1 ) , as does eliminating the thiophene ring from cephalothin for -lactamase ( table 5 ) . molecules representing the core of the substrate , containing the key reactive group , and representing over 70% of its atoms and functionality , typically had little or no measurable activity against their respective enzymes ( figure 3 ) . whereas fragment - based approaches are sometimes suitable for substrate discovery , as observed earlier for purine nucleoside phosphorylase and atu3266 , they seem unreliable as a general strategy . even in that work , however , substrate recognition was often decreased by 5 orders of magnitude compared to that of canonical substrates . this activity loss can be accepted when one knows the fundamental activity for which one is probing ; it is harder to tolerate when seeking to identify the core enzyme activity from a screen of a diverse library of potential substrates . it is challenging to reconcile the large effects on substrate recognition with those of inhibitors , where it is uncommon to find that removal of a single atom or small moiety reduces affinity by 79 kcal / mol , as was observed here . not only did small changes in the substrate have pronounced effects , the residual pieces often did not measurably bind to the enzymes . at least partly , this may reflect the limits of detection over background , and more importantly the compounding effects of reduced substrate recognition on catalytic competence . the idea of integrated , nonadditive recognition of substrates has precedence in experimental and theoretical enzymology returning to jencks idea that groups distal to the reactive center may provide crucial binding energy for catalytic recognition and these results may have implications for promiscuity on the substrate side of enzyme substrate interactions , providing a counterpoint to the promiscuity of enzyme recognition and evolution . enzyme promiscuity and moonlighting activities seem central to the evolution of new activities , and noncanonical substrates and ligands may be specifically recognized . what this study suggests is that enzyme recognition of optimal substrates for any given reaction is mostly punctate , tolerating little diminution in substrate functional groups . physically , this may reflect the integrated chemistry of small molecules , where each atom contributes to the electronic properties of the entire substrate . thus , while enzymes remain plastic to evolution overall , and may be promiscuous with unrelated substrates , they may be selected for high fidelity around the structure of their cognate substrate , in which little change is tolerated ; this idea has also been suggested for signaling molecules and their receptors . for instance , there are -lactamase substrates that are substantially larger than cephalothin , and for these the removal of functional groups to yield a substrate just below the size of cephalothin only marginally affects their reactivity to -lactamase . however , the larger side chains of these molecules do not make strong interactions with -lactamase , and these may be seen as extraneous groups that are not germane to substrate recognition by the enzyme . the tolerance to diversity of substrate sizes may be more common for a detoxification enzyme like -lactamase , which operates by itself and not in a pathway . whereas we suspect that in most cases substrates do not tolerate fragmenting , and fragments will not be a pragmatic general strategy for function discovery , there will be cases where such a strategy may well be fruitful . finally , using substrate fragments as probes for new inhibitors and fragments as leads for inhibitor discovery is unaffected by this study ; these approaches to inhibitor discovery continue to hold great promise . most substrate functional groups may be essential for recognition and turnover by a cognate enzyme , and removal of even a small portion of the substrate , distal to the reactive center , can dramatically reduce the activity . once deconstructed into smaller parts , the resulting fragments show little to no activity , and often do not measurably bind to the enzyme active site . enzymes typically did not evolve for inhibitor binding as they did for substrates , and fidelity requirements appear much higher for the latter . the idea that the stringent specificity constraints on substrates , as opposed to inhibitors , reflects downstream affects of products on the cell is more speculative . pragmatically , this study supports programs to synthesize and collect a relatively full repertoire of the core metabolites recognized by biology with which to interrogate protein function . structures were determined between 1.37 and 1.71 resolution , and the phases for all structures were determined by difference fourier methods ( methods in the si ) .

middle ear cholesteatoma is a benign cystic lesion of aggressive behavior which requiring prompt surgical treatment to prevent local and intracranial complications . surgery is the mainstay treatment of cholesteatoma however , the risk of residual cholesteatoma and recurrence occurs in a relatively high numbers of patients , whatever the type of surgery used , . the detection of cholesteatoma in patients who have undergone middle ear surgery is often difficult for the follow - up of patients operated for cholesteatoma . high resolution computerized tomography ( ct ) scan is considered the basic method for imaging the nonoperated middle ear and can be complemented by magnetic resonance imaging ( mri ) , , , . however , following mastoid surgery , both imaging modalities can not reliably distinguish residual or recurrent disease from postoperative changes such as granulation tissue , fluid or fibrous tissue . diffusion - weighted ( dw ) sequences are highly promising in differentiating recurrent cholesteatoma from granulation tissue dw mri depends on the difference in diffusion of water molecules in different biological tissues . in granulation tissue , water molecules are more mobile and thus appear less intense on dw sequence , . currently , single - shot echo - planar dwi is the most used dwi technique because of its short imaging time ( about 2 min ) . on the other hand , multi - shot non - echo - planar dwi sequences requiring a longer imaging time are associated with decreased susceptibility artifacts , . the adc map is free from t1 or t2 effects , and provides a true quantitative display of water diffusion at each voxel , . adcs , have not yet been assessed as a diagnostic criterion for the detection of recurrent middle ear cholesteatoma with no clear - cut values exist for differentiation of recurrent cholesteatoma from granulation tissue or fibrosis , , , , . in this study , we aim to determine the diagnostic accuracy of dwi and the difference between the adcs of postoperative middle ear cleft cholesteatoma and those of non - cholesteatomatous tissue on single - shot echo planar dw images . the study was approved by the institutional review board and informed consent was obtained from all patients . we included 30 consecutive patients in our study , previously subjected to tympanomastoid surgery for the treatment of cholesteatoma with clinical and ct suspicion of recurrence and scheduled for second look operation , during the period from may 2015 to october 2016 . twenty patients were females and 10 were males , with an age ranging from 9 to 45 years old ( mean = 27 years ) . canal wall - up technique was already done for 17 ears , while 14 ears had canal wall - down technique . the main clinical presentation of the patients was a recurrent purulent discharge ( n = 25 ) , perforated tympanic membrane with and without discharge ( n = 3 ) , chronic ear discharge with sign of increase intracranial tension ( n = 1 ) , chronic discharge with facial paresis ( n = 1 ) ( table 1 ) . all patients were scheduled for revision surgery and the intraoperative findings were compared to imaging results . the mean interval between the first tympanomastoid surgery and ct examination was 1230 months ; all patients were further assessed by mri imaging of the petrous temporal bone on an average of 12 weeks after ct examination . positive cases of recurrent cholesteatoma were referred for second - look surgery , while ve cases received medical treatment . inclusion criteria include : patient with history of tympano - mastoid surgery for cholesteatoma with clinical suspicion of recurrent cholesteatoma or infection during follow - up . exclusion criteria include : patient with unclear operative history , active infection or mastoid abscess . 1.full history taking including : age , sex , chronic ear discharge , conductive hearing loss , headache , vomiting ( signs of increased intracranial tension ) and facial palsy.2.through clinical examination : all patients are subjected to otoscopic examination at the ent department.3.operative history : ( mastoidectomy , otoscope and otoendoscope ) full history taking including : age , sex , chronic ear discharge , conductive hearing loss , headache , vomiting ( signs of increased intracranial tension ) and facial palsy . through clinical examination : all patients are subjected to otoscopic examination at the ent department . operative history : ( mastoidectomy , otoscope and otoendoscope ) 4.1 : mdct examination using multi - detector ct scanner 16 channel ( ge ct / bright speed elite scanner , general electric medical system , usa ) . ct technique : the images were obtained with 0.5 mm collimation , 0.5 mm thickness , 320 mas , and 120kvp . the obtained data was reconstructed in the axial plane using high resolution bone algorithm ( extended window sitting at around 4000hu and window level around 300hu ) with 0.5 mm section thickness , 0.05 mm increments , and a fov of 100 , with a matrix size of 512 512 . at this collimation , an isotropic voxel ( which measure 0.5 mm per side ) was obtained . the axial data were then transferred to a separate workstation for post - processing , with a commercially available 3d reformatting software ( baxara 3d ) . sections was taken at 1 mm increments beginning at the level of the floor of hypotympanum and jugular fossa extending to the level of arcuate eminence using line for localization . 4.2 : mri examinations were performed with 1.5-t mri unit ( philips , achiva , philips medical systems , eindhoven , netherlands ) following patients informed consent and exclusion of contraindications . the following sequences were acquired on the middle ear using an 8 channel head coil and applying the parallel sense imaging is used to obtain better resolution , faster dynamic scans and to reduce susceptibility artifacts . dwi parameters includes ( fov , 230 230 mm ; epi factor , 61 ; thickness / gap , 5/1 mm ; acquisition matrix , 112 89 ) was performed by using b values of 800 and 1000 s / mm in axial , multisection , single - shot , echo - planar imaging sequence . te was taken as short as possible for the acquisition of the highest signal - to - noise ratio . the following sequences were acquired on the middle ear.1.3d ciss ( constructive interference in steady state ) with a gradient echo component tilt angle : 70 , slice thickness : 0.7 mm , repetition time ( tr ) : 11.5 ms , echo time ( te ) : 5.75 ms , sense factor 2 , fov : 230 mm . acquisition time 3 min and 50 s.2.axial and coronal echo planar single shot turbo spin echo diffusion - weighted imaging : 2.5 mm thick axial slices , te : 98 ms , tr : 2000 ms , sense factor 2 , diffusion factor : b 1000 s / mm , fov : 230 mm . acquisition time 2 min and 50 s,3.axial t1 and t2-weighted coronal spin echo ( tse ) . tr / te , 5270/119 ms ; matrix , 512 ; section thickness , sense factor 2 , 3 mm ; field of view , 230 mm . acquisition time 3 min and 12 s. 3d ciss ( constructive interference in steady state ) with a gradient echo component tilt angle : 70 , slice thickness : 0.7 mm , repetition time ( tr ) : 11.5 ms , echo time ( te ) : 5.75 ms , sense factor 2 , fov : 230 mm . acquisition time 3 min and 50 s. axial and coronal echo planar single shot turbo spin echo diffusion - weighted imaging : 2.5 mm thick axial slices , te : 98 ms , tr : 2000 ms , sense factor 2 , diffusion factor : b 1000 s / mm , fov : 230 mm . acquisition time 2 min and 50 s , axial t1 and t2-weighted coronal spin echo ( tse ) . tr / te , 5270/119 ms ; matrix , 512 ; section thickness , sense factor 2 , 3 mm ; field of view , 230 mm . acquisition time 3 min and 12 s. the adc value of the cholesteatoma was calculated on the adc map from the selected region of interest ( roi ) that matched the lesion . the image section for roi placement was selected and the roi drawn by the reading radiologist . the image for roi placement was selected to avoid the edges of the lesion and partial volume averaging , so the central aspect of the lesion was targeted . if the lesion is of small size , the mean adc of the lesion was taken from rois on the two contiguous sections . repeated roi measurements were done two to three times on the image for each case , and the median adc was obtained . all imaging , whether ct or mri , was prospectively interpreted by the same radiologist , specifically trained in petrous bone imaging for about 15 years experience . on ct , recurrent cholesteatoma was diagnosed by identifying the presence of new bone erosion not detected in the preoperative evaluation , the presence of tissue density mass with well - defined borders and an air space between the mass and walls of the tympanic cavity , expansion of the mastoidectomy cavity and erosion of the walls including the tegmen , ossicular chain amputation , or labyrinthine or facial nerve sheath destruction that could not be attributed to past surgery . images were considered negative for cholesteatoma when middle ear cavities were either air filled or showed minimal thickening . interpretation was indeterminate , whenever complete opacification of the tympano - mastoid cavity . on mri sequences , the diagnosis of cholesteatoma was based on identifying area of restricted diffusion in the form of marked hyperintense signal in comparison with brain tissue on the b = ( 800 , 1000 ) images of diffusion - weighted . the radiologist classified mri results into two groups ( recurrent cholesteatoma or non - cholesteatomas ) . lesion dimension at maximum diameter on axial and coronal planes was also recorded on mri and ct and compared with that of operative finding . the surgical findings were obtained from the surgical reports of one team of otology surgeons . the surgical results were classified as recurrence of cholesteatoma or no recurrence of cholesteatoma ( granulation tissue ) . all patients were scheduled for revision surgery and the intraoperative findings were compared to imaging results . the sensitivity , specificity , positive and negative predictive values of different mr sequences used and adcs , was assessed using ssps ( version 12 ) . 1.full history taking including : age , sex , chronic ear discharge , conductive hearing loss , headache , vomiting ( signs of increased intracranial tension ) and facial palsy.2.through clinical examination : all patients are subjected to otoscopic examination at the ent department.3.operative history : ( mastoidectomy , otoscope and otoendoscope ) full history taking including : age , sex , chronic ear discharge , conductive hearing loss , headache , vomiting ( signs of increased intracranial tension ) and facial palsy . through clinical examination : all patients are subjected to otoscopic examination at the ent department . operative history : ( mastoidectomy , otoscope and otoendoscope ) 4.1 : mdct examination using multi - detector ct scanner 16 channel ( ge ct / bright speed elite scanner , general electric medical system , usa ) . ct technique : the images were obtained with 0.5 mm collimation , 0.5 mm thickness , 320 mas , and 120kvp . the obtained data was reconstructed in the axial plane using high resolution bone algorithm ( extended window sitting at around 4000hu and window level around 300hu ) with 0.5 mm section thickness , 0.05 mm increments , and a fov of 100 , with a matrix size of 512 512 . at this collimation , an isotropic voxel ( which measure 0.5 mm per side ) was obtained . the axial data were then transferred to a separate workstation for post - processing , with a commercially available 3d reformatting software ( baxara 3d ) . sections was taken at 1 mm increments beginning at the level of the floor of hypotympanum and jugular fossa extending to the level of arcuate eminence using line for localization . 4.2 : mri examinations were performed with 1.5-t mri unit ( philips , achiva , philips medical systems , eindhoven , netherlands ) following patients informed consent and exclusion of contraindications . the following sequences were acquired on the middle ear using an 8 channel head coil and applying the parallel sense imaging is used to obtain better resolution , faster dynamic scans and to reduce susceptibility artifacts . dwi parameters includes ( fov , 230 230 mm ; epi factor , 61 ; thickness / gap , 5/1 mm ; acquisition matrix , 112 89 ) was performed by using b values of 800 and 1000 s / mm in axial , multisection , single - shot , echo - planar imaging sequence . te was taken as short as possible for the acquisition of the highest signal - to - noise ratio . the following sequences were acquired on the middle ear.1.3d ciss ( constructive interference in steady state ) with a gradient echo component tilt angle : 70 , slice thickness : 0.7 mm , repetition time ( tr ) : 11.5 ms , echo time ( te ) : 5.75 ms , sense factor 2 , fov : 230 mm . acquisition time 3 min and 50 s.2.axial and coronal echo planar single shot turbo spin echo diffusion - weighted imaging : 2.5 mm thick axial slices , te : 98 ms , tr : 2000 ms , sense factor 2 , diffusion factor : b 1000 s / mm , fov : 230 mm . acquisition time 2 min and 50 s,3.axial t1 and t2-weighted coronal spin echo ( tse ) . tr / te , 5270/119 ms ; matrix , 512 ; section thickness , sense factor 2 , 3 mm ; field of view , 230 mm . acquisition time 3 min and 12 s. 3d ciss ( constructive interference in steady state ) with a gradient echo component tilt angle : 70 , slice thickness : 0.7 mm , repetition time ( tr ) : 11.5 ms , echo time ( te ) : 5.75 ms , sense factor 2 , fov : 230 mm . acquisition time 3 min and 50 s. axial and coronal echo planar single shot turbo spin echo diffusion - weighted imaging : 2.5 mm thick axial slices , te : 98 ms , tr : 2000 ms , sense factor 2 , diffusion factor : b 1000 s / mm , fov : 230 mm . acquisition time 2 min and 50 s , axial t1 and t2-weighted coronal spin echo ( tse ) . tr / te , 5270/119 ms ; matrix , 512 ; section thickness , sense factor 2 , 3 mm ; field of view , 230 mm . acquisition time 3 min and 12 s. the adc value of the cholesteatoma was calculated on the adc map from the selected region of interest ( roi ) that matched the lesion . the image section for roi placement was selected and the roi drawn by the reading radiologist . the image for roi placement was selected to avoid the edges of the lesion and partial volume averaging , so the central aspect of the lesion was targeted . if the lesion is of small size , the mean adc of the lesion was taken from rois on the two contiguous sections . repeated roi measurements were done two to three times on the image for each case , and the median adc was obtained . all imaging , whether ct or mri , was prospectively interpreted by the same radiologist , specifically trained in petrous bone imaging for about 15 years experience . on ct , recurrent cholesteatoma was diagnosed by identifying the presence of new bone erosion not detected in the preoperative evaluation , the presence of tissue density mass with well - defined borders and an air space between the mass and walls of the tympanic cavity , expansion of the mastoidectomy cavity and erosion of the walls including the tegmen , ossicular chain amputation , or labyrinthine or facial nerve sheath destruction that could not be attributed to past surgery . images were considered negative for cholesteatoma when middle ear cavities were either air filled or showed minimal thickening . interpretation was indeterminate , whenever complete opacification of the tympano - mastoid cavity . on mri sequences , the diagnosis of cholesteatoma was based on identifying area of restricted diffusion in the form of marked hyperintense signal in comparison with brain tissue on the b = ( 800 , 1000 ) images of diffusion - weighted . the radiologist classified mri results into two groups ( recurrent cholesteatoma or non - cholesteatomas ) . lesion dimension at maximum diameter on axial and coronal planes was also recorded on mri and ct and compared with that of operative finding . the surgical findings were obtained from the surgical reports of one team of otology surgeons . the surgical results were classified as recurrence of cholesteatoma or no recurrence of cholesteatoma ( granulation tissue ) . all patients were scheduled for revision surgery and the intraoperative findings were compared to imaging results . the sensitivity , specificity , positive and negative predictive values of different mr sequences used and adcs , was assessed using ssps ( version 12 ) . this prospective study was conducted in 30 patients with 31ears as one patient had bilateral tympanomastoid surgery for cholesteatoma . we evaluated 30 consecutive patients ( 31 ears ) , 20 were females and 10 males , their age ranged from 945 years , mean age 27 years , who had undergone a canal wall - down mastoidectomy or a canal wall - up mastoidectomy for a cholesteatoma of the middle ear and underwent an mdct and mr dwi examination before second - look operation . the main clinical presentation of the patients were recurrent purulent discharge ( n = 25 ) , perforated tympanic membrane with and without discharge ( n = 3 ) , chronic ear discharge with sign of increase intra cranial tension ( n = 1 ) , chronic discharge with facial paresis ( n = 1 ) . canal wall - up technique was done for 17 ears , while 14 ears had canal wall - down technique . 3 , fig . 5 ) and complete opacification in 21 ears ( figs . 1 , 2 , 4 , 6 and 8) . 6 ) and the right was affected in 5patients ( fig . 1 , fig . 5 ) . ct criteria of recurrent cholesteatoma detected in 10 cases in the form of localized nondependent tissue density mass with new bone erosion not detected in previous ct or pre - operative study ( fig . 10 ) , while the remaining 20 cases the mastoiedctomy cavity was completely opacified . the size of cholesteatoma mass lesion ranged from 10 to 30 mm ( mean17.8 5.7 mm ) . ossicular chains were intact in 6 ears and not visualized ( eroded or amputated ) in 25 ears . the tegmen tympani was eroded in one case , sigmoid sinus plate eroded in one case , tympanic segment of the facial nerve canal was eroded in one case , and lateral mastoid wall was eroded in one case ( fig . the other ear was normally aerated in 25 ears and revealed chronic suppurative otitis media in 4 ears ( figs . 3 , 4 and 6 ) , bilateral cholesteatoma detected in one case ( fig . the included patients were classified into two groups , ( cholesteatoma group include 20 cases ) and non cholesteatoma group ( include 10 cases ) . all patients showed low signal intensity on unenhanced t1wi , high signal intensity on t2wi and 3dciss sequences . based on mr dwi sequence , the cholesteatoma group ( 20 patients with 21 ears ) revealed recurrent cholesteatoma in the form of area of restricted diffusion on dw images at b factor of 800 , 1000 s / mm ( figs . patients without recurrent cholesteatoma ( 10 patients ) , all diffusion - weighted fast se mr images obtained with b of 800,1000 s / mm were correctly interpreted as showing no restriction and were diagnosed as granulation or infection ( fig . the size of the recurrent cholesteatoma mass was ranged from 1030mmat ct , 715 mm at mr dwi and 714 mm at operative finding ( table 9 ) . there was one false - positive case measures 9 6-mm related to lsc that was not a recurrent cholesteatoma but bone graft placed during the initial surgery to seal a lateral semicircular canal fistula ( fig . the adc values of cholesteatoma group ( 21 ears ) were ranged from 553 to 759 10 mm / s ) , mean sd611.8 51.8 10 mm / s ) and median 595 10 mm / s ) . the adcs of non cholesteatoma group ( 10 ears ) was ranged from 1495.8 to 1766.8 10 mm / s , mean 1638.3 93.5 10 mm / s and median 1653.5 10 mm / s . large recurrence seen in attico - antral region were found in 10 ears , the largest lesion reached 30 mm at ct , 14 mm at mr dwi and 14.4 mm operative size ( fig . 3 ) , medium sized recurrence in 8 cases , while smaller recurrences were seen in the middle ear cavity in 3 ears , reaching 10 mm at ct , 7 mm at mr dwi and 7.3 mm at the operation ( fig . the lesion sizes at ct was not correlated with that of the mr dwi and operative because ct gives apparent size of cholesteatoma including the surrounding granulation tissue while the size at mr dwi and operative was more accurate excluding the surrounding granulation tissue with significant ( p value = 0.001 * ) . granulation tissue , fibrosis and secretions were found in the remaining 10 ears ( fig . good correlation between mr dwi finding and operative confirmation , the sensitivity was 100% , the specificity was 90% , the positive predictive value 95.2% and p value is 0.001 . mdct had low sensitivity , detect only 10 cases out of 30 cases , the sensitivity was 47.6% , specificity 100% , and negative predictive value 47.6% and p value is 0.5 ( table 5 ) . we evaluated 30 consecutive patients ( 31 ears ) , 20 were females and 10 males , their age ranged from 945 years , mean age 27 years , who had undergone a canal wall - down mastoidectomy or a canal wall - up mastoidectomy for a cholesteatoma of the middle ear and underwent an mdct and mr dwi examination before second - look operation . the main clinical presentation of the patients were recurrent purulent discharge ( n = 25 ) , perforated tympanic membrane with and without discharge ( n = 3 ) , chronic ear discharge with sign of increase intra cranial tension ( n = 1 ) , chronic discharge with facial paresis ( n = 1 ) . canal wall - up technique was done for 17 ears , while 14 ears had canal wall - down technique . 3 , fig . 5 ) and complete opacification in 21 ears ( figs . 1 , 2 , 4 , 6 and 8) . fig . 6 ) and the right was affected in 5patients ( fig . 1 , fig . 5 ) . ct criteria of recurrent cholesteatoma detected in 10 cases in the form of localized nondependent tissue density mass with new bone erosion not detected in previous ct or pre - operative study ( fig . 10 ) , while the remaining 20 cases the mastoiedctomy cavity was completely opacified . the size of cholesteatoma mass lesion ranged from 10 to 30 mm ( mean17.8 5.7 mm ) . ossicular chains were intact in 6 ears and not visualized ( eroded or amputated ) in 25 ears . the tegmen tympani was eroded in one case , sigmoid sinus plate eroded in one case , tympanic segment of the facial nerve canal was eroded in one case , and lateral mastoid wall was eroded in one case ( fig . the other ear was normally aerated in 25 ears and revealed chronic suppurative otitis media in 4 ears ( figs . 3 , 4 and 6 ) , bilateral cholesteatoma detected in one case ( fig . based on mri findings the included patients were classified into two groups , ( cholesteatoma group include 20 cases ) and non cholesteatoma group ( include 10 cases ) . all patients showed low signal intensity on unenhanced t1wi , high signal intensity on t2wi and 3dciss sequences . based on mr dwi sequence , the cholesteatoma group ( 20 patients with 21 ears ) revealed recurrent cholesteatoma in the form of area of restricted diffusion on dw images at b factor of 800 , 1000 s / mm ( figs . patients without recurrent cholesteatoma ( 10 patients ) , all diffusion - weighted fast se mr images obtained with b of 800,1000 s / mm were correctly interpreted as showing no restriction and were diagnosed as granulation or infection ( fig . the size of the recurrent cholesteatoma mass was ranged from 1030mmat ct , 715 mm at mr dwi and 714 mm at operative finding ( table 9 ) . there was one false - positive case measures 9 6-mm related to lsc that was not a recurrent cholesteatoma but bone graft placed during the initial surgery to seal a lateral semicircular canal fistula ( fig . the adc values of cholesteatoma group ( 21 ears ) were ranged from 553 to 759 10 mm / s ) , mean sd611.8 51.8 10 mm / s ) and median 595 10 mm / s ) . the adcs of non cholesteatoma group ( 10 ears ) was ranged from 1495.8 to 1766.8 10 mm / s , mean 1638.3 93.5 10 mm / s and median 1653.5 10 mm / s . the cut off value of cholesteatoma is 759 10 mm / s ( table 8) . large recurrence seen in attico - antral region were found in 10 ears , the largest lesion reached 30 mm at ct , 14 mm at mr dwi and 14.4 mm operative size ( fig . 3 ) , medium sized recurrence in 8 cases , while smaller recurrences were seen in the middle ear cavity in 3 ears , reaching 10 mm at ct , 7 mm at mr dwi and 7.3 mm at the operation ( fig . the lesion sizes at ct was not correlated with that of the mr dwi and operative because ct gives apparent size of cholesteatoma including the surrounding granulation tissue while the size at mr dwi and operative was more accurate excluding the surrounding granulation tissue with significant ( p value = 0.001 * ) . granulation tissue , fibrosis and secretions were found in the remaining 10 ears ( fig . good correlation between mr dwi finding and operative confirmation , the sensitivity was 100% , the specificity was 90% , the positive predictive value 95.2% and p value is 0.001 . mdct had low sensitivity , detect only 10 cases out of 30 cases , the sensitivity was 47.6% , specificity 100% , and negative predictive value 47.6% and p value is 0.5 ( table 5 ) . high resolution ct scan and conventional mri have low specificity when it comes to differentiating granulation tissue from relapsing cholesteatoma due to lack of characterization of the detected soft tissue in the surgical cavity , , , , ( fig . our study depict low specificity and accuracy of mdct ( 47.6% ) , this results is near the result of adam et al . who studied 41 patients underwent revision tympanomastoidectomy with sensitivity 50% and specificity 43% for the recurrent well defined mass . ct could not confirm the diagnosis of recurrent cholesteatoma in completely opacified tympanomastoid cavity and could not differentiate between recurrence , residual and granulation tissue . ct has widely been accepted for assessing the extent and location of disease and evaluating complications of cholesteatomas . preoperative imaging is especially important for demonstrating disease in hidden areas ( such as the sinus tympani and facial recess ) and extension of disease into the epitympanum ( attic ) and mastoid antrum , and for detecting normal anatomic variations ( such as an aberrant course of the facial nerve ) . ct is useful if no soft tissue is seen in the middle ear ( or petrous apex or mastoid depending on original location of disease ) . if rounded soft tissue is present , then findings are suggestive of recurrent disease . however , if amorphous soft tissue or complete middle ear opacification is present , ct is nonspecific and can not distinguish granulation tissue or scar tissue from recurrent disease . the importance of diffusion - weighted mr imaging in the detection of recurrent cholesteatoma has been reported in several previous studies , . cholesteatoma has high signal intensity on diffusion - weighted images obtained with b factors of 800 or 1000 s / mm , whereas granulation tissue has low signal intensity . several interpretations to explain the high signal intensity of cholesteatoma on diffusion - weighted images have been proposed . two mechanisms have been discussed for this high signal intensity : restricted molecular diffusion and t2 shine - through effects . the reason for the increased signal intensity of cholesteatoma on diffusion - weighted mr images is still unknown but seems to be similar to that for the increased signal intensity of epidermoid cysts on diffusion - weighted images , , . reduced diffusion in cholesteatoma compared with granulation tissue , fibrous tissue , or mucoid secretion is assumed to contribute to high signal intensity of cholesteatoma on diffusion - weighted imaging . in the present study , diffusion - weighted mr imaging depicted 21ears of surgically proven 20 patients with cholesteatoma . in our series of cases , we did not have any false negative cases but one false positive case in which bone graft used to seal a lateral semicircular canal fistula . this can be explained by the absence of infection in the surgical cavity as all patients with ear drainage received antimicrobials both local and systemic before imaging . this can help eliminate the false positive results that can be met with due to the presence of abscess or infection within the surgical cavity . artifacts from air - bone and air - tissue interfaces are one of the main drawbacks of dw mri . fast se dw sequences proved better in reducing artifacts observed with echo - planner dw sequences , giving a better spatial resolution and better analysis of the lesions . in recent literature , aikele et al . reported a sensitivity of 77% ( 10/13 ) in detecting residual or recurrent cholesteatoma using the combination of standard mr imaging sequences and dwi missing three small residual cholesteatoma pearls ( < 5 mm ) . specificity , positive and negative predictive values of 100% , 100% and 75% was reported . another recent report by stasolla et al . reported a sensitivity of 86% ( 6/7 ) detecting recurrent cholesteatoma using echo - planar dwi . in their series only one small cholesteatoma of 2 mm was missed , while the size of the other cholesteatomas varied from 4 to 14 mm . specificity , positive and negative predictive values of 100% , 100% and 92% were reported . in both studies , included patients were clinically suspected of having residual of recurrent cholesteatoma although the results of previous studies , have proved that comparison of diffusion - weighted images obtained with a b of 800 s / mm without measurement of the apparent diffusion coefficient is sufficient for analysis of the diffusion - weighted sequence we try to assess the benefit of adding adc values in detecting recurrent cholesteatoma . the current study showed a significant difference in adcs between postoperative recurrent cholesteatoma ( mean 611.8 51.8 ) and non - cholesteatoma patients ( mean 1638.3 93.5 ) , with the adc value of cholesteatoma significantly lower than that of non - cholesteatoma . at an optimum adc cut off value of 759 10 mm / s , it was possible to detect cholesteatoma with good diagnostic performance . this method had good sensitivity and marginally higher specificity with the subjective qualitative method when cholesteatoma demonstrated a signal on the dw images . maheshwari concluded the adc of a case report of cholesteatoma patient as 0.58 10 mm / s . detect the concordance between the surgical findings and the calculated adc value of cholesteatoma and suggests that adc values could be used to allow greater specificity to differentiate cholesteatoma from middle ear abscess or mixed - pattern lesions . our results are matched with ravei k et al . who concluded that patient with cholesteatoma were found to have much lower adcs ( median , 707 10 mm / s ; interquartile range , 539858 10 mm / s ; p < 0.001 ) compared with those that did not have cholesteatoma ( median , 1849 10 mm / s ; interquartile range , 15741982 10 mm / s ; p < 0.001 ) . our results also concordant with dr.n.kailasanathan study , who stated that most of cholesteatomas shows adc values higher than 0.55 however infected cholesteatoma can show low adc value . dw mri has 100% sensitivity , 75% specificity , 97.3% ppv and 100% npv in detecting cholesteatoma the main limitation of in the sensitivity of the subjective qualitative method is with small recurrent cholesteatoma less than 5 mm , which do not demonstrate a signal high enough to be detected on the dw imaging images . if cholesteatoma is too small to be detected , adc values can not be measured or used as a diagnostic tool . however , when an abnormal soft tissue demonstrates a dw imaging signal , our study shows that the use of adc values can not only detect cholesteatoma with a high sensitivity , but can also improve the confidence levels of the qualitative method when used together . although specificity was 100% and positive predictive value 95.2% in our study , diffusion - weighted imaging may potentially cause false positive results . high signal intensity in the area of the petrous bone is seen not only with cholesteatomas . susceptibility artifacts at the air - bone border at the base of the skull , chordomas , cholesterol granulomas , and abscesses may also appear markedly hyperintense on diffusion - weighted imaging . care should be taken to be aware of the surgical procedures used during the initial surgery before analyzing mr images , especially when a special material such as bone powder has been used . in conclusion , the advances in mri techniques are currently changing the preoperative evaluation and the postoperative follow - up protocols for cholesteatoma . dw mri appears to be an accurate method , as opposed to a standard second - look operation , for the follow - up of patients who have undergone a canal wall up procedure for a chronic otitis media with cholesteatoma . dw mri fused with ct is a helpful and effective method for distinguishing cholesteatoma from other soft tissues in post - operative ears . mr dwi sensitivity was 100% and negative predictive value was 100% , which means that patients who show no signs of recurrent cholesteatoma on dw fast se images may not need second- or third - look surgery . fusion imaging using hrct and dw mri appears to be a promising technique for both the diagnosis and precise localization of cholesteatomas . the apparent diffusion coefficient ( adc ) of postoperative middle ear cholesteatoma is significantly lower than that of noncholesteatomatous tissue . nasr mohamed m. osman : data collection , technique , image revision , interpretation and final editing .

objectiveto evaluate the accuracy and sensitivity of diffusion - weighted magnetic resonance imaging with adc value combined with mdct in evaluating recurrent cholesteatoma.patientsthirty patients ( 20 females and 10 males ) , their age ranged from 10 to 40years , had undergone a tympanomastoid surgery for a cholesteatoma of the middle ear underwent mdct and mr dwi examination before second- or third - look surgery from may 2015 to october 2016.resultsct showed partial opacification of the tympanomastoid cavity in 10 ears and complete opacification in 21 ears . ct detects 10 cases out of 20 cases of recurrent cholesteatoma with sensitivity 47.6% , specificity 100% , and npp 47.6% . dwi depicted 21 out of 20 cases proved cholesteatoma patients ( sensitivity 100% , specificity 90% , ppv 95.2% and p value is 0.001 ) . all mri of patients without cholesteatoma were correctly interpreted as showing negative findings for cholesteatoma ( specificity = 100% ) . the adc of cholesteatoma group ( 21 ears ) were ranged from 553 to 759 103 mm2/s and the adcs of non cholesteatoma group ( 10 ears ) was ranged from 1495.8 to 1766.8 103 mm2/s . cut off value of cholesteatoma is 759 103 mm2/s.conclusionmr dwi with adc combined with mdct has high sensitivity , specificity , accuracy in detecting recurrent cholesteatoma .

Introduction Patients and methods All patients are subjected Imaging evaluation Results Demographic and clinical data MDCT findings ( MRI and MR DWI findings ( CECs values: MR DWI, MDCT versus operative diagnosis of recurrent cholesteatoma Discussion Conclusion Conflict of interest Funding Author contributions

middle ear cholesteatoma is a benign cystic lesion of aggressive behavior which requiring prompt surgical treatment to prevent local and intracranial complications . surgery is the mainstay treatment of cholesteatoma however , the risk of residual cholesteatoma and recurrence occurs in a relatively high numbers of patients , whatever the type of surgery used , . the detection of cholesteatoma in patients who have undergone middle ear surgery is often difficult for the follow - up of patients operated for cholesteatoma . high resolution computerized tomography ( ct ) scan is considered the basic method for imaging the nonoperated middle ear and can be complemented by magnetic resonance imaging ( mri ) , , , . diffusion - weighted ( dw ) sequences are highly promising in differentiating recurrent cholesteatoma from granulation tissue dw mri depends on the difference in diffusion of water molecules in different biological tissues . adcs , have not yet been assessed as a diagnostic criterion for the detection of recurrent middle ear cholesteatoma with no clear - cut values exist for differentiation of recurrent cholesteatoma from granulation tissue or fibrosis , , , , . in this study , we aim to determine the diagnostic accuracy of dwi and the difference between the adcs of postoperative middle ear cleft cholesteatoma and those of non - cholesteatomatous tissue on single - shot echo planar dw images . we included 30 consecutive patients in our study , previously subjected to tympanomastoid surgery for the treatment of cholesteatoma with clinical and ct suspicion of recurrence and scheduled for second look operation , during the period from may 2015 to october 2016 . twenty patients were females and 10 were males , with an age ranging from 9 to 45 years old ( mean = 27 years ) . positive cases of recurrent cholesteatoma were referred for second - look surgery , while ve cases received medical treatment . inclusion criteria include : patient with history of tympano - mastoid surgery for cholesteatoma with clinical suspicion of recurrent cholesteatoma or infection during follow - up . the following sequences were acquired on the middle ear using an 8 channel head coil and applying the parallel sense imaging is used to obtain better resolution , faster dynamic scans and to reduce susceptibility artifacts . the following sequences were acquired on the middle ear.1.3d ciss ( constructive interference in steady state ) with a gradient echo component tilt angle : 70 , slice thickness : 0.7 mm , repetition time ( tr ) : 11.5 ms , echo time ( te ) : 5.75 ms , sense factor 2 , fov : 230 mm . acquisition time 3 min and 50 s.2.axial and coronal echo planar single shot turbo spin echo diffusion - weighted imaging : 2.5 mm thick axial slices , te : 98 ms , tr : 2000 ms , sense factor 2 , diffusion factor : b 1000 s / mm , fov : 230 mm . acquisition time 3 min and 50 s. axial and coronal echo planar single shot turbo spin echo diffusion - weighted imaging : 2.5 mm thick axial slices , te : 98 ms , tr : 2000 ms , sense factor 2 , diffusion factor : b 1000 s / mm , fov : 230 mm . acquisition time 3 min and 12 s. the adc value of the cholesteatoma was calculated on the adc map from the selected region of interest ( roi ) that matched the lesion . if the lesion is of small size , the mean adc of the lesion was taken from rois on the two contiguous sections . interpretation was indeterminate , whenever complete opacification of the tympano - mastoid cavity . on mri sequences , the diagnosis of cholesteatoma was based on identifying area of restricted diffusion in the form of marked hyperintense signal in comparison with brain tissue on the b = ( 800 , 1000 ) images of diffusion - weighted . the sensitivity , specificity , positive and negative predictive values of different mr sequences used and adcs , was assessed using ssps ( version 12 ) . the following sequences were acquired on the middle ear using an 8 channel head coil and applying the parallel sense imaging is used to obtain better resolution , faster dynamic scans and to reduce susceptibility artifacts . the following sequences were acquired on the middle ear.1.3d ciss ( constructive interference in steady state ) with a gradient echo component tilt angle : 70 , slice thickness : 0.7 mm , repetition time ( tr ) : 11.5 ms , echo time ( te ) : 5.75 ms , sense factor 2 , fov : 230 mm . acquisition time 3 min and 12 s. the adc value of the cholesteatoma was calculated on the adc map from the selected region of interest ( roi ) that matched the lesion . if the lesion is of small size , the mean adc of the lesion was taken from rois on the two contiguous sections . repeated roi measurements were done two to three times on the image for each case , and the median adc was obtained . on ct , recurrent cholesteatoma was diagnosed by identifying the presence of new bone erosion not detected in the preoperative evaluation , the presence of tissue density mass with well - defined borders and an air space between the mass and walls of the tympanic cavity , expansion of the mastoidectomy cavity and erosion of the walls including the tegmen , ossicular chain amputation , or labyrinthine or facial nerve sheath destruction that could not be attributed to past surgery . interpretation was indeterminate , whenever complete opacification of the tympano - mastoid cavity . on mri sequences , the diagnosis of cholesteatoma was based on identifying area of restricted diffusion in the form of marked hyperintense signal in comparison with brain tissue on the b = ( 800 , 1000 ) images of diffusion - weighted . the sensitivity , specificity , positive and negative predictive values of different mr sequences used and adcs , was assessed using ssps ( version 12 ) . this prospective study was conducted in 30 patients with 31ears as one patient had bilateral tympanomastoid surgery for cholesteatoma . we evaluated 30 consecutive patients ( 31 ears ) , 20 were females and 10 males , their age ranged from 945 years , mean age 27 years , who had undergone a canal wall - down mastoidectomy or a canal wall - up mastoidectomy for a cholesteatoma of the middle ear and underwent an mdct and mr dwi examination before second - look operation . the main clinical presentation of the patients were recurrent purulent discharge ( n = 25 ) , perforated tympanic membrane with and without discharge ( n = 3 ) , chronic ear discharge with sign of increase intra cranial tension ( n = 1 ) , chronic discharge with facial paresis ( n = 1 ) . 5 ) and complete opacification in 21 ears ( figs . ct criteria of recurrent cholesteatoma detected in 10 cases in the form of localized nondependent tissue density mass with new bone erosion not detected in previous ct or pre - operative study ( fig . 10 ) , while the remaining 20 cases the mastoiedctomy cavity was completely opacified . the size of cholesteatoma mass lesion ranged from 10 to 30 mm ( mean17.8 5.7 mm ) . the tegmen tympani was eroded in one case , sigmoid sinus plate eroded in one case , tympanic segment of the facial nerve canal was eroded in one case , and lateral mastoid wall was eroded in one case ( fig . the included patients were classified into two groups , ( cholesteatoma group include 20 cases ) and non cholesteatoma group ( include 10 cases ) . based on mr dwi sequence , the cholesteatoma group ( 20 patients with 21 ears ) revealed recurrent cholesteatoma in the form of area of restricted diffusion on dw images at b factor of 800 , 1000 s / mm ( figs . patients without recurrent cholesteatoma ( 10 patients ) , all diffusion - weighted fast se mr images obtained with b of 800,1000 s / mm were correctly interpreted as showing no restriction and were diagnosed as granulation or infection ( fig . the size of the recurrent cholesteatoma mass was ranged from 1030mmat ct , 715 mm at mr dwi and 714 mm at operative finding ( table 9 ) . the adc values of cholesteatoma group ( 21 ears ) were ranged from 553 to 759 10 mm / s ) , mean sd611.8 51.8 10 mm / s ) and median 595 10 mm / s ) . the adcs of non cholesteatoma group ( 10 ears ) was ranged from 1495.8 to 1766.8 10 mm / s , mean 1638.3 93.5 10 mm / s and median 1653.5 10 mm / s . large recurrence seen in attico - antral region were found in 10 ears , the largest lesion reached 30 mm at ct , 14 mm at mr dwi and 14.4 mm operative size ( fig . 3 ) , medium sized recurrence in 8 cases , while smaller recurrences were seen in the middle ear cavity in 3 ears , reaching 10 mm at ct , 7 mm at mr dwi and 7.3 mm at the operation ( fig . the lesion sizes at ct was not correlated with that of the mr dwi and operative because ct gives apparent size of cholesteatoma including the surrounding granulation tissue while the size at mr dwi and operative was more accurate excluding the surrounding granulation tissue with significant ( p value = 0.001 * ) . good correlation between mr dwi finding and operative confirmation , the sensitivity was 100% , the specificity was 90% , the positive predictive value 95.2% and p value is 0.001 . mdct had low sensitivity , detect only 10 cases out of 30 cases , the sensitivity was 47.6% , specificity 100% , and negative predictive value 47.6% and p value is 0.5 ( table 5 ) . we evaluated 30 consecutive patients ( 31 ears ) , 20 were females and 10 males , their age ranged from 945 years , mean age 27 years , who had undergone a canal wall - down mastoidectomy or a canal wall - up mastoidectomy for a cholesteatoma of the middle ear and underwent an mdct and mr dwi examination before second - look operation . 5 ) and complete opacification in 21 ears ( figs . ct criteria of recurrent cholesteatoma detected in 10 cases in the form of localized nondependent tissue density mass with new bone erosion not detected in previous ct or pre - operative study ( fig . the size of cholesteatoma mass lesion ranged from 10 to 30 mm ( mean17.8 5.7 mm ) . the tegmen tympani was eroded in one case , sigmoid sinus plate eroded in one case , tympanic segment of the facial nerve canal was eroded in one case , and lateral mastoid wall was eroded in one case ( fig . based on mri findings the included patients were classified into two groups , ( cholesteatoma group include 20 cases ) and non cholesteatoma group ( include 10 cases ) . based on mr dwi sequence , the cholesteatoma group ( 20 patients with 21 ears ) revealed recurrent cholesteatoma in the form of area of restricted diffusion on dw images at b factor of 800 , 1000 s / mm ( figs . patients without recurrent cholesteatoma ( 10 patients ) , all diffusion - weighted fast se mr images obtained with b of 800,1000 s / mm were correctly interpreted as showing no restriction and were diagnosed as granulation or infection ( fig . the size of the recurrent cholesteatoma mass was ranged from 1030mmat ct , 715 mm at mr dwi and 714 mm at operative finding ( table 9 ) . the adc values of cholesteatoma group ( 21 ears ) were ranged from 553 to 759 10 mm / s ) , mean sd611.8 51.8 10 mm / s ) and median 595 10 mm / s ) . the adcs of non cholesteatoma group ( 10 ears ) was ranged from 1495.8 to 1766.8 10 mm / s , mean 1638.3 93.5 10 mm / s and median 1653.5 10 mm / s . the cut off value of cholesteatoma is 759 10 mm / s ( table 8) . large recurrence seen in attico - antral region were found in 10 ears , the largest lesion reached 30 mm at ct , 14 mm at mr dwi and 14.4 mm operative size ( fig . 3 ) , medium sized recurrence in 8 cases , while smaller recurrences were seen in the middle ear cavity in 3 ears , reaching 10 mm at ct , 7 mm at mr dwi and 7.3 mm at the operation ( fig . the lesion sizes at ct was not correlated with that of the mr dwi and operative because ct gives apparent size of cholesteatoma including the surrounding granulation tissue while the size at mr dwi and operative was more accurate excluding the surrounding granulation tissue with significant ( p value = 0.001 * ) . good correlation between mr dwi finding and operative confirmation , the sensitivity was 100% , the specificity was 90% , the positive predictive value 95.2% and p value is 0.001 . mdct had low sensitivity , detect only 10 cases out of 30 cases , the sensitivity was 47.6% , specificity 100% , and negative predictive value 47.6% and p value is 0.5 ( table 5 ) . ct could not confirm the diagnosis of recurrent cholesteatoma in completely opacified tympanomastoid cavity and could not differentiate between recurrence , residual and granulation tissue . the importance of diffusion - weighted mr imaging in the detection of recurrent cholesteatoma has been reported in several previous studies , . cholesteatoma has high signal intensity on diffusion - weighted images obtained with b factors of 800 or 1000 s / mm , whereas granulation tissue has low signal intensity . several interpretations to explain the high signal intensity of cholesteatoma on diffusion - weighted images have been proposed . the reason for the increased signal intensity of cholesteatoma on diffusion - weighted mr images is still unknown but seems to be similar to that for the increased signal intensity of epidermoid cysts on diffusion - weighted images , , . reduced diffusion in cholesteatoma compared with granulation tissue , fibrous tissue , or mucoid secretion is assumed to contribute to high signal intensity of cholesteatoma on diffusion - weighted imaging . reported a sensitivity of 77% ( 10/13 ) in detecting residual or recurrent cholesteatoma using the combination of standard mr imaging sequences and dwi missing three small residual cholesteatoma pearls ( < 5 mm ) . specificity , positive and negative predictive values of 100% , 100% and 75% was reported . reported a sensitivity of 86% ( 6/7 ) detecting recurrent cholesteatoma using echo - planar dwi . in their series only one small cholesteatoma of 2 mm was missed , while the size of the other cholesteatomas varied from 4 to 14 mm . specificity , positive and negative predictive values of 100% , 100% and 92% were reported . in both studies , included patients were clinically suspected of having residual of recurrent cholesteatoma although the results of previous studies , have proved that comparison of diffusion - weighted images obtained with a b of 800 s / mm without measurement of the apparent diffusion coefficient is sufficient for analysis of the diffusion - weighted sequence we try to assess the benefit of adding adc values in detecting recurrent cholesteatoma . the current study showed a significant difference in adcs between postoperative recurrent cholesteatoma ( mean 611.8 51.8 ) and non - cholesteatoma patients ( mean 1638.3 93.5 ) , with the adc value of cholesteatoma significantly lower than that of non - cholesteatoma . at an optimum adc cut off value of 759 10 mm / s , it was possible to detect cholesteatoma with good diagnostic performance . maheshwari concluded the adc of a case report of cholesteatoma patient as 0.58 10 mm / s . detect the concordance between the surgical findings and the calculated adc value of cholesteatoma and suggests that adc values could be used to allow greater specificity to differentiate cholesteatoma from middle ear abscess or mixed - pattern lesions . who concluded that patient with cholesteatoma were found to have much lower adcs ( median , 707 10 mm / s ; interquartile range , 539858 10 mm / s ; p < 0.001 ) compared with those that did not have cholesteatoma ( median , 1849 10 mm / s ; interquartile range , 15741982 10 mm / s ; p < 0.001 ) . dw mri has 100% sensitivity , 75% specificity , 97.3% ppv and 100% npv in detecting cholesteatoma the main limitation of in the sensitivity of the subjective qualitative method is with small recurrent cholesteatoma less than 5 mm , which do not demonstrate a signal high enough to be detected on the dw imaging images . however , when an abnormal soft tissue demonstrates a dw imaging signal , our study shows that the use of adc values can not only detect cholesteatoma with a high sensitivity , but can also improve the confidence levels of the qualitative method when used together . although specificity was 100% and positive predictive value 95.2% in our study , diffusion - weighted imaging may potentially cause false positive results . susceptibility artifacts at the air - bone border at the base of the skull , chordomas , cholesterol granulomas , and abscesses may also appear markedly hyperintense on diffusion - weighted imaging . dw mri appears to be an accurate method , as opposed to a standard second - look operation , for the follow - up of patients who have undergone a canal wall up procedure for a chronic otitis media with cholesteatoma . mr dwi sensitivity was 100% and negative predictive value was 100% , which means that patients who show no signs of recurrent cholesteatoma on dw fast se images may not need second- or third - look surgery . the apparent diffusion coefficient ( adc ) of postoperative middle ear cholesteatoma is significantly lower than that of noncholesteatomatous tissue .

diabetes mellitus ( dm ) is responsible for 1% of worldwide blindness and is the leading cause of vision loss among working aged individuals in industrialized countries.13 diabetes adversely affects all parts of the eyes and visual pathways , but most vision loss results from diabetic retinopathy ( dr).4 a subclinical retinal neuropathy is the earliest manifestation of dr , and retinal vascular abnormalities due to progressive capillary endothelial cell damage frequently follow . capillary closure results in retinal ischemia , which in severe cases produces retinal neovascularization and proliferative dr . severe fibrovascular proliferation with traction retinal detachment and vitreous hemorrhage is the most common cause of severe vision loss among diabetics , but moderate vision loss from diabetic macular edema ( dme ) occurs more commonly.5 dme affects approximately 7.5% of diabetics ( 750,000 people in the united states).6 among type 1 diabetics , 0% have dme at 5 years after being diagnosed with dm and 29% have it by 20 years , whereas among type 2 diabetics , dme affects 3% at 5 years and 28% at 20 years.7,8 other studies suggest that the 10-year incidence of dme varies from 20% to 40% , depending upon the patient s age and the type and severity of the diabetes.9 risk factors for dme include male sex , duration of diabetes , poor glucose control , use of insulin , diuretic use , systemic arterial hypertension , cardiovascular disease , hyperlipidemia , proteinuria , impaired renal function , and vitreomacular traction.1012 risk factors for dme , such as serum lipid concentrations , may be different from those responsible for the development of dr.13,14 the incidence of blindness due to neovascular age - related macular degeneration ( amd ) is falling because of the successful implementation of anti - vascular endothelial growth factor ( vegf ) therapy.15 since the worldwide prevalence of dm is expected to increase from 4.0% in 1995 to 5.4% in 2025,16 with the number of patients projected to reach 430 million by 2030,17 the number of patients affected with dr and dme will increase significantly , thereby shifting the epidemiologic focus from amd to dr . improved treatment of dme with advanced pharmacotherapies will be critical in the fight against a diabetes - induced epidemic of worldwide blindness . this paper discusses the efficacy and safety of aflibercept , the newest anti - vegf drug , in the treatment of dme . dme represents a collection of fundus abnormalities microaneurysms , hemorrhages , and exudates , with associated thickening of the macula.18 for most of the past three decades , center - threatening and center - involving dme referred to as clinically significant macular edema ( csme ) has been diagnosed by binocular examination of the macula.19 the recent introduction of optical coherence tomography ( oct ) together with the widespread availability of anti - vegf drugs for center - involving dme has made the use of the early treatment of diabetic retinopathy study ( etdrs ) classification system less useful and the current classification of dme is generally limited to center - involving or not center - involving edema . intraretinal fluid accumulation may reversibly decrease vision in the short term , but over longer time periods it causes permanent loss of vision.20 to combat dme - related vision loss , the early treatment of diabetic retinopathy study established focal / grid laser photocoagulation as the gold standard for the treatment of csme.21 eyes with csme had a 32% risk of moderate vision loss over 3 years , but this was reduced by 50% with the timely application of laser.22,23 unfortunately , only 3% of patients in the etdrs trial improved by 15 letters , although subsequent analyses showed that 30% of eyes originally worse than 20/32 improved by 10 letters , with an average gain of + 4 letters . in a more recent drcr.net trial , 15% of laser - treated patients experienced 15 letter improvements.24 the reason for the laser s efficacy remains unknown , but it may be due to improved retinal oxygenation.25 laser decreases hypoxia in an animal model of retinal vein occlusion,26 and supplemental oxygen has been shown to decrease dme in human subjects.27 unfortunately , vision gains following macular laser are frequently disappointing , and photocoagulation may be complicated by laser creep and choroidal neovascularization,28 both of which decrease visual acuity in the long term . retinal barrier by phosphorylation of junctional proteins represents a key event in the development of dme,29,30 but despite a voluminous body of clinical knowledge regarding the formation and treatment of dme , the molecular trigger for its development long remained unknown . michaelson31 postulated the existence of an intraocular substance that promoted vascular growth and folkman32 proposed that a soluble vasoproliferative molecule was necessary for tumor growth . critical advances in our understanding of ocular neovascularization began with the discovery ( in 1983 ) of vascular permeability factor ( vpf)33 and the subsequent discovery of vegf ( in 1989 ) by two independent research groups.34,35 protein sequencing showed that vpf and vegf were identical molecules , thereby enabling scientists to focus their initial development efforts on a single molecular target . vegf is a dimeric glycoprotein with a molecular weight of 3646 kda that segregates into seven families : vegf - a , vegf - b , vegf - c , vegf - d , vegf - e , vegf - f , and placental growth factor ( plgf).36 isoforms of vegf - a , of which there are at least six major ( vegf121 , vegf145 , vegf165 , vegf183 , vegf189 , and vegf206 ) and eight minor,37,38 are the most important promoters of intraocular neovascularization and hyperpermeability . diffusible vegf binds to and dimerizes three transmembrane receptors ( vegfr1 , vegfr2 , and vegfr3),36 and although vegfr1 binds vegf165 with greater affinity , vegfr2 regulates the blood retinal barrier and controls endothelial cell mitogenesis.39 vegf upregulation occurs largely in response to localized oxidative stress with stabilization of hypoxia inducible factor-1.40 several cells within the retina produce vegf ( capillary endothelial cells , pericytes , pigment epithelial cells , neurons , and astrocytes)41,42 and though all cell types respond to vegf , the capillary endothelial cell is its primary target . blocking vegf with ranibizumab restores claudin-1 levels within 24 hours.44 plgf upregulation occurs in diabetic eyes,45 but its role in the development of dr remains unclear . retinal barrier in vitro,44 but animal models suggest that pigf plays a critical role in the development of dr.46 genetic deletion of pigf in a diabetic mouse strain prevents diabetes - induced retinal cell death , capillary degeneration , pericyte loss , and blood retinal barrier breakdown.47 several lines of evidence implicate vegf in the development of dr . intravitreal injections of vegf produce the characteristic findings of dr ( microaneurysms , hemorrhages , macular edema , and neovascularization),48,49 and elevated intraocular vegf levels have been detected in eyes with active dme.50 aqueous vegf concentrations in patients with dme are three times those in the plasma,51 and aqueous levels correlate with dme severity.51 laser remained the standard of care for center - involving dme for over two decades , but investigators continually sought more effective therapies . intensive research and innovative drug development produced five drugs ( pegaptanib , macugen , eyetech , new york , ny , usa ; bevacizumab , avastin , genentech , south san francisco , ca , usa / roche , basel , switzerland ; ranibizumab , lucentis , genentech / roche ; aflibercept , eylea , regeneron , tarrytown , ny , usa ; and conbercept , chengdu kanghong biotech , chengdu , people s republic of china ) that specifically bind diffusible vegf . pegaptanib ( an aptamer to vegf165 ) improved bcva better than sham / laser ( 10 letter improvement : 34% versus 10% , p=0.003 ; mean change in bcva : + 6.1 versus + 1.3 letters ) in a multicenter phase ii trial.52 patients receiving bevacizumab in the bevacizumab or laser therapy in the management of diabetic macular edema ( bolt ) trial improved by a mean of + 8.6 etdrs letters compared to 0.5 letters for those treated with laser.53 small pilot studies of ten patients each showed that ranibizumab decreased dme and improved bcva.54,55 the value of anti - vegf therapy in the treatment of dme emerged from the phase ii ranibizumab trials ( resolve and read-2)5659 and was solidified in several phase iii trials ( restore , drcr.net protocol i , and rise / ride).6064 the rise and ride trials demonstrated that monthly injections of ranibizumab produced 2-year bcva improvements of approximately + 10 letters and accelerated a shift toward establishing intravitreal anti - vegf injections as first - line therapy for center - involving dme . unfortunately , treatment regimens that rely on monthly clinic visits and injections challenge patients compliance and signal the need for strategies with lower injection frequencies . aflibercept , previously referred to as the vegf - trap , is a 115 kd , recombinant , high - affinity , vegf - binding fusion protein . it contains all human protein sequences with the second extracellular binding domain from vegfr1 and the third extracellular binding domain from vegfr2 fused to the fc fragment of a human immunoglobulin igg molecule.65 aflibercept attaches to the receptor binding sites of all isomers of vegf - a , vegf - b , and plgf with a vegf165 binding affinity ( 0.45 pm ) that is 100-fold greater than ranibizumab and bevacizumab.66 this tenacious attachment results from its favorable three - dimensional configuration that brings each of its fab binding segments into contact with each vegf subunit , thereby creating a nearly irreversible two - fisted grasp.67 in capillary endothelial cell assays , aflibercept inhibits cellular migration and calcium uptake 10126 times more than ranibizumab and bevacizumab.66 aflibercept has an intravitreal half - life of 4.7 days in rabbits68 longer than either ranibizumab ( 2.88 days)69 or bevacizumab ( 4.32 days)70 but its half - life in human eyes has not been determined . pharmacokinetic models suggest that the intraocular half - life of aflibercept in human eyes is intermediate between that of ranibizumab and bevacizumab ( approximately 9 days).71 after intravitreal injection , aflibercept ( unaltered ) passes into the systemic circulation where its half - life is approximately 6 days . aflibercept binds plasma vegf and lowers serum concentrations to below 10 pg / ml ( the lower detectable limit of some assays ) for at least 7 days.72 important findings from the key aflibercept dme trials are detailed in table 1 . a phase i study25 assessed the safety and efficacy of aflibercept in five patients with dme . subjects with central subfield thickness ( cst ) > 250 m and bcva from 20/32 to 20/320 were enrolled . single injections of 4 mg aflibercept were administered , followed by a 6-week observation period . the most common ocular complications , minor irritation and conjunctival injection , were unrelated to the injections . by 4 weeks after the injections , the mean bcva improved by 9 letters and the mean cst improved by 49 m . at 6 weeks , four of five patients had mean cst improvements of 74 m ( p=0.0625 ) and four of five experienced improved bcva ( median of 3 letters ) . fluorescein angiography at 6 weeks showed no change in leakage in three patients and decreased leakage in two others . the authors concluded that a single injection of aflibercept was well tolerated and further studies in patients with dme were warranted . the phase ii dme and vegf trap - eye : investigation of clinical impact ( da vinci ) study73 determined whether different doses and dosing intervals of aflibercept are superior to laser . the 52-week ( primary endpoint at 24 weeks ) , multi - center , randomized , double - masked trial enrolled 221 patients ( 200 completed the trial ) from the united states , canada , and austria . major inclusion criteria included crt > 250 m and bcva from 73 to 24 etdrs letters ; major ocular exclusion criteria included previous macular or panretinal laser photocoagulation , and intravitreal corticosteroids or antiangiogenesis drugs administered within 3 months of screening . systemic exclusionary criteria included uncontrolled arterial hypertension , renal failure requiring dialysis , or a thromboembolic event within the previous 6 months . patients were randomized to five treatment groups : 0.5 mg aflibercept q4wk , 2 mg q4wk , 2 mg q4wk 3 followed by q8wk , 2 mg q4wk 3 followed by pro re nata ( prn ) , and laser photocoagulation with sham injections . patients in the prn arm were eligible for repeat intravitreal injections if the crt was > 250 m , the crt increased by > 50 m compared to the previous least measurement , or the bcva decreased by 5 etdrs letters from the previous measurement with any accompanying increase in crt . patients in the laser / sham group were eligible for repeat laser every 16 weeks if csme was detected . patients in the aflibercept groups experienced average gains of + 8.5 to + 11.4 etdrs letters compared to + 2.5 letters in the laser group ( p<0.0085 for each aflibercept group compared to laser ) . the 2 mg q8wk group gained fewer letters than the q4wk group , but the groups experienced different bcva gains after the first injections , suggesting that differences in the composition of the enrolled groups rather than the treatment regimens were responsible . gains of + 0 , + 10 , and + 15 letters were seen in up to 93% , 64% , and 34% of eyes in the aflibercept groups compared to 68% , 32% , and 21% of eyes in the laser group . mean changes in crt ranged from 127.3 to 194.5 m in the aflibercept groups to only 67.9 m in the laser arm ( p=0.0066 for each aflibercept group versus laser ) . patients in the 2 mg prn arm received a mean of 1.5 ( out of a possible 3 ) injections during the prn phase and patients in the laser arm received a mean of 1.7 ( out of a possible 2 ) procedures . aflibercept was well tolerated with adverse event rates similar to those seen in other anti - vegf trials . four patients ( all receiving aflibercept ) developed severe systemic arterial hypertension , though all carried previous diagnoses of hypertension . the main 52-week outcomes of the da vinci trial74 were the proportion of patients improving by 15 letters bcva and the mean improvements in crt . the proportion of eyes gaining 15 letters was 40.9% , 45.5% , 23.8% , and 42.2% respectively , compared to 11.4% for the laser group . the mean changes in bcva in the aflibercept groups increased from + 9.7 to + 13.1 letters compared to 1.3 letters for the laser group ( p<0.0001 versus laser ) . mean changes in crt were 165.4 to 227.4 m versus 58.4 m . dr severity scores improved in 40% , 31% , 64% , and 32% of patients respectively in the aflibercept groups but in only 12% of patients in the laser group ; dr worsening was seen in 0%13% of eyes treated with aflibercept and in 24% of eyes treated with laser / sham . patients receiving 2 mg q8wk and 2 mg prn received similar numbers of injections ( 7.2 and 7.4 ) as those in the restore trial ( 7).60 eyes receiving aflibercept were eligible for rescue laser beginning at week 24 . the mean number of lasers given to patients randomized to aflibercept was less than 1 , whereas patients in the laser / sham group received a mean of 2.5 lasers . the incidence of endophthalmitis ( 2% ) was similar to that in the resolve trial.56 the study of intravitreal administration of vegf trap - eye in patients with dme ( vista ; nct01363440 ) and the vegf trap - eye in vision impairment due to dme ( vivid ; nct01331681 ) trials75 were similarly designed , double - blind , randomized , phase iii trials that enrolled 872 patients ( eyes ) ( vista : 466 ; vivid : 406 ) with center - involving dme . vista - dme was run in the united states , whereas vivid - dme was run in australia , europe , and japan . eligible patients were type i or ii diabetics with bcva of 7324 letters ( 20/4020/320 ) and crt thickening on oct . eyes were randomized 1:1:1 to receive intravitreal aflibercept injection ( iai ) 2 mg q4wk , iai 2 mg q8wk after 5 monthly loading doses , or laser photocoagulation / sham injection . patients were eligible for laser retreatment every 12 weeks if etdrs - defined edema was present . all study eyes were eligible for additional ( rescue ) treatment beginning at 24 weeks if they lost 10 letters of bcva on two consecutive visits or 15 letters at any visit from the previous best measurement , and bcva was worse than baseline . for laser - treated eyes , additional treatment consisted of 5 monthly doses of 2 mg iai , followed by injections every 8 weeks , and for iai - treated eyes , active laser therapy was performed . the primary temporal endpoint was at 52 weeks , but patients receiving iai will be treated through 148 weeks . patients randomized to laser / sham will be eligible to crossover to iai during year 3 . secondary efficacy endpoints included the proportion of patients gaining 15 letters , the proportion of patients gaining 10 letters , the proportion of eyes experiencing a two - step improvement in the etdrs diabetic retinopathy severity scale ( drss ) score , the mean changes in central retinal thickness ( crt ) as measured by oct , the change from baseline in the national eye institute visual function questionnaire-25 ( nei vfq-25 ) near activities subscale score , and the change from baseline in the nei vfq-25 distance activities subscale score . vista enrolled a greater proportion of black patients and vivid enrolled a greater proportion of asian patients . more eyes in vista , compared to vivid , had previously received anti - vegf injections ( 42.9% versus 8.9% ) . mean bcva changes from baseline to 52 weeks for the groups receiving iai 2 mg q4wk , iai 2 mg q8wk , and laser / sham were + 12.5 , + 10.7 , and + 0.2 letters ( p<0.0001 ) in vista and + 10.5 , + 10.7 , and + 1.2 letters ( p<0.0001 ) in vivid . when eyes receiving additional therapy were included in the analysis , those in the iai groups changed by + 10.7 to + 12.4 letters from baseline , whereas those in the laser groups changed by + 4.2 and + 3.5 letters . visual acuity gains were significantly greater in the iai groups in both patients who had and had not received prior anti - vegf therapy . the corresponding proportions improving by 10 letters were 64.9% , 58.3% , and 19.5% respectively ( p<0.0001 ) in vista and 54.4% , 53.3% , and 25.8% respectively ( p<0.0001 ) in vivid . the corresponding proportions improving by 15 letters were 41.6% , 31.1% , and 7.8% ( p<0.0001 ) in vista and 32.4% , 33.3% , and 9.1% ( p<0.0001 ) in vivid . the corresponding proportions that lost 15 letters were 0.6% , 0.7% , and 9.1% respectively ( p<0.0001 ) in vista and 0.7% , 0% , and 10.6% respectively ( p<0.0001 ) in vivid . compared to laser , most patients receiving iai did not lose any letters from baseline : 94.2% , 92.7% , and 57.1% in vista , and 94.1% , 91.9% , and 62.9% ( p<0.0001 ) in vivid . significantly more patients treated with iai q4wk and q8wk than laser experienced a two - step improvement in drss in both vista ( 33.8% and 29.1% versus 14.3% ) and vivid ( 33.3% and 27.7% versus 7.5% ) . mean changes in crt were 185.9 , 183.1 , and 73.3 m in vista and 195.0 , 192.4 , and 66.2 m in vivid . the mean sd in nei vfq-25 scores for the iai q4wk groups were significantly different from the laser groups only for the near activities subscale scores in vista ( 9.020.6 versus 5.420.4 ; p=0.0168 ) . for patients treated with laser / sham , the mean numbers of procedures were 2.7 and 2.1 in vista and vivid , respectively . more patients in the laser group than the iai groups received additional ( rescue ) therapy ( vista : 31.2% versus 0.7% and 2.6% ; vivid : 24.1% versus 4.4% and 8.1% ) . incidences of ocular and nonocular adverse events and serious adverse events including anti - platelets trialists collaborative defined vascular events and deaths were similar among all groups . serious nonocular adverse events were uncommon ( hypertension : 9.7% ; cerebrovascular accidents : 1.1% ; and myocardial infarction : 1.1% ) . incidences of intraocular inflammation were 0.2% ( 4/1,832 injections ) , 0.1% ( 1/1,284 injections ) , and 0.5% ( 1/212 injections ) in vista and 0.2% ( 4/1,566 injections ) , 0.4% ( 5/1,186 injections ) , and 0.7% ( 1/135 injections ) in vivid . the incidences of congestive heart failure and anemia were higher in the aflibercept groups , and the incidences of myocardial infarction and osteoarthritis were higher in the laser groups . the total numbers of vascular deaths were 2 , 2 , and 2 , and the total numbers of deaths were 2 , 4 , and 2 due to additional deaths from b - cell lymphoma and lung carcinoma in the 2 mg q8wk group . a bayesian network meta - analysis was used to indirectly compare the literature - reported efficacies of ranibizumab and aflibercept on the treatment of dme . for 10 letter gains , the results slightly favored ranibizumab ( relative risk : 1.59 , 95% credible interval : 0.615.37).76 the only trial to directly compare iai with bevacizumab or ranibizumab for the treatment of dme was the recently reported drcr.net protocol t trial.77 this prospective , comparison trial randomized 660 patients at 89 sites to receive 1.25 mg bevacizumab , 0.3 mg ranibizumab , or 2 mg aflibercept . entry criteria included bcva from 20/32 to 20/320 with center - involving dme by clinical examination and oct . patients were treated every 4 weeks unless the bcva reached 20/20 or better with a cst below the eligibility threshold , or there was no bcva change of 5 letters or more or a 10% change in cst over the past two injections . beginning at week 24 , injections were withheld if the bcva change was < 5 letters and the cst change was < 10% over two injections irrespective of bcva . mean numbers of injections were 9 ( aflibercept ) , 10 ( bevacizumab ) , and 10 ( ranibizumab ) ( p=0.045 ) . laser photocoagulation was performed in 37% , 56% , and 46% of eyes respectively ( p<0.001 ) . mean changes in bcva at 1 year were + 13.3 letters ( aflibercept ) , + 9.7 letters ( bevacizumab ) , and + 11.2 letters ( ranibizumab ) ( p<0.001 : aflibercept versus bevacizumab ; p=0.03 : aflibercept versus ranibizumab ) . subgroup analysis was critical in uncovering significant differences in efficacy among the drugs . for eyes with baseline bcva of 20/32 to 20/40 , mean changes were + 8.0 ( aflibercept ) , + 7.5 ( bevacizumab ) , and + 8.3 letters ( ranibizumab ) . when baseline va was 20/50 , mean changes in bcva were + 18.9 ( aflibercept ) , + 11.8 ( bevacizumab ) , and + 14.2 letters ( ranibizumab ) . only two eyes developed endophthalmitis . there were no significant differences in the rates of serious adverse events ( p=0.40 ) , hospitalization ( p=0.51 ) , death ( p=0.72 ) , or major cardiovascular events . aflibercept has been approved by the united states food and drug administration ( fda ) for the treatment of neovascular amd and macular edema due to retinal vein occlusions . the fda also approved aflibercept for the treatment of center - involving macular edema due to dme ( 2014 ) and dr with associated dme ( 2015 ) . in 2014 , the european union approved aflibercept for the treatment of dme . ongoing aflibercept trials for the treatment of dr include vivid - japan , a phase iii , open - label study evaluating the safety and tolerability of intravitreal aflibercept in japanese patients with dme , the act trial , a two - dose trial evaluating the effects of intravitreal aflibercept on proliferative dr , and drcr.net protocol v , that compares aflibercept with laser photocoagulation for eyes with dme but excellent bcva . apart from the recently published drcr.net protocol t trial,77 no randomized trials have compared aflibercept with other anti - vegf drugs for the treatment of dme . physicians will be tempted to compare the results of the pivotal vista / vivid75 trials with other completed phase iii trials ( particularly rise / ride61 ) , but should do so with caution because of differences in patient populations and treatment strategies . vista / vivid enrolled a large asian subpopulation ( 20% ) compared to rise / ride ( 5% ) and also studied an active laser control group ( treated at baseline ) , whereas rise / ride used a sham control group that was eligible for laser only after 3 months . ranibizumab - treated patients in rise / ride were eligible for laser after 3 months , whereas iai - treated patients could not receive laser for at least 24 weeks . patients receiving aflibercept in vista but not vivid reported significant improvements in near visual function on the nei vfq-25 questionnaires . the differences in visual outcomes in vista and vivid were similar , so these reported quality differences may have been due to the different study populations . the north america based vista trial had a significant proportion of african american subjects ( 11.1% ) , whereas the eastern hemisphere - based vivid trial had a large proportion of asian subjects ( 19.3% ) . patients receiving iai q8wk in vivid / vista developed a saw tooth pattern of crt measurements after the five loading doses , but there were no corresponding changes in bcva . the crt patterns suggest that 8 weeks approaches the average effective treatment interval for dme and amd study populations . unfortunately , vivid and vista were not able to determine if this saw tooth pattern of macular thickening causes long - term compromise of macular morphology or visual acuity . physicians should be aware that carefully selected individual patients may be extended to much longer intervals , whereas others will require more frequent injections . study developers required five monthly doses of aflibercept before extending the intertreatment interval to 8 weeks . unfortunately , we do not know if a shorter initiation sequence is sufficient or if a longer sequence is required for optimal results . aflibercept possesses a much longer systemic half - life than ranibizumab , causing serum accumulation and depression of vegf levels after intravitreal injections.72 some investigators worry that aflibercept s longer half - life may increase its risk of vegf - associated vascular occlusive events such as stroke , but pivotal trials with both aflibercept and ranibizumab were underpowered to detect infrequent complications , so results vary and firm conclusions can not be reached . ranibizumab was associated with a dose - dependent increase in stroke rate compared to the sham / laser group in rise / ride , but the results from protocol t suggested that there were no differences in stroke rates among the anti - vegf drugs . treatment guidelines for dme are evolving rapidly and experts frequently disagree on optimal strategies , but the phase iii trials suggest that several principles are reasonable to follow . rise / ride and vista / vivid were designed according to the pharmacokinetic profiles of ranibizumab and aflibercept to optimize visual outcomes . excellent visual results were obtained with monthly ranibizumab injections through 2 years and monthly or bimonthly ( following 5 monthly injections ) injections through 52 weeks . mean improvements in bcva with monthly injections are slightly better than those achieved in the more flexible restore and drcr.net protocol i trials , but comparing the results from these different trials must be done carefully because of their different entry criteria . regimens with monthly injections impose significant hardships upon both patients and physicians , and so the bimonthly injection regimen from vivid / vista provides some relief without compromising visual outcomes . more flexible treatment regimens with reinjections based on visual acuity ( restore ) or visual acuity together with oct findings ( protocol i ) allow for fewer injections while still producing excellent visual outcomes . the need for macular laser photocoagulation in patients receiving monthly anti - vegf injections is not clear . vivid and vista allowed for laser photocoagulation at 6 months in eyes with incomplete responses to pharmacotherapy , but eyes receiving ranibizumab monotherapy in restore had excellent visual acuity results . hopefully , future studies will better characterize the need for laser photocoagulation in patients receiving aflibercept . the aggregate results from protocol t suggest that small but statistically different bcva improvements result from the use of the three anti - vegf drugs , but important additional conclusions were gleaned from the subgroup analyses . for eyes with reasonably good baseline va ( 20/40 ) , monthly injections of bevacizumab , ranibizumab , or aflibercept can be expected to produce excellent improvements in bcva ( approximately + 8 letters ) . for eyes with va 20/50 , monthly aflibercept provides a clear advantage over bevacizumab ( of 7.1 letters ) and ranibizumab ( of 4.7 letters ) . a tiered approach to eyes with dme based on for patients with poor va ( 20/50 ) and increased crt , a cost - effective analysis prior to the completion of vista / vivid and protocol t recommended that intravitreal pharmacotherapy with triamcinolone and less expensive anti - vegf injections ( bevacizumab ) should be considered.78 not surprisingly , less frequently administered ( q8wk ) aflibercept is more cost - effective than monthly injections since it reduces cost by 39% . the recently published results of vista / vivid and particularly protocol t invite an updated analysis . the treatment of eyes with good va ( > 20/32 ) has not been systematically studied with anti - vegf agents , and at this time , the less expensive laser photocoagulation may be the best choice . intravitreal aflibercept is superior to laser photocoagulation for eyes with center - involving dme and may be more effective than bevacizumab and ranibizumab for eyes with bcva 20/50 .

the treatment of center - involving diabetic macular edema ( dme ) has improved because of the proven efficacy of drugs that inhibit the effects of vascular endothelial growth factor ( vegf ) . the newest anti - vegf drug , aflibercept , has recently been approved by the united states food and drug administration for the treatment of center - involving dme and for diabetic retinopathy in eyes with dme . in the pivotal phase iii vista and vivid trials , intravitreal aflibercept 2 mg injections every 4 or 8 weeks ( after 5 monthly loading doses ) produced superior gains in bcva compared to laser / sham injections . in the diabetic retinopathy clinical research network protocol t trial , which featured monthly anti - vegf monotherapy for 6 months , followed by monthly pro re nata anti - vegf injections with laser rescue therapy from months 6 through 12 , aflibercept 2 mg monthly was superior to bevacizumab 1.25 mg and ranibizumab 0.5 mg in eyes with bcva of 20/50 or worse ( aflibercept versus bevacizumab : p<0.001 ; aflibercept versus ranibizumab : p=0.003 ) , but the three regimens were comparable for eyes with va of 20/40 or better . only in the 20/50 or worse subgroup did aflibercept achieve clinical superiority ( > 5 letter difference ) to bevacizumab . each treatment regimen led to significant macular thinning , with aflibercept being superior to bevacizumab in both visual acuity subgroups ( p<0.001 for each ) , but it was not statistically superior to ranibizumab in either group . in diabetic patients , aflibercept has an excellent safety profile that does not appear to differ from laser / sham or other vegf inhibitory drugs .

Introduction Historical perspective and rationale for VEGF blockade Aflibercept structure and biochemistry Clinical trials Analysis and future considerations Conclusion

diabetes mellitus ( dm ) is responsible for 1% of worldwide blindness and is the leading cause of vision loss among working aged individuals in industrialized countries.13 diabetes adversely affects all parts of the eyes and visual pathways , but most vision loss results from diabetic retinopathy ( dr).4 a subclinical retinal neuropathy is the earliest manifestation of dr , and retinal vascular abnormalities due to progressive capillary endothelial cell damage frequently follow . severe fibrovascular proliferation with traction retinal detachment and vitreous hemorrhage is the most common cause of severe vision loss among diabetics , but moderate vision loss from diabetic macular edema ( dme ) occurs more commonly.5 dme affects approximately 7.5% of diabetics ( 750,000 people in the united states).6 among type 1 diabetics , 0% have dme at 5 years after being diagnosed with dm and 29% have it by 20 years , whereas among type 2 diabetics , dme affects 3% at 5 years and 28% at 20 years.7,8 other studies suggest that the 10-year incidence of dme varies from 20% to 40% , depending upon the patient s age and the type and severity of the diabetes.9 risk factors for dme include male sex , duration of diabetes , poor glucose control , use of insulin , diuretic use , systemic arterial hypertension , cardiovascular disease , hyperlipidemia , proteinuria , impaired renal function , and vitreomacular traction.1012 risk factors for dme , such as serum lipid concentrations , may be different from those responsible for the development of dr.13,14 the incidence of blindness due to neovascular age - related macular degeneration ( amd ) is falling because of the successful implementation of anti - vascular endothelial growth factor ( vegf ) therapy.15 since the worldwide prevalence of dm is expected to increase from 4.0% in 1995 to 5.4% in 2025,16 with the number of patients projected to reach 430 million by 2030,17 the number of patients affected with dr and dme will increase significantly , thereby shifting the epidemiologic focus from amd to dr . this paper discusses the efficacy and safety of aflibercept , the newest anti - vegf drug , in the treatment of dme . dme represents a collection of fundus abnormalities microaneurysms , hemorrhages , and exudates , with associated thickening of the macula.18 for most of the past three decades , center - threatening and center - involving dme referred to as clinically significant macular edema ( csme ) has been diagnosed by binocular examination of the macula.19 the recent introduction of optical coherence tomography ( oct ) together with the widespread availability of anti - vegf drugs for center - involving dme has made the use of the early treatment of diabetic retinopathy study ( etdrs ) classification system less useful and the current classification of dme is generally limited to center - involving or not center - involving edema . intraretinal fluid accumulation may reversibly decrease vision in the short term , but over longer time periods it causes permanent loss of vision.20 to combat dme - related vision loss , the early treatment of diabetic retinopathy study established focal / grid laser photocoagulation as the gold standard for the treatment of csme.21 eyes with csme had a 32% risk of moderate vision loss over 3 years , but this was reduced by 50% with the timely application of laser.22,23 unfortunately , only 3% of patients in the etdrs trial improved by 15 letters , although subsequent analyses showed that 30% of eyes originally worse than 20/32 improved by 10 letters , with an average gain of + 4 letters . in a more recent drcr.net trial , 15% of laser - treated patients experienced 15 letter improvements.24 the reason for the laser s efficacy remains unknown , but it may be due to improved retinal oxygenation.25 laser decreases hypoxia in an animal model of retinal vein occlusion,26 and supplemental oxygen has been shown to decrease dme in human subjects.27 unfortunately , vision gains following macular laser are frequently disappointing , and photocoagulation may be complicated by laser creep and choroidal neovascularization,28 both of which decrease visual acuity in the long term . vegf is a dimeric glycoprotein with a molecular weight of 3646 kda that segregates into seven families : vegf - a , vegf - b , vegf - c , vegf - d , vegf - e , vegf - f , and placental growth factor ( plgf).36 isoforms of vegf - a , of which there are at least six major ( vegf121 , vegf145 , vegf165 , vegf183 , vegf189 , and vegf206 ) and eight minor,37,38 are the most important promoters of intraocular neovascularization and hyperpermeability . intravitreal injections of vegf produce the characteristic findings of dr ( microaneurysms , hemorrhages , macular edema , and neovascularization),48,49 and elevated intraocular vegf levels have been detected in eyes with active dme.50 aqueous vegf concentrations in patients with dme are three times those in the plasma,51 and aqueous levels correlate with dme severity.51 laser remained the standard of care for center - involving dme for over two decades , but investigators continually sought more effective therapies . intensive research and innovative drug development produced five drugs ( pegaptanib , macugen , eyetech , new york , ny , usa ; bevacizumab , avastin , genentech , south san francisco , ca , usa / roche , basel , switzerland ; ranibizumab , lucentis , genentech / roche ; aflibercept , eylea , regeneron , tarrytown , ny , usa ; and conbercept , chengdu kanghong biotech , chengdu , people s republic of china ) that specifically bind diffusible vegf . pegaptanib ( an aptamer to vegf165 ) improved bcva better than sham / laser ( 10 letter improvement : 34% versus 10% , p=0.003 ; mean change in bcva : + 6.1 versus + 1.3 letters ) in a multicenter phase ii trial.52 patients receiving bevacizumab in the bevacizumab or laser therapy in the management of diabetic macular edema ( bolt ) trial improved by a mean of + 8.6 etdrs letters compared to 0.5 letters for those treated with laser.53 small pilot studies of ten patients each showed that ranibizumab decreased dme and improved bcva.54,55 the value of anti - vegf therapy in the treatment of dme emerged from the phase ii ranibizumab trials ( resolve and read-2)5659 and was solidified in several phase iii trials ( restore , drcr.net protocol i , and rise / ride).6064 the rise and ride trials demonstrated that monthly injections of ranibizumab produced 2-year bcva improvements of approximately + 10 letters and accelerated a shift toward establishing intravitreal anti - vegf injections as first - line therapy for center - involving dme . it contains all human protein sequences with the second extracellular binding domain from vegfr1 and the third extracellular binding domain from vegfr2 fused to the fc fragment of a human immunoglobulin igg molecule.65 aflibercept attaches to the receptor binding sites of all isomers of vegf - a , vegf - b , and plgf with a vegf165 binding affinity ( 0.45 pm ) that is 100-fold greater than ranibizumab and bevacizumab.66 this tenacious attachment results from its favorable three - dimensional configuration that brings each of its fab binding segments into contact with each vegf subunit , thereby creating a nearly irreversible two - fisted grasp.67 in capillary endothelial cell assays , aflibercept inhibits cellular migration and calcium uptake 10126 times more than ranibizumab and bevacizumab.66 aflibercept has an intravitreal half - life of 4.7 days in rabbits68 longer than either ranibizumab ( 2.88 days)69 or bevacizumab ( 4.32 days)70 but its half - life in human eyes has not been determined . single injections of 4 mg aflibercept were administered , followed by a 6-week observation period . the phase ii dme and vegf trap - eye : investigation of clinical impact ( da vinci ) study73 determined whether different doses and dosing intervals of aflibercept are superior to laser . the 52-week ( primary endpoint at 24 weeks ) , multi - center , randomized , double - masked trial enrolled 221 patients ( 200 completed the trial ) from the united states , canada , and austria . patients were randomized to five treatment groups : 0.5 mg aflibercept q4wk , 2 mg q4wk , 2 mg q4wk 3 followed by q8wk , 2 mg q4wk 3 followed by pro re nata ( prn ) , and laser photocoagulation with sham injections . patients in the laser / sham group were eligible for repeat laser every 16 weeks if csme was detected . patients in the aflibercept groups experienced average gains of + 8.5 to + 11.4 etdrs letters compared to + 2.5 letters in the laser group ( p<0.0085 for each aflibercept group compared to laser ) . the 2 mg q8wk group gained fewer letters than the q4wk group , but the groups experienced different bcva gains after the first injections , suggesting that differences in the composition of the enrolled groups rather than the treatment regimens were responsible . the mean changes in bcva in the aflibercept groups increased from + 9.7 to + 13.1 letters compared to 1.3 letters for the laser group ( p<0.0001 versus laser ) . dr severity scores improved in 40% , 31% , 64% , and 32% of patients respectively in the aflibercept groups but in only 12% of patients in the laser group ; dr worsening was seen in 0%13% of eyes treated with aflibercept and in 24% of eyes treated with laser / sham . the mean number of lasers given to patients randomized to aflibercept was less than 1 , whereas patients in the laser / sham group received a mean of 2.5 lasers . the incidence of endophthalmitis ( 2% ) was similar to that in the resolve trial.56 the study of intravitreal administration of vegf trap - eye in patients with dme ( vista ; nct01363440 ) and the vegf trap - eye in vision impairment due to dme ( vivid ; nct01331681 ) trials75 were similarly designed , double - blind , randomized , phase iii trials that enrolled 872 patients ( eyes ) ( vista : 466 ; vivid : 406 ) with center - involving dme . vista - dme was run in the united states , whereas vivid - dme was run in australia , europe , and japan . eyes were randomized 1:1:1 to receive intravitreal aflibercept injection ( iai ) 2 mg q4wk , iai 2 mg q8wk after 5 monthly loading doses , or laser photocoagulation / sham injection . for laser - treated eyes , additional treatment consisted of 5 monthly doses of 2 mg iai , followed by injections every 8 weeks , and for iai - treated eyes , active laser therapy was performed . patients randomized to laser / sham will be eligible to crossover to iai during year 3 . secondary efficacy endpoints included the proportion of patients gaining 15 letters , the proportion of patients gaining 10 letters , the proportion of eyes experiencing a two - step improvement in the etdrs diabetic retinopathy severity scale ( drss ) score , the mean changes in central retinal thickness ( crt ) as measured by oct , the change from baseline in the national eye institute visual function questionnaire-25 ( nei vfq-25 ) near activities subscale score , and the change from baseline in the nei vfq-25 distance activities subscale score . more eyes in vista , compared to vivid , had previously received anti - vegf injections ( 42.9% versus 8.9% ) . mean bcva changes from baseline to 52 weeks for the groups receiving iai 2 mg q4wk , iai 2 mg q8wk , and laser / sham were + 12.5 , + 10.7 , and + 0.2 letters ( p<0.0001 ) in vista and + 10.5 , + 10.7 , and + 1.2 letters ( p<0.0001 ) in vivid . visual acuity gains were significantly greater in the iai groups in both patients who had and had not received prior anti - vegf therapy . compared to laser , most patients receiving iai did not lose any letters from baseline : 94.2% , 92.7% , and 57.1% in vista , and 94.1% , 91.9% , and 62.9% ( p<0.0001 ) in vivid . for patients treated with laser / sham , the mean numbers of procedures were 2.7 and 2.1 in vista and vivid , respectively . the total numbers of vascular deaths were 2 , 2 , and 2 , and the total numbers of deaths were 2 , 4 , and 2 due to additional deaths from b - cell lymphoma and lung carcinoma in the 2 mg q8wk group . for 10 letter gains , the results slightly favored ranibizumab ( relative risk : 1.59 , 95% credible interval : 0.615.37).76 the only trial to directly compare iai with bevacizumab or ranibizumab for the treatment of dme was the recently reported drcr.net protocol t trial.77 this prospective , comparison trial randomized 660 patients at 89 sites to receive 1.25 mg bevacizumab , 0.3 mg ranibizumab , or 2 mg aflibercept . entry criteria included bcva from 20/32 to 20/320 with center - involving dme by clinical examination and oct . mean changes in bcva at 1 year were + 13.3 letters ( aflibercept ) , + 9.7 letters ( bevacizumab ) , and + 11.2 letters ( ranibizumab ) ( p<0.001 : aflibercept versus bevacizumab ; p=0.03 : aflibercept versus ranibizumab ) . for eyes with baseline bcva of 20/32 to 20/40 , mean changes were + 8.0 ( aflibercept ) , + 7.5 ( bevacizumab ) , and + 8.3 letters ( ranibizumab ) . when baseline va was 20/50 , mean changes in bcva were + 18.9 ( aflibercept ) , + 11.8 ( bevacizumab ) , and + 14.2 letters ( ranibizumab ) . aflibercept has been approved by the united states food and drug administration ( fda ) for the treatment of neovascular amd and macular edema due to retinal vein occlusions . the fda also approved aflibercept for the treatment of center - involving macular edema due to dme ( 2014 ) and dr with associated dme ( 2015 ) . in 2014 , the european union approved aflibercept for the treatment of dme . ongoing aflibercept trials for the treatment of dr include vivid - japan , a phase iii , open - label study evaluating the safety and tolerability of intravitreal aflibercept in japanese patients with dme , the act trial , a two - dose trial evaluating the effects of intravitreal aflibercept on proliferative dr , and drcr.net protocol v , that compares aflibercept with laser photocoagulation for eyes with dme but excellent bcva . apart from the recently published drcr.net protocol t trial,77 no randomized trials have compared aflibercept with other anti - vegf drugs for the treatment of dme . physicians will be tempted to compare the results of the pivotal vista / vivid75 trials with other completed phase iii trials ( particularly rise / ride61 ) , but should do so with caution because of differences in patient populations and treatment strategies . the differences in visual outcomes in vista and vivid were similar , so these reported quality differences may have been due to the different study populations . patients receiving iai q8wk in vivid / vista developed a saw tooth pattern of crt measurements after the five loading doses , but there were no corresponding changes in bcva . ranibizumab was associated with a dose - dependent increase in stroke rate compared to the sham / laser group in rise / ride , but the results from protocol t suggested that there were no differences in stroke rates among the anti - vegf drugs . treatment guidelines for dme are evolving rapidly and experts frequently disagree on optimal strategies , but the phase iii trials suggest that several principles are reasonable to follow . mean improvements in bcva with monthly injections are slightly better than those achieved in the more flexible restore and drcr.net protocol i trials , but comparing the results from these different trials must be done carefully because of their different entry criteria . the need for macular laser photocoagulation in patients receiving monthly anti - vegf injections is not clear . vivid and vista allowed for laser photocoagulation at 6 months in eyes with incomplete responses to pharmacotherapy , but eyes receiving ranibizumab monotherapy in restore had excellent visual acuity results . the aggregate results from protocol t suggest that small but statistically different bcva improvements result from the use of the three anti - vegf drugs , but important additional conclusions were gleaned from the subgroup analyses . for eyes with reasonably good baseline va ( 20/40 ) , monthly injections of bevacizumab , ranibizumab , or aflibercept can be expected to produce excellent improvements in bcva ( approximately + 8 letters ) . for eyes with va 20/50 , monthly aflibercept provides a clear advantage over bevacizumab ( of 7.1 letters ) and ranibizumab ( of 4.7 letters ) . a tiered approach to eyes with dme based on for patients with poor va ( 20/50 ) and increased crt , a cost - effective analysis prior to the completion of vista / vivid and protocol t recommended that intravitreal pharmacotherapy with triamcinolone and less expensive anti - vegf injections ( bevacizumab ) should be considered.78 not surprisingly , less frequently administered ( q8wk ) aflibercept is more cost - effective than monthly injections since it reduces cost by 39% . the treatment of eyes with good va ( > 20/32 ) has not been systematically studied with anti - vegf agents , and at this time , the less expensive laser photocoagulation may be the best choice . intravitreal aflibercept is superior to laser photocoagulation for eyes with center - involving dme and may be more effective than bevacizumab and ranibizumab for eyes with bcva 20/50 .

local canonical correlation analysis ( cca ) is a multivariate statistical method in fmri that uses the joint time course of a group of neighboring voxels , usually in a 3 3 in - plane voxel grid , to determine the significance of activation . the value of a suitable test statistic is used as a measure of activation . since the joint time course of the neighborhood is used , it is not immediately clear to which voxel the measure of activation should be assigned . for example , if a 3 3 voxel neighborhood is chosen and the measure of activation is significant , without further assumptions one can only conclude that activation occurred somewhere within the 3 3 voxel neighborhood . if the activation is assigned to all voxels of the neighborhood , loss of spatial specificity will occur . to increase spatial specificity , it has been proposed to assign the measure of activation to the center voxel of the 3 3 neighborhood [ 1 , 2 ] . a center voxel assignment is usually justified by mathematical convenience but can also be reasoned on the fact that the fmri bold response leads to patches of activation patterns that are most likely of convex shape and simple connectivity ( without any holes in the interior neighborhood ) . however , this center voxel assignment proved to be prone to yield artifacts as activations tend to bleed to the neighboring voxels of strongly active voxels . the smoothing artifact is not only common in conventional cca , but also in any analysis technique that involves spatial low - pass filter kernels , such as univariate ( single voxel ) analysis where the data have been preprocessed using gaussian spatial smoothing . in conventional data smoothing , the smoothing artifact has been intentionally induced to increase the signal - to - noise ratio at the cost of reduced specificity and occurrence of typical spatial low - pass artifacts such as blurring of edges of activation patterns . to compensate for the smoothing artifact in conventional cca for example , a minimum relative weight for the center voxel was used to restrict false activations . in another study using a more adaptive approach , the smoothing artifact was reduced by utilizing the spatial dependence among voxels as much as possible and assigning the significance of activation to the dominant voxel of local maxima . this method was shown to be effective in eliminating the smoothing artifact in motor activation data that is known to have large contrast - to - noise ratio ( cnr ) , however , in data where the activation is more subtle ( such as hippocampal activation using an episodic memory paradigm ) , the method has the disadvantage of being less sensitive , according to our studies . to reduce the smoothing artifact in cca , it is necessary to constrain the spatial weights properly and impose the condition that the center voxel always has the largest weight . use nonnegative spatial weights with maximum weight of the center voxel in order to ensure spatial low - pass filter properties of ccca . this has the additional benefit of constraining cca to eliminate spurious correlations occurring in conventional cca where spatial filters can have positive and negative coefficients . recently , we provided a mathematical framework for ccca and computed roc properties of ccca with different linear constraints and a nonlinear constraint for activation patterns of motor data and episodic memory data [ 7 , 8 ] . in this paper we expand our previous research and investigate in detail the smoothing artifact that is associated with each spatial constraint in ccca . furthermore , we provide a novel approach of how to correct the measure of activation for the smoothing artifact . parts of this paper have been published in abstract form ( one page ) at a recent conference . in the following we briefly review cca and ccca , and explicitly consider the constraints introduced recently . mathematically , cca is a generalization of the general linear model ( glm ) by allowing the incorporation of spatial basis functions according to ( 1)(1f1()++sfs())y(,t ) = 1x1(t)++rxr(t)+(t ) , where the data are given by y( , t ) , is the vector representing the spatial coordinates x , y , and z , and t is time . the functions fi( ) , i = 1 , , s represent the spatial basis functions modeling the activation pattern in a neighborhood . the functions xj(t ) , j = 1 , , r are the temporal basis functions modeling the signal observed ( which is the result of a convolution of the hemodynamic response function and the stimulus function ) . the coefficients i and j are the spatial and temporal weights , respectively , that are being determined and optimized by the data for each individual neighborhood using an optimization routine . the symbol denotes spatial convolution and (t ) is a gaussian - distributed random error term . if the number of spatial basis functions is reduced to a single function , ( 1 ) becomes ( 2)f1()y(,t)=1x1(t)++rxr(t)+(t ) . when f1( ) is a simple gaussian function , we obtain the conventional glm used frequently in fmri . we assume that the functions fi( ) , i = 1 , , s are spatial dirac delta functions defining a local neighborhood within a 3 3 pixel neighborhood ( s 9 ) . let y be the matrix representing s voxel time courses with dimension t s and x the conventional design matrix of size t r for the r temporal regressors . furthermore , let and be two unknown vectors of size s 1 and r 1 , respectively . in cca , we look for the linear combinations of voxel time courses y and temporal regressors x such that the correlation between both quantities is maximum . this leads to an eigenvalue problem with min(s , r ) solutions from which the solution with the largest eigenvalue ( i.e. , maximum canonical correlation ) is being chosen . without constraints on the i , the specificity of the activation pattern obtained by cca is low and could result in artifacts ( see e.g. , ) . to put constraints on the spatial weights in order to restrict the space of unreasonable solutions for fmri , we consider the following four scenarios for the components i of , where 1 is the weight for the center voxel and the other i 's represent the weights for the s neighborhood voxels . one has ( 6)1max(i)>0 , i0 i2.note that the neighborhood size s is not a fixed quantity , but is determined from the data by ccca and can differ for each center voxel . one has ( 4)1i=2si>0 , i0 i2 . constraint 3 ( average constraint ) . one has ( 6)1max(i)>0 , i0 i2 . the smoothing artifact in cca is defined as the probability of incorrectly declaring the center voxel of a configuration of size s ( s 9 for a 3 3 neighborhood ) to be active . in the following , we outline how to compute the posterior probability to detect the smoothing artifact in real data using a bayesian framework . the posterior probability , p , that a center voxel is not active when it was in fact declared active , is given by ( 7)p = p(center voxel is not active >0,cnr , m , cnr , s ) , where > 0 indicates that the center voxel was declared active ( statistic > threshold 0 with [ 0 , ) ) , cnr is the univariate contrast - to - noise ratio of the center voxel , m labels the method of data analysis , cnr is the contrast - to - noise ratio of the entire configuration defining the neighborhood within a 3 3 pixel region , and s is the size of the configuration ( i.e. , number of declared active voxels 9 within the neighborhood ) . for abbreviation , we define the set of parameters , , to be ( 8)={cnr , m , cnr , s}. then , according to bayes ' theorem for conditional probabilities , ( 7 ) can be written as ( 9)p = p(>0 center voxel is not active,)p(center voxel is not active )(p(>0 ))1 , which is of the form ( 10)p = p1p3p2 , where ( 11)p1=p(>0 center voxel is not active,),(12)p2=p(>0 ),(13)p3=p(center voxel is not active ) . the term p1 is called the bleeding artifact because it represents the probability that an inactive voxel is declared as active . we determine p1 as a function of the size , s , of the configuration only and not as a function of the geometrical shape of the configuration . note that the dependence on s is an approximation , because in reality there are 2 = 256 possible configurations that can contain 0 to 8 active voxels ( corresponding to s { 1 , , 9 } since s labels the neighborhood size within a 3 3 pixel grid , which always includes the center voxel , independent if the center voxel is active or not ) . each configuration of size s has , depending on its distance of all voxel members to the center voxel , a slightly different value for p1 . for example , configurations with s = 7 leads to 3 different classes based on a distance measure , that is , class 1 = { center voxel , 4 corner voxels , and 2 midedge voxels } , class 2 = { center voxel , 3 corner voxels , and 3 midedge voxels } , class 3 = { center voxel , 2 corner voxels , and 4 midedge voxels}. according to our simulations , p1 is strongly dependent on s but not on a particular configuration of s. only a weak dependence based on different class memberships exist , which we neglect for the purpose of this research . to estimate p1 , it is thus reasonable to group all configurations for a particular s together and compute an average value of p1 over all possible configurations with size s. the term p1 can be estimated from simulations using a mixture of resampled resting - state data and activation data at given using kernel density estimation . resampled resting - state data are considered null data with respect to any task fmri function since the temporal structure is destroyed by resampling using the wavelet transform . this resampling , however , does not destroy the autocorrelations inherent in resting - state data . furthermore , the resampling does not affect the spatial correlations within the data because the permutations of the wavelet coefficients are kept the same for each voxel time series in a particular simulation ; however , different simulations use different permutations [ 11 , 12 ] . the simulated data are superpositions of time series from a 3 3 pixel neighborhood of null data and activation data . since the entire neighborhoods are used from resting - state data , realistic spatial correlations of the simulated data are obtained . in particular , for a configuration of s active voxels in the 3 3 neighborhood , the simulated voxel time courses , yi(t ) , are obtained by ( 14)yi(t)={yi(0)(t),for i=1,x(t)+yi(0)(t),for i {2, ,s } , where i = 1 refers to the center voxel and all other i to the surrounding voxels of the configuration of size s within the 3 3 neighborhood . all yi(t ) correspond to resampled resting - state time courses and represent spatially and temporally correlated null ( noise ) data . thus , p1 is a strong function of cnr of the configuration but not of the value cnr ( which is the contrast - to - noise ratio of the inactive center voxel ) , and the dependence of p1 on cnr can be neglected . the activation is determined by the hemodynamic response function , x(t ) , of interest multiplied by factor so that the configuration has a given cnr . in order to compute the cnr we use the general definition ( 15)cnr=(ii)1/2 , where i and i are the eigenvalues of the covariance matrix of the activation signal and noise , respectively . note that ( 15 ) can be used for a single voxel time series or an entire neighborhood of arbitrary size . to determine the activation signal and noise of a configuration using ccca , we convert the ccca problem into a multivariate multiple regression problem of the form ( 16)y=xb+e , where y are the data ( size t s ) , is the optimum spatial weight vector ( size s 1 ) , x is the design matrix ( size t r ) , b is the matrix of regression weights ( size r s ) , and e is a residual error matrix ( size t s ) . for a given contrast vector c , we reparameterize the design matrix x and obtain a transformed design matrix x~ such that ( 17)x~=[xeff x ] , where ( 18)xeff = x(xx)1c(c(xx)1c)1 is the first regressor of the new design matrix x~ that is associated with a parameter estimate equivalent to the original contrast cb [ 8 , 14 ] . the matrix x is perpendicular to xeff and plays no role in the estimation of c. then , the signal s(t ) is obtained by ( 19)s = xeffb , and the noise n(t ) is obtained by ( 20)n=(yxeffb). this term can be estimated directly from the real data . in this case , for each m and s > 1 , p2 is a 2d function of cnr and cnr , but depends strongly only on cnr so that the dependence on cnr can be neglected . note that for s = 1 , cnr = cnr , and in this case p2 is a 1d function of cnr only . it is possible to determine first the joint probability density p( , cnr | s , m ) using 2d kernel density estimation with a 2d gaussian kernel , which then can be integrated numerically to obtain p2 according to ( 21)p2(0,cnr , s , m)=0p(,cnr s , m)d0p(,cnr s , m)d. note that p2(0 , cnr , s , m ) for fixed { 0 , s , m } has a sigmoidal shape approaching the value 1 for cnr > 0.6 . thus , voxels that are declared active at a family - wise error rate ( fwe ) < 0.05 have necessarily a large cnr for which p2(0 , cnr , s , m ) 1 ( see section 4 ) . the term p3 is less difficult to determine because it is independent of the value of the statistic and depends strongly on the univariate cnr of the center voxel ( configuration with size s = 1 , and m = 1 ) , that is , ( 22)p3=p(center voxel is not active )p(center voxel is not active cnr , s=1,m=1)=p(center voxel is not active , cnr s=1,m=1)p(cnr s=1,m=1 ) , where m = 1 labels the univariate single voxel analysis method without smoothing . then , p3 is only a function of cnr and can be estimated from linear mixture modeling of the real data assuming that the data consists only of active and inactive voxels with unknown fractions . with this assumption , we define the cnr distribution of the data as h(cnr ) , consisting of the mixtures f(cnr ) and g,(cnr ) using ( 23)h(x)=a1df(cnrd)+(1a)g,(cnr ) . the distribution f(cnr ) is estimated from resampled resting - state data and the scaled distribution ( 1/d)f(cnr / d ) reflects the null distribution in activation data . the fact that f(cnr ) is scaled by constant d is rooted in the observation that in activation data more neural activity exists and maybe by spatial correlations or other hemodynamic means the distribution of the signal corresponding to inactive voxels is shifted to slightly larger values of cnr . the second term on the right in ( 23 ) , g,(cnr ) , represents the cnr distribution of active voxels modeled by a gaussian distribution with mean and variance . all the parameters a , d , , and are obtained from least squares fitting using activation data . then , ( 24)p3(cnr)=a(1/d)f(cnr / d ) h(cnr ) . overall , the posterior probability that a center voxel is not active is given by ( 25)p = p(center voxel is not active >0,)p(>0 center voxel is not active,)p3(cnr)p(>0 )p1(0,cnr , m , s)p3(cnr ) , since p( > highly active ( i.e. , fwe < 0.05 ) . in the following , we call the function p1(0 , cnr , m , s ) the smoothing artifact function . to correct for the smoothing artifact we propose the rule : ( 26)voxel is assigned to be inactive if p>0.5 and assign zero to the measure of activation if this statement is true . fmri was performed for 6 normal subjects with irb approval ( according to institutional requirements ) in a 3.0 t ge hdx mri scanner equipped with an 8-channel head coil and parallel imaging acquisition using epi with imaging parameters : asset = 2 , ramp sampling , tr / te = 2 sec/30 ms , fa = 70 deg , fov = 22 cm 22 cm , thickness / gap = 4 mm/1 mm , 25 slices , and resolution 96 96 . we briefly describe the paradigms and refer the reader for more detail to our previous article . the first data set was collected during resting - state where the subject tried to relax and refrain from executing any overt task with eyes closed . the second data set was collected while the subject was performing an episodic memory task with oblique coronal slices collected perpendicular to the long axis of the hippocampus . specifically , this task consisted of memorization of novel faces paired with occupations and contained 6 periods of encoding , distraction , and recognition tasks as well as short instructions where words on the screen reminded subjects of the task ahead . the third data set was obtained by performing an event - related motor task involving bilateral finger tapping while the subject was looking at a screen . all fmri data were realigned using statistical parametric mapping ( spm5 , http://www.fil.ion.ucl.ac.uk/spm/ ) and maximum motion components were found to be less than 0.6 mm in all directions . in a preprocessing step , all voxel time series were corrected for different slice timings and high - pass filtered by regression using a discrete cosine basis with cut - off frequency 1/120 hz . no temporal low - pass filtering was carried out . all voxels with intensity larger than 10% of the mean intensity this threshold effectively eliminated all nonbrain voxels leading to an average of about 4500 voxels per slice . all activation maps were thresholded using a fwe < 0.05 determined by using nonparametric methods [ 7 , 16 ] with wavelet resampled resting - state data [ 11 , 12 ] . all voxel time courses and temporal regressors were mean subtracted ( over time ) and variance normalized . as local spatial basis functions we use dirac delta functions in each 3 3 in - slice neighborhood . for the temporal modeling , we specified design matrices as in spm5 containing all conditions of the paradigms . in particular , for the memory paradigm we modeled instruction ( i ) , encoding ( e ) , recognition ( r ) , and control ( c ) by temporal reference functions whereas for the motor paradigm , fixation ( f ) , and motor task ( m ) were modeled according to the paradigm timings . all reference functions were convolved as usual with the standard spm5 two - gamma hemodynamic response function . for the motor task we computed activation maps for the contrast m - f , and for the memory task we used the contrast e - c . we used reparameterization of the design matrix x ( see ( 18 ) ) to incorporate the contrast of interest and optimized the spatial coefficients for each spatial constraint using the methods proposed in our previous publication . using simulated data , the smoothing artifact function p1 ( see ( 11 ) ) was determined for the motor paradigm with contrast m - f and memory paradigm with contrast e - c , respectively . simulations were carried out for all methods m ( single voxel analysis , single voxel analysis with gaussian spatial smoothing , unconstrained cca , cca with the simple constraint , cca with the sum constraint , cca with the average constraint , and cca with the maximum constraint ) , cnr in the range in steps of 0.1 , and configuration sizes 1 to 9 . all possible 256 configurations in a 3 3 neighborhood with inactive center voxel were simulated 1000 times and then regrouped according to the sizes s = { 1 , , 9}. figure 1 shows the smoothing artifact function for the motor paradigm for a typical subject . please note that this figure is a composition of nine different images where each image belongs to a configuration of a particular size ( 1 to 9 ) and each abscissa is the cnr ranging from 0 to 1 in steps of 0.1 . the vertical axis labels the different analysis methods applied and the color determines the value of p1 , ranging from 0 to 1 . bluish color indicates that the smoothing artifact is negligible whereas red color indicates that the smoothing artifact is significant . it is obvious that single - voxel analysis without gaussian smoothing does not show any smoothing artifact and single - voxel analysis with spatial smoothing leads to a significant smoothing artifact , the larger the cnr and the larger the neighborhood is . it is also obvious that unconstrained cca has the largest smoothing artifact and this artifact is already large for configuration sizes of s = 2 and cnr = 0.2 . however , choosing the simple nonnegativity constraint for ccca almost completely eliminates the smoothing artifact ( p1 < 0.35 ) . similarly , ccca with the sum constraint has a smoothing artifact that is even lower ( p1 < 0.3 ) and should be considered the method of choice if a high specificity is desirable . the ccca methods with the more complicated constraints ( avg constraint and max constraint ) show a significant smoothing artifact for configuration sizes of s 3 , as long as the cnr is large ( cnr > 0.6 ) . these two ccca methods have very high sensitivity but can lead to false activations when the configuration size is large . the function p2(0 , cnr , s , m ) = 0p( , cnr | s , m)d/0p( , cnr | s , m)d was calculated in matlab ( http://www.mathworks.com/ ) by 2d kernel density estimation of p( , cnr | s , m ) using an optimum bandwidth estimator according to sheather and jones . in general , p( , cnr | s , m ) has a bimodal distribution for configuration sizes s { 2,3 , 4,5 , 6,7}. for lower s , the larger mode of the density occurs at lower values of { , cnr } , whereas for larger values of s , the larger mode occurs at higher values of { , cnr}. for s { 8,9 } , the density becomes unimodal with mode located at large values of { , cnr}. also note , that is strongly correlated with cnr , which is expected . an example of p( , cnr | s , m ) is given in figure 2 for s = 5 and ccca with the maximum constraint . the shape of p2(0 , cnr , s , m ) obtained by numerical integration of 0p( , cnr | s , m)d/0p( , cnr | s , m)d and density smoothing is shown in figure 3 for all s and 0 cnr 1 . note the s - shaped form obtained for p2(0 , cnr , s , m ) for all integrations of bimodal distributions involving p( , cnr | s , m ) , whereas for s = 1 the function p2 is zero for cnr 1 and for s { 8,9 } p2 has the value 1 for 0 < cnr 1 . the function p2 for size s = 1 plays no role in determining the posterior probability p because the smoothing artifact is zero by definition , since a single - voxel - neighborhood can not have any bleeding of signal strength . in figure 4 we computed the null cnr density function ( 1/d)f(cnr / d ) using real motor activation data of a typical subject and obtained a dilation parameter d = 1.26 , indicating that the null distribution of the cnr obtained from resampled resting - state data is slightly inflated in activation data . the overall fit of the density functions ( 1/d)f(cnr / d ) and g,(cnr ) is good leading to a small residual mean squared error = 0.014 0.114 ( compare the light blue curve and the dark blue curve in figure 4 ) . a very similar curve was obtained for the memory paradigm using data from a different subject . here the dilation parameter was found to be d = 1.27 and mean squared error = 0.011 0.105 ( figure 5 ) . we show examples of the severity of the smoothing artifact for activation data thresholded at the p < 0.05 level , corrected for multiple comparison ( i.e. , fwe < 0.05 ) . the number of voxels affected by the smoothing artifact can be considerable for single voxel with gaussian smoothing and ccca with the average constraint as well as ccca with the max constraint , as seen in motor data ( figure 6 ) . for ccca with the sum constraint , however , there is no correction for the smoothing artifact necessary because the sum constraint produces a sufficiently dominant weight for the center voxel so that inactive voxels can not obtain a dominant weight in the neighborhood of active voxels . we did not find any voxel with a smoothing artifact > 0.1 confirming that ccca with the sum constraint has largest specificity of the proposed ccca methods . the activation patterns that are corrected for the smoothing artifact show small changes compared to the uncorrected ones , however , these changes can provide important information of the activation profile . for example , in figures 8 and 9 we show a magnified region of the left motor cortex and the right hippocampus , respectively , for selected analysis methods ( single voxel with and without gaussian smoothing , ccca with the maximum constraint ) . here we see that correction for the smoothing artifact leads to a separation of the right motor cortex ( see green arrows in figure 8) . this result is consistent with the activation pattern from single voxel analysis without gaussian smoothing . we believe that for the motor activation data , single - voxel analysis is already accurate due to the high cnr of the bold response for motor activation . regarding the hippocampal activation , we see that the correction for the smoothing artifact leads to a clear separation of hippocampal activation into three focal regions ( see blue arrow in figure 9 ) . it is conceivable that the corrected activation maps are more accurate representations of true hippocampal activations in this high - resolution study because it is known that the hippocampus is composed of the ca fields ( ca1 , ca2 , ca3 , and ca4 ) , the dentate gyrus and subiculum , and each of these subregions has a specific function in memory . the obtained corrections of the activation pattern are more probable than a continuous elongated activation pattern obtained with ccca without correction for the smoothing artifact . this condition is still a conservative correction for activation maps . to obtain better specificity but at a cost of losing sensitivity tables 1 and 2 show the number of voxels affected by the smoothing artifact for thresholds 0.1 to 0.5 . note that lowering the threshold for p to 0.2 leads to a dramatic increase in the number of voxels . thus , p > 0.2 should be avoided . the choice p > 0.3 is probably a good compromise of achieving better specificity and still maintaining high sensitivity for the examples shown here . however , the decision to use a lower threshold than 0.5 will primarily dependent on the particular application of the research . we preferred p > 0.5 which lead to a relatively small number of voxels that needed to be corrected . with this threshold the sensitivity of the methods is still very large and mostly voxel configurations of sizes 4 to 8 in motor data and 3 to 7 in memory data were affected by the smoothing artifact ( figure 10 ) . note that ccca with the max constraint leads to larger configuration sizes ( mean value s = 5.6 ) that are affected by the smoothing artifact than ccca with the average constraint ( mean value s = 4.3 ) . this fact is expected due to the increased freedom of the spatial constraints in ccca with the maximum constraint leading , on average , to larger configuration sizes which are more probable to induce a smoothing artifact than the other constrained ccca methods . we summarize the ideas introduced in this study and results obtained as follows.we investigated the smoothing artifact in cca and proposed a new technique to reduce this artifact in fmri data analysis . using data from a motor activation paradigm and an episodic memory paradigm , we showed examples of activation maps obtained with constrained cca methods , the corresponding magnitude of the smoothing artifact , and activation maps corrected for the smoothing artifact.for all data studied , we found no appreciable smoothing artifact for ccca with the sum constraint . the best overall performance was obtained by ccca with the maximum constraint corrected for the smoothing artifact . we recommend this technique for fmri data analysis to obtain high sensitivity and good specificity . we investigated the smoothing artifact in cca and proposed a new technique to reduce this artifact in fmri data analysis . using data from a motor activation paradigm and an episodic memory paradigm , we showed examples of activation maps obtained with constrained cca methods , the corresponding magnitude of the smoothing artifact , and activation maps corrected for the smoothing artifact . for all data studied , we found no appreciable smoothing artifact for ccca with the sum constraint . the best overall performance was obtained by ccca with the maximum constraint corrected for the smoothing artifact . we recommend this technique for fmri data analysis to obtain high sensitivity and good specificity .

a wide range of studies show the capacity of multivariate statistical methods for fmri to improve mapping of brain activations in a noisy environment . an advanced method uses local canonical correlation analysis ( cca ) to encompass a group of neighboring voxels instead of looking at the single voxel time course . the value of a suitable test statistic is used as a measure of activation . it is customary to assign the value to the center voxel ; however , this is a choice of convenience and without constraints introduces artifacts , especially in regions of strong localized activation . to compensate for these deficiencies , different spatial constraints in cca have been introduced to enforce dominance of the center voxel . however , even if the dominance condition for the center voxel is satisfied , constrained cca can still lead to a smoothing artifact , often called the bleeding artifact of cca , in fmri activation patterns . in this paper a new method is introduced to measure and correct for the smoothing artifact for constrained cca methods . it is shown that constrained cca methods corrected for the smoothing artifact lead to more plausible activation patterns in fmri as shown using data from a motor task and a memory task .

1. Introduction 2. Theory 3. Materials and Methods 4. Results and Discussion 5. Conclusions

local canonical correlation analysis ( cca ) is a multivariate statistical method in fmri that uses the joint time course of a group of neighboring voxels , usually in a 3 3 in - plane voxel grid , to determine the significance of activation . the value of a suitable test statistic is used as a measure of activation . since the joint time course of the neighborhood is used , it is not immediately clear to which voxel the measure of activation should be assigned . for example , if a 3 3 voxel neighborhood is chosen and the measure of activation is significant , without further assumptions one can only conclude that activation occurred somewhere within the 3 3 voxel neighborhood . if the activation is assigned to all voxels of the neighborhood , loss of spatial specificity will occur . to increase spatial specificity , it has been proposed to assign the measure of activation to the center voxel of the 3 3 neighborhood [ 1 , 2 ] . a center voxel assignment is usually justified by mathematical convenience but can also be reasoned on the fact that the fmri bold response leads to patches of activation patterns that are most likely of convex shape and simple connectivity ( without any holes in the interior neighborhood ) . however , this center voxel assignment proved to be prone to yield artifacts as activations tend to bleed to the neighboring voxels of strongly active voxels . the smoothing artifact is not only common in conventional cca , but also in any analysis technique that involves spatial low - pass filter kernels , such as univariate ( single voxel ) analysis where the data have been preprocessed using gaussian spatial smoothing . in conventional data smoothing , the smoothing artifact has been intentionally induced to increase the signal - to - noise ratio at the cost of reduced specificity and occurrence of typical spatial low - pass artifacts such as blurring of edges of activation patterns . to compensate for the smoothing artifact in conventional cca for example , a minimum relative weight for the center voxel was used to restrict false activations . in another study using a more adaptive approach , the smoothing artifact was reduced by utilizing the spatial dependence among voxels as much as possible and assigning the significance of activation to the dominant voxel of local maxima . this method was shown to be effective in eliminating the smoothing artifact in motor activation data that is known to have large contrast - to - noise ratio ( cnr ) , however , in data where the activation is more subtle ( such as hippocampal activation using an episodic memory paradigm ) , the method has the disadvantage of being less sensitive , according to our studies . to reduce the smoothing artifact in cca , it is necessary to constrain the spatial weights properly and impose the condition that the center voxel always has the largest weight . use nonnegative spatial weights with maximum weight of the center voxel in order to ensure spatial low - pass filter properties of ccca . in this paper we expand our previous research and investigate in detail the smoothing artifact that is associated with each spatial constraint in ccca . furthermore , we provide a novel approach of how to correct the measure of activation for the smoothing artifact . mathematically , cca is a generalization of the general linear model ( glm ) by allowing the incorporation of spatial basis functions according to ( 1)(1f1()++sfs())y(,t ) = 1x1(t)++rxr(t)+(t ) , where the data are given by y( , t ) , is the vector representing the spatial coordinates x , y , and z , and t is time . the functions xj(t ) , j = 1 , , r are the temporal basis functions modeling the signal observed ( which is the result of a convolution of the hemodynamic response function and the stimulus function ) . if the number of spatial basis functions is reduced to a single function , ( 1 ) becomes ( 2)f1()y(,t)=1x1(t)++rxr(t)+(t ) . let y be the matrix representing s voxel time courses with dimension t s and x the conventional design matrix of size t r for the r temporal regressors . in cca , we look for the linear combinations of voxel time courses y and temporal regressors x such that the correlation between both quantities is maximum . without constraints on the i , the specificity of the activation pattern obtained by cca is low and could result in artifacts ( see e.g. to put constraints on the spatial weights in order to restrict the space of unreasonable solutions for fmri , we consider the following four scenarios for the components i of , where 1 is the weight for the center voxel and the other i 's represent the weights for the s neighborhood voxels . the smoothing artifact in cca is defined as the probability of incorrectly declaring the center voxel of a configuration of size s ( s 9 for a 3 3 neighborhood ) to be active . in the following , we outline how to compute the posterior probability to detect the smoothing artifact in real data using a bayesian framework . the posterior probability , p , that a center voxel is not active when it was in fact declared active , is given by ( 7)p = p(center voxel is not active >0,cnr , m , cnr , s ) , where > 0 indicates that the center voxel was declared active ( statistic > threshold 0 with [ 0 , ) ) , cnr is the univariate contrast - to - noise ratio of the center voxel , m labels the method of data analysis , cnr is the contrast - to - noise ratio of the entire configuration defining the neighborhood within a 3 3 pixel region , and s is the size of the configuration ( i.e. then , according to bayes ' theorem for conditional probabilities , ( 7 ) can be written as ( 9)p = p(>0 center voxel is not active,)p(center voxel is not active )(p(>0 ))1 , which is of the form ( 10)p = p1p3p2 , where ( 11)p1=p(>0 center voxel is not active,),(12)p2=p(>0 ),(13)p3=p(center voxel is not active ) . the term p1 is called the bleeding artifact because it represents the probability that an inactive voxel is declared as active . note that the dependence on s is an approximation , because in reality there are 2 = 256 possible configurations that can contain 0 to 8 active voxels ( corresponding to s { 1 , , 9 } since s labels the neighborhood size within a 3 3 pixel grid , which always includes the center voxel , independent if the center voxel is active or not ) . each configuration of size s has , depending on its distance of all voxel members to the center voxel , a slightly different value for p1 . to estimate p1 , it is thus reasonable to group all configurations for a particular s together and compute an average value of p1 over all possible configurations with size s. the term p1 can be estimated from simulations using a mixture of resampled resting - state data and activation data at given using kernel density estimation . furthermore , the resampling does not affect the spatial correlations within the data because the permutations of the wavelet coefficients are kept the same for each voxel time series in a particular simulation ; however , different simulations use different permutations [ 11 , 12 ] . in particular , for a configuration of s active voxels in the 3 3 neighborhood , the simulated voxel time courses , yi(t ) , are obtained by ( 14)yi(t)={yi(0)(t),for i=1,x(t)+yi(0)(t),for i {2, ,s } , where i = 1 refers to the center voxel and all other i to the surrounding voxels of the configuration of size s within the 3 3 neighborhood . thus , p1 is a strong function of cnr of the configuration but not of the value cnr ( which is the contrast - to - noise ratio of the inactive center voxel ) , and the dependence of p1 on cnr can be neglected . note that ( 15 ) can be used for a single voxel time series or an entire neighborhood of arbitrary size . to determine the activation signal and noise of a configuration using ccca , we convert the ccca problem into a multivariate multiple regression problem of the form ( 16)y=xb+e , where y are the data ( size t s ) , is the optimum spatial weight vector ( size s 1 ) , x is the design matrix ( size t r ) , b is the matrix of regression weights ( size r s ) , and e is a residual error matrix ( size t s ) . for a given contrast vector c , we reparameterize the design matrix x and obtain a transformed design matrix x~ such that ( 17)x~=[xeff x ] , where ( 18)xeff = x(xx)1c(c(xx)1c)1 is the first regressor of the new design matrix x~ that is associated with a parameter estimate equivalent to the original contrast cb [ 8 , 14 ] . it is possible to determine first the joint probability density p( , cnr | s , m ) using 2d kernel density estimation with a 2d gaussian kernel , which then can be integrated numerically to obtain p2 according to ( 21)p2(0,cnr , s , m)=0p(,cnr s , m)d0p(,cnr s , m)d. note that p2(0 , cnr , s , m ) for fixed { 0 , s , m } has a sigmoidal shape approaching the value 1 for cnr > 0.6 . the term p3 is less difficult to determine because it is independent of the value of the statistic and depends strongly on the univariate cnr of the center voxel ( configuration with size s = 1 , and m = 1 ) , that is , ( 22)p3=p(center voxel is not active )p(center voxel is not active cnr , s=1,m=1)=p(center voxel is not active , cnr s=1,m=1)p(cnr s=1,m=1 ) , where m = 1 labels the univariate single voxel analysis method without smoothing . in the following , we call the function p1(0 , cnr , m , s ) the smoothing artifact function . to correct for the smoothing artifact we propose the rule : ( 26)voxel is assigned to be inactive if p>0.5 and assign zero to the measure of activation if this statement is true . the second data set was collected while the subject was performing an episodic memory task with oblique coronal slices collected perpendicular to the long axis of the hippocampus . specifically , this task consisted of memorization of novel faces paired with occupations and contained 6 periods of encoding , distraction , and recognition tasks as well as short instructions where words on the screen reminded subjects of the task ahead . in a preprocessing step , all voxel time series were corrected for different slice timings and high - pass filtered by regression using a discrete cosine basis with cut - off frequency 1/120 hz . in particular , for the memory paradigm we modeled instruction ( i ) , encoding ( e ) , recognition ( r ) , and control ( c ) by temporal reference functions whereas for the motor paradigm , fixation ( f ) , and motor task ( m ) were modeled according to the paradigm timings . for the motor task we computed activation maps for the contrast m - f , and for the memory task we used the contrast e - c . using simulated data , the smoothing artifact function p1 ( see ( 11 ) ) was determined for the motor paradigm with contrast m - f and memory paradigm with contrast e - c , respectively . simulations were carried out for all methods m ( single voxel analysis , single voxel analysis with gaussian spatial smoothing , unconstrained cca , cca with the simple constraint , cca with the sum constraint , cca with the average constraint , and cca with the maximum constraint ) , cnr in the range in steps of 0.1 , and configuration sizes 1 to 9 . all possible 256 configurations in a 3 3 neighborhood with inactive center voxel were simulated 1000 times and then regrouped according to the sizes s = { 1 , , 9}. figure 1 shows the smoothing artifact function for the motor paradigm for a typical subject . please note that this figure is a composition of nine different images where each image belongs to a configuration of a particular size ( 1 to 9 ) and each abscissa is the cnr ranging from 0 to 1 in steps of 0.1 . the vertical axis labels the different analysis methods applied and the color determines the value of p1 , ranging from 0 to 1 . it is obvious that single - voxel analysis without gaussian smoothing does not show any smoothing artifact and single - voxel analysis with spatial smoothing leads to a significant smoothing artifact , the larger the cnr and the larger the neighborhood is . it is also obvious that unconstrained cca has the largest smoothing artifact and this artifact is already large for configuration sizes of s = 2 and cnr = 0.2 . however , choosing the simple nonnegativity constraint for ccca almost completely eliminates the smoothing artifact ( p1 < 0.35 ) . similarly , ccca with the sum constraint has a smoothing artifact that is even lower ( p1 < 0.3 ) and should be considered the method of choice if a high specificity is desirable . in figure 4 we computed the null cnr density function ( 1/d)f(cnr / d ) using real motor activation data of a typical subject and obtained a dilation parameter d = 1.26 , indicating that the null distribution of the cnr obtained from resampled resting - state data is slightly inflated in activation data . the overall fit of the density functions ( 1/d)f(cnr / d ) and g,(cnr ) is good leading to a small residual mean squared error = 0.014 0.114 ( compare the light blue curve and the dark blue curve in figure 4 ) . a very similar curve was obtained for the memory paradigm using data from a different subject . we show examples of the severity of the smoothing artifact for activation data thresholded at the p < 0.05 level , corrected for multiple comparison ( i.e. the number of voxels affected by the smoothing artifact can be considerable for single voxel with gaussian smoothing and ccca with the average constraint as well as ccca with the max constraint , as seen in motor data ( figure 6 ) . for ccca with the sum constraint , however , there is no correction for the smoothing artifact necessary because the sum constraint produces a sufficiently dominant weight for the center voxel so that inactive voxels can not obtain a dominant weight in the neighborhood of active voxels . we did not find any voxel with a smoothing artifact > 0.1 confirming that ccca with the sum constraint has largest specificity of the proposed ccca methods . the activation patterns that are corrected for the smoothing artifact show small changes compared to the uncorrected ones , however , these changes can provide important information of the activation profile . for example , in figures 8 and 9 we show a magnified region of the left motor cortex and the right hippocampus , respectively , for selected analysis methods ( single voxel with and without gaussian smoothing , ccca with the maximum constraint ) . here we see that correction for the smoothing artifact leads to a separation of the right motor cortex ( see green arrows in figure 8) . we believe that for the motor activation data , single - voxel analysis is already accurate due to the high cnr of the bold response for motor activation . regarding the hippocampal activation , we see that the correction for the smoothing artifact leads to a clear separation of hippocampal activation into three focal regions ( see blue arrow in figure 9 ) . it is conceivable that the corrected activation maps are more accurate representations of true hippocampal activations in this high - resolution study because it is known that the hippocampus is composed of the ca fields ( ca1 , ca2 , ca3 , and ca4 ) , the dentate gyrus and subiculum , and each of these subregions has a specific function in memory . the obtained corrections of the activation pattern are more probable than a continuous elongated activation pattern obtained with ccca without correction for the smoothing artifact . to obtain better specificity but at a cost of losing sensitivity tables 1 and 2 show the number of voxels affected by the smoothing artifact for thresholds 0.1 to 0.5 . with this threshold the sensitivity of the methods is still very large and mostly voxel configurations of sizes 4 to 8 in motor data and 3 to 7 in memory data were affected by the smoothing artifact ( figure 10 ) . note that ccca with the max constraint leads to larger configuration sizes ( mean value s = 5.6 ) that are affected by the smoothing artifact than ccca with the average constraint ( mean value s = 4.3 ) . this fact is expected due to the increased freedom of the spatial constraints in ccca with the maximum constraint leading , on average , to larger configuration sizes which are more probable to induce a smoothing artifact than the other constrained ccca methods . we summarize the ideas introduced in this study and results obtained as follows.we investigated the smoothing artifact in cca and proposed a new technique to reduce this artifact in fmri data analysis . using data from a motor activation paradigm and an episodic memory paradigm , we showed examples of activation maps obtained with constrained cca methods , the corresponding magnitude of the smoothing artifact , and activation maps corrected for the smoothing artifact.for all data studied , we found no appreciable smoothing artifact for ccca with the sum constraint . the best overall performance was obtained by ccca with the maximum constraint corrected for the smoothing artifact . we investigated the smoothing artifact in cca and proposed a new technique to reduce this artifact in fmri data analysis . using data from a motor activation paradigm and an episodic memory paradigm , we showed examples of activation maps obtained with constrained cca methods , the corresponding magnitude of the smoothing artifact , and activation maps corrected for the smoothing artifact . for all data studied , we found no appreciable smoothing artifact for ccca with the sum constraint . the best overall performance was obtained by ccca with the maximum constraint corrected for the smoothing artifact .

limited data exist on the frequency of non - severe hypoglycaemic events in people with type 1 or type 2 diabetes in real - world practice , as non - severe hypoglycaemic events , by definition , do not require healthcare professional interactions ( are not routinely registered ) . the frequency of non - severe hypoglycaemic events in real - world practice may differ from that observed in clinical trials because of the characteristics of clinical trial designs . to our knowledge , this is the first study reporting the frequency of non - severe hypoglycaemic events in real - world practice in the seven countries involved in our study . non - severe hypoglycaemic events are common amongst people with type 1 or insulin - treated type 2 diabetes . many people with diabetes rarely or never inform their general practitioner / specialist about their hypoglycaemia and the real burden may be underestimated . the goal of diabetes management for people with type 1 or type 2 diabetes mellitus is to maintain normoglycaemia so as to reduce diabetic complications and the risk of mortality ; however , the intensification of therapy to achieve this goal may increase the incidence of hypoglycaemic episodes . hypoglycaemia remains a common and unpredictable side effect of insulin therapy , and has a negative physical and emotional impact on people with diabetes 1 . hypoglycaemic episodes are characterized as either severe or non - severe according to whether assistance is required from another individual , or whether the person with diabetes can manage the event alone , respectively 2,3 . non - severe hypoglycaemic events account for 8898% of all hypoglycaemic events 46 and have been shown to affect functioning 7 , health - related quality of life 4,8 , healthcare resource use 4 and work productivity 7 . furthermore , hypoglycaemia presents a significant barrier to optimum diabetes management , as fear of hypoglycaemic events may cause exaggerated avoidance behaviour and consequently suboptimum insulin therapy and poor glycaemic control 9,10 . whilst the importance of education about the recognition and treatment of hypoglycaemia is acknowledged in the current european association for the study of diabetes and american diabetes association consensus statement 11 , the real - world levels of communication between healthcare professionals and people with diabetes regarding hypoglycaemia are not fully understood . data on the frequency of hypoglycaemia , specifically non - severe hypoglycaemic events , outside of clinical trial settings are limited and varied 1,5,6,8 . the variability of data is probably attributable to differing study populations ( degree of selection , type 1 diabetes and/or insulin - treated type 2 diabetes ) , targets for glycaemic control , duration of treatment , methods of data collection and country coverage within these studies . our aim was to investigate the real - world frequency of self - reported non - severe hypoglycaemic events , levels of impaired hypoglycaemia awareness and discussion of hypoglycaemic events within physician consultations . we used a multi - country questionnaire - based survey in a large non - interventional cohort of people with type 1 diabetes or insulin - treated type 2 diabetes . the questionnaire also explored the health - related impact and economic burden of hypoglycaemia , the results of which are to be provided in a follow - on publication . the questionnaire - based survey was conducted between november 2011 and may 2012 and recruited respondents from austria , denmark , finland , norway , sweden , switzerland and the netherlands . respondents were primarily recruited via existing large consumer panels that were established to reflect a representative sample of the general diabetes population , based on age , gender and other demographic characteristics . where sufficient numbers of respondents could not be identified via consumer panels , other methods of recruitment were initiated , including the use of advertisements on diabetes - related websites and patient association websites ( with a link to the screener for inclusion in the survey ) , face - to - face recruitment , telephone recruitment and subsequent referrals from friends / family . in addition , some respondents were directly recruited at general practitioner clinics by nurses who were asked to identify participants and seek consent for participation , before providing contact details for those eligible to take part in the survey . all respondents completed a screening stage to determine eligibility for study inclusion . before study entry , respondents were unaware that the survey related to hypoglycaemia . a target of 600 respondents per country was set with an expectation that the probability of a hypoglycaemic event would have a 95% ci of 4% . the inclusion criteria were a diagnosis of either type 1 diabetes or type 2 diabetes from a healthcare professional , current insulin treatment and age > 15 years . in addition , respondents were required to read and speak the native language of the country in which they resided and have an email address in order to complete the questionnaire online . respondents were offered a small incentive for completion of the entire survey ( 525 ) , in line with current market research guidelines and to ensure there was no undue incentive to participate . all respondents were anonymous according to the regulations and practice of the market research governing bodies , the european society for opinion and marketing research 12 and the european pharmaceutical market research association 13 . eligible respondents were invited by email to complete an online questionnaire , in four waves . they received invitations for the second , third and fourth questionnaires 7 days after they had completed the previous questionnaire . questionnaires were adapted from those used in a previous study 7 , which had been designed using insights collected during focus groups on the impact of hypoglycaemia reported by people with diabetes 14 . data collected in the first questionnaire included respondent demographics , previous experience with and awareness of hypoglycaemia , the impact of hypoglycaemia and the number of non - severe hypoglycaemic events and severe hypoglycaemic events . respondents were also asked about hypoglycaemia - related discussions during general practitioner / specialist consultations . respondents who had experienced a non - severe hypoglycaemic event were asked whether they normally informed their general practitioner / specialist after they had had a hypoglycaemic event . a non - severe hypoglycaemic event was defined as symptoms of hypoglycaemia ( e.g. sweating , shaking , headache ) with or without a blood glucose measurement , or a low blood glucose measurement ( 3.1 mmol / l ) without symptoms , that the individual managed without assistance from another person . a severe hypoglycaemic event was defined as an event of low blood glucose level needing help from a third party to manage ( e.g. help from a family member or a healthcare professional , including emergency room visits and hospitalization ) . questions also referred to non - severe hypoglycaemic events occurring during the daytime or the night - time ( while the respondent was in bed / asleep ) . the subsequent questionnaires focused only on the number of non - severe hypoglycaemic events and the impact of these events . completion of the survey in four waves provided data for the number of non - severe hypoglycaemic events occurring over the past 4 weeks , whilst minimizing the recall period ( i.e. four 7-day periods were reported ) . questionnaires were completed anonymously but responses could be tracked across the four waves by an identification number assigned at study initiation . limits for upper and lower entry values were included within the questionnaire to minimize erroneous values . in addition , data were cleaned using a logical consistency check that allowed the removal of individual answers for which incorrect calculations had been made by a respondent ( e.g. where longer treatment duration than diabetes duration was reported ) , or the removal of the respondent from the entire analysis in instances where type of diabetes was not known or where erroneous reporting of simple demographic variables occurred ( e.g. diabetes duration longer than current age ) . the rate of non - severe hypoglycaemic events was calculated using data from all respondents who completed at least one wave of the survey . the first questionnaire collected data for non - severe hypoglycaemic events in the last 4 weeks and the last 7 days . all subsequent waves reported only the number of non - severe hypoglycaemic events in the last 7 days , so the estimated weekly rates from the 4-week rate provided in wave one could be matched with the weekly rates reported by the four times 7-day rates across waves one , two , three and four . annual event rates were calculated using the subsequent wave mean event rate per respondent - week multiplied by 52 . the relationships between demographic factors and the annual rate of non - severe hypoglycaemic events were analysed in regression models . the continuous dependent variable of the annual event rate was estimated by combining two variables : the 4-weekly non - severe hypoglycaemia event rate and , for respondents who did not experience a hypoglycaemic event in the previous 4 weeks , answers to the question , how often do you normally have non - severe hypoglycaemic events? this analysis used data collected during the first wave of the survey . regression analyses were conducted for the whole study population , as the study was not designed for cross - country comparisons . the classification system for awareness of hypoglycaemia was based on a prospectively validated study by pedersen - bjergaard et al . never to the question , can you feel when your blood sugar is low? was classified as being unaware of hypoglycaemia , those who answered usually were classified as having impaired awareness and those who answered always were classified as aware . standard descriptive methods ( means / percentage and standard deviations ) were used to report results for respondents in the following four groups : people with type 1 diabetes , people with type 2 diabetes receiving basal - only / long - acting insulin - only therapy , people with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy , or people with type 2 diabetes receiving another form of insulin therapy . comparisons were performed using t - tests and a p value < 0.05 was considered to indicate statistical significance . a total of 3959 respondents across seven countries were recruited to the study and 132 ( 3.3% ) were excluded as a result of inadequate questionnaire completion . the remaining 3827 respondents completed the initial survey , with 76 , 66 and 57% completing waves two , three and four , respectively , resulting in a total of 11 440 respondent - week records . the demographics for respondents with type 1 diabetes and those with type 2 diabetes are shown in table1 and were similar across countries ( data not shown ) . differences between respondents with type 1 diabetes and respondents with type 2 diabetes were consistent with those expected ( age , diabetes duration etc . ) . age and bmi were negatively correlated with the annual rate of non severe hypoglycaemic events ( p < 0.05 ) . female gender and duration of insulin treatment were positively correlated with the annual event rate ( p < 0.05 ) . respondent - related characteristics significant negative correlation with yearly number of non - severe hypoglycaemic events ( for both type 1 diabetes and type 2 diabetes , according to regression analysis ; p < 0.05 ) . significant positive correlation with yearly number of non - severe hypoglycaemic events ( for both type 1 diabetes and type 2 diabetes , according to regression analysis ; p < 0.05 ) . significant ( p < 0.05 ) negative correlation with yearly number of non - severe hypoglycaemic events ( type 2 diabetes only ) . duration of insulin treatment was correlated with diabetes duration , and thus duration of treatment was included in the regression analysis . a significant positive correlation was found between duration of treatment and yearly number of non - severe hypoglycaemic events ( for both type 1 diabetes and type 2 diabetes ; p < 0.05 ) . medical complications were not significantly associated with yearly number of non - severe hypoglycaemic events , independent of their association with age . questionnaire options for medical complications included : none , eye problems , neuropathy , cardiovascular disease , renal disease , amputations , other ( please specify ) . the mean self - reported non - severe hypoglycaemic event rate was 1.8 per respondent - week for respondents with type 1 diabetes and 0.5 for respondents with type 2 diabetes ( table2 ) . rates for respondents with type 2 diabetes were 0.4 ( respondents receiving basal - only / long - acting insulin - only therapy ) , 0.7 ( respondents receiving basal - bolus / both short- and long - acting insulin therapy ) and 0.5 ( respondents receiving another form of insulin therapy ; table2 ) . the calculated mean annual event rates were therefore 91.0 , 20.3 , 35.4 and 27.0 in the four groups ( table2 ) . the proportion of nocturnal non - severe hypoglycaemic events were slightly greater in respondents with type 2 diabetes than in respondents with type 1 diabetes : 22% ( type 1 diabetes ) , 32% ( respondents with type 2 diabetes receiving basal - only / long - acting insulin - only therapy ) , 22% ( respondents with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy ) and 27% ( respondents with type 2 diabetes receiving another form of insulin therapy ; table2 ) . four - week non - severe hypoglycaemic event rates recalled by respondents in questionnaire one were similar to , although slightly lower than , those collected over the four waves of the study ( table2 ) . self - reported , recalled rates of hypoglycaemic events rw , respondent week ; nshe , non - severe hypoglycaemic event ; bot , basal - only / long - acting insulin - only therapy ; bb , basal bolus / short- and long - acting insulin therapy ; she , severe hypoglycaemic event ; other , e.g. mixed insulin . all respondent - weeks reported ; includes all respondents regardless of whether completing all four questionnaires . all respondents completing wave 1 ( two responses were removed because of erroneous answers for this question ) . all respondents completing wave 1 ( seven responses were removed because of erroneous answers for this question ) . the mean number of self - reported severe hypoglycaemic events experienced in the last year was 0.7 for respondents with type 1 diabetes , 0.1 for respondents with type 2 diabetes receiving basal - only / long - acting insulin - only therapy , 0.2 for respondents with type 2 diabetes receiving basal - bolus /both short- and long - acting insulin therapy and 0.2 for respondents with type 2 diabetes receiving another form of insulin therapy ( table2 ) . overall , 76% of study respondents ( 87% of respondents with type 1 and 5978% of respondents with type 2 diabetes ) had previously experienced a hypoglycaemic event at any point ( i.e. not just in the study recall period ) . in respondents who had previous experience of hypoglycaemic events , impaired awareness was reported by 53% of respondents with type 1 diabetes , 45% of respondents with type 2 diabetes receiving basal - only / long - acting insulin therapy only , 43% of respondents with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy and 43% of respondents with type 2 diabetes receiving another form of insulin therapy ( table3 ) . a further 10 , 19 , 6 and 8% were classified as unaware for each respondent type , respectively . respondents with type 1 diabetes who were unaware had significantly higher rates of non - severe hypoglycaemic events than those who were always aware ( p < 0.05 ; table3 ) . among respondents with type 2 diabetes receiving basal - only / long - acting insulin - only therapy and respondents with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy , respondents with impaired awareness had significantly higher non - severe hypoglycaemic event rates than those who were aware ( p < 0.05 ) . in respondents with type 2 diabetes receiving another form of insulin therapy , significantly lower non - severe hypoglycaemic event rates were observed in unaware respondents than in respondents who were always aware ( p < 0.05 ; table3 ) . significantly higher rates of severe hypoglycaemic events were reported by respondents with type 1 diabetes classified either as unaware or as having impaired awareness , compared with aware respondents ( p < 0.05 ) . self - reported respondent awareness of hypoglycaemia and corresponding event rates in respondents who had previously experienced a hypoglycaemic event bot , basal - only / long - acting insulin - only therapy ; bb , basal - bolus / short- and long - acting insulin therapy ; other , e.g. mixed insulin ; nshe , non - severe hypoglycaemic event ; she , severe hypoglycaemic event . all respondents who had previously experienced a nshe at any point ( i.e. not just in the study recall period ; n = 2925 ) . a high proportion of respondents who had experienced a non - severe hypoglycaemic event stated that they never informed their general practitioner / specialist about their hypoglycaemia : 65% ( respondents with type 1 diabetes ) , 50% ( respondents with type 2 diabetes receiving basal - only / long - acting insulin - only therapy ) , 59% ( respondents with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy ) and 53% ( respondents with type 2 diabetes receiving another form of insulin therapy ; table4 ) . the lowest level of communication was reported in the netherlands ( data not shown ) . the proportion of respondents in the netherlands who rarely or never informed their general practitioner / specialist about their hypoglycaemic events was 86% ( respondents with type 1 diabetes ) , 64% ( respondents with type 2 diabetes receiving basal - only therapy / long - acting insulin - only therapy ) , 77% ( respondents with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy ) and 79% ( respondents with type 2 diabetes receiving another form of insulin therapy ) . event rates for non - severe hypoglycaemic events were significantly higher for respondents with type 1 diabetes or type 2 diabetes who rarely or never informed a physician about their non - severe hypoglycaemic events ( p < 0.05 ) . when respondents were asked about topics discussed during general practitioner / specialist consultations , 17% ( type 1 diabetes ) , 28% ( type 2 diabetes receiving basal - only / long - acting insulin - only therapy ) , 26% ( type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy ) and 21% ( type 2 diabetes receiving another form of insulin therapy ) of respondents stated that their general practitioner / specialist did not ask about hypoglycaemia during routine appointments ( table4 ) . communication between respondents and general practitioners / specialists bot , basal - only / long - acting insulin - only therapy ; bb , basal - bolus / short- and long - acting insulin therapy ; other , e.g. mixed insulin ; nshe , non - severe hypoglycaemic event . this study captures the self - reported , recalled rates of non - severe hypoglycaemic events and severe hypoglycaemic events in both people with type 1 diabetes and those with insulin - treated type 2 diabetes , and shows that hypoglycaemic events remain a common adverse event of insulin therapy in both groups . the majority of the published literature on hypoglycaemic event rates includes only people with type 1 diabetes , or is focused on reporting severe hypoglycaemic events only , and may not adequately reflect the frequency of hypoglycaemic events ( especially non - severe hypoglycaemic events ) across the insulin - treated diabetes population . in contrast , the present study explored the frequency of non - severe hypoglycaemic events and severe hypoglycaemic events in people with type 1 diabetes and people with insulin - treated type 2 diabetes across seven european countries . the recalled rates of non - severe hypoglycaemic events for respondents with type 1 diabetes ( 1.8 per respondent , per week ) in this study are comparable with results from three previously conducted studies in northern europe , which reported non - severe hypoglycaemic event rates of 1.8 , 2.0 and 2.2 per respondent , per week 6,16,17 . rates of non - severe hypoglycaemic events for respondents with type 2 diabetes in the current study are higher than those reported in a prospective single - centre study in scotland , uk ( 0.40.7 vs 0.3 per respondent , per week ) but this variation may be attributable to differences in the geographical region , type 2 diabetes treatment regimen , and study sample size , or the way in which hypoglycaemic events had been defined 5 . hypoglycaemic events occurred less frequently in respondents with insulin - treated type 2 diabetes compared with respondents with type 1 diabetes , and previous studies suggest the frequency of severe hypoglycaemic events in type 2 diabetes to be approximately one - third of that experienced by people with type 1 diabetes 5,18 . the results reported in the present study for severe hypoglycaemic events are consistent with this trend ( type 1 diabetes 0.7 ; type 2 0.10.2 ) and suggest a similar ratio for non - severe hypoglycaemic events ( type 1 diabetes 1.8 ; type 2 0.40.7 ) . it should be noted that the frequency of hypoglycaemic events in respondents with type 2 diabetes varies according to the treatment regimen ( basal - only / long - acting insulin - only therapy , basal - bolus / both short- and long - acting insulin therapy , or another form of insulin therapy ) ; however , this was to be expected given the different insulin coverage they provide 11 . overall , nocturnal events represented between one quarter and one third of all non - severe hypoglycaemic events . in the present study , the proportion of overall non - severe hypoglycaemic events occurring at night was 22% ( respondents with type 1 diabetes ) , 32% ( respondents with type 2 diabetes receiving basal - only / long acting insulin - only therapy ) , 22% ( respondents with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy ) , and 27% ( respondents with type 2 diabetes receiving another form of insulin therapy ) . few other studies have reported rates of nocturnal events , although the proportion of nocturnal events would be expected to vary between insulin regimens . in the present study , we investigated levels of hypoglycaemia awareness and reported 10% ( type 1 diabetes ) and 619% ( type 2 diabetes ) of respondents to be classified as unaware and 53% ( type 1 diabetes ) and 4345% ( type 2 diabetes ) to have impaired awareness of hypoglycaemia ( based on respondents with experience of hypoglycaemic events ) . a comparable proportion of respondents with type 1 diabetes were found to have impaired awareness ( 47% ) or be classified as unaware ( 13% ) in a 1-year prospective study that used the validated question , do you recognise symptoms when you have a hypo? 15 . furthermore , a cross - sectional study in a cohort of 401 people with type 2 diabetes , also using this question , reported a similar proportion of respondents with impaired awareness ( 46% ) to that in the current study ( 4345% ) 18 . there is no consensus on how to classify awareness , but our method benefits from the use of three categories ( instead of two , aware or in addition , it is the only method proven to perform similarly across language barriers 21 . for example , symptoms of hypoglycaemia have been shown to decline with increasing age and the prevalence of impaired awareness of hypoglycaemia is reported to increase with duration of type 1 diabetes 6 ; results may be confounded by these factors . 18 reported impaired awareness of hypoglycaemia to be the most important risk factor for severe hypoglycaemia . results of the current study show that respondents with type 1 diabetes classified as unaware or as having impaired awareness of hypoglycaemia reported significantly higher rates ( p < 0.05 ) of severe hypoglycaemic events than respondents who were always aware . unaware respondents with type 1 diabetes also reported significantly higher rates of non - severe hypoglycaemic events compared with aware respondents ( p < 0.05 ) . this could be explained by unaware respondents failing to take action to prevent the onset of an event because of an inability to recognize the symptoms of low blood sugar . additionally , this inability may cause respondents to overcompensate by testing their blood glucose more frequently , resulting in the identification of more events ; however , this is an area that requires further investigation , especially as these trends were not observed in respondents with type 2 diabetes . an important finding of the current study was the high proportion of respondents with type 1 diabetes ( 65% ) and type 2 diabetes ( 5059% ) who rarely or never informed their general practitioner / specialist about their hypoglycaemic events . despite these results , only 17% of respondents with type 1 diabetes and 2128% of respondents with type 2 diabetes said that their general practitioner / specialist did not ask them about hypoglycaemia during routine appointments , suggesting some level of communication regarding hypoglycaemic events is taking place . the reluctance of people with diabetes to discuss their hypoglycaemia may be caused by wider factors such as concerns regarding driving privileges 9 , implications for employment , or fear that they may be perceived by their general practitioner / specialist to have poor control of their diabetes . further research is needed to understand the reasoning behind why people may not actively be reporting their hypoglycaemic events . along with discussions on the frequency of non - severe hypoglycaemic events and severe hypoglycaemic events , other important aspects such as impaired hypoglycaemia awareness 18 and fear of hypoglycaemia 9,10 should be addressed , given that these are associated with an increased risk of severe hypoglycaemic events 18 and a risk of suboptimum glycaemic control 9,10 , respectively . an opportunity exists for more standardized measures of these self - reported outcomes , which may also help to improve understanding for people with diabetes , and improve communication levels . with the endorsement by both the american diabetes association and the european association for the study of diabetes of education regarding recognition and treatment of hypoglycaemia 11 , it is hoped that communication between people with diabetes and their physicians will increase further . whilst greater education could be expected to improve blood glucose management , there will still be an underlying increase in hypoglycaemic complications as insulin treatment regimens are intensified over time 1 . this is supported by our current regression analysis , where the number of non - severe events increased with duration of insulin treatment . the frequency of hypoglycaemic events reported during randomized trials , such as the diabetes control and complications trial 22 , and the united kingdom prospective diabetes study 23 , may not be reflective of the incidence in real - world practice because of trial inclusion and exclusion criteria and because observational studies have reported a higher incidence of hypoglycaemic events in unselected populations 1 . in addition , there are key benefits to obtaining data directly from people with diabetes , particularly since a high number of them are not reporting non - severe hypoglycaemic events to their doctor . respondent demographics show that 8% of respondents with type 1 diabetes receive long - acting insulin - only therapy . it is likely that this figure may be the result of incorrect reporting of diabetes type by respondents with type 2 diabetes . as a result , given that respondents with type 2 diabetes have fewer hypoglycaemic events , our study may underestimate the frequency of events for respondents with type 1 diabetes . the survey is based upon the recall of both severe hypoglycaemic events and non - severe hypoglycaemic events and the interpretation of symptoms is open to bias . a previous study showed that a respondent s ability to remember non - severe hypoglycaemic events during the previous week was not significantly different from the prospective recording of events over 1 week 6 . the current study was therefore designed to maximize the optimum recall period , by asking respondents to record events occurring in the previous week for each of the four questionnaires over 4 consecutive weeks . also , a previous study has shown that people with type 1 diabetes and people with type 2 diabetes are able to accurately recall severe hypoglycaemic events within a 1-year period ( corresponding to the recall period in the current study ) 15 . there is also the potential that the duration of the study may over- or underestimate the annual frequency of hypoglycaemia , given that seasonal variation was not considered ( the study was conducted december may ) . the recruitment of respondents , mostly via online panels and the requirement of an email address in order to participate in the study could have introduced selection bias ; however , the internet penetration rates for all of the countries studied are high ( 8097% ) 24 . the anonymous nature of the online panel may allow a better means of obtaining self - reported data on areas such as communication levels with physicians . recruitment was via broad panels reflective of the general population and respondents were invited via email to participate in the survey by following a link , and were not informed that the survey was about hypoglycaemia before they clicked on the link to enter the survey . there are therefore no reasons to suggest any selection bias towards people struggling with hypoglycaemia in the first wave of the study ; however , since the response rates for subsequent waves diminished ( 76 , 66 and 57% of respondents completed waves two , three and four , respectively ) we can not exclude the possibility that later waves were completed by respondents who had more experience of hypoglycaemic events . nevertheless , a subsequent analysis comparing event rates for the different waves did not suggest any trends towards higher frequency in later waves . the target recruitment rate of 600 respondents per country was not reached in austria , norway and switzerland because of difficulties in accessing people with diabetes ; however , results were remarkably consistent across the countries . some respondents did not complete all four waves , but only small changes in the non - severe hypoglycaemic event rates ( 1.09 in wave 1 to 0.93 in wave 4 ) were seen when comparing data across waves . this was a descriptive study , therefore , few comparisons were explored and no adjustments were made for multiple cross - country comparisons ; however , variations in non - severe hypoglycaemic event rates across countries were 1.32.0 per respondent , per week in type 1 diabetes , and 0.21.0 per respondent , per week in type 2 diabetes ( 0.20.5 in type 2 diabetes respondents receiving basal - only / long - acting insulin - only therapy , 0.51.0 in type 2 diabetes respondents receiving basal - bolus / both short- and long - acting insulin therapy and 0.20.9 in type 2 diabetes respondents receiving another form of insulin therapy ) . this might reflect other demographic differences which were not captured , such as local differences in treatment regimens , different patient education levels or targets for glycaemic control . additionally , the recruitment method does not differentiate between primary and secondary care patients , which may also have an impact . despite these limitations , the present study reports the real - world rates of hypoglycaemic events in a large number of people with type 1 diabetes and people with type 2 diabetes across seven european countries and provides evidence for a need to minimize the frequency of hypoglycaemia . it is acknowledged that both severe and non - severe hypoglycaemic events are more frequent in people with type 1 diabetes , but the associated social and economic burden of events in people with type 2 diabetes is likely to be substantial given the global epidemic of type 2 diabetes 25 . hypoglycaemia presents a barrier to optimum glycaemic control , increasing the risk of diabetic complications and mortality ; therefore , discussion during physician consultations and education on the recognition and treatment of hypoglycaemic events for people with diabetes are imperative to encourage greater communication with physicians . r. weitgasser has received honoraria for lectures and as a member of the advisory board for novo nordisk , eli lilly , sanofi , novartis , medtronic , msd , takeda , astra zeneca / bms , boehringer - ingelheim and roche diagnostics . j. lahtela has received honoraria as a symposium speaker for novartis , sanofi , novo nordisk , msd symposia . c. geelhoed - duijvestijn has received honoraria for lectures from novo nordisk and as a member of the advisory boards of medtronic , sanofi aventis and elli lilly . u. pedersen - bjergaard has received honoraria for lectures and consultancy from novo nordisk , sanofi aventis , and bms and has served as a member of advisory board for novo nordisk . r. weitgasser has received honoraria for lectures and as a member of the advisory board for novo nordisk , eli lilly , sanofi , novartis , medtronic , msd , takeda , astra zeneca / bms , boehringer - ingelheim and roche diagnostics . j. lahtela has received honoraria as a symposium speaker for novartis , sanofi , novo nordisk , msd symposia . c. geelhoed - duijvestijn has received honoraria for lectures from novo nordisk and as a member of the advisory boards of medtronic , sanofi aventis and elli lilly . u. pedersen - bjergaard has received honoraria for lectures and consultancy from novo nordisk , sanofi aventis , and bms and has served as a member of advisory board for novo nordisk .

aimshypoglycaemia presents a barrier to optimum diabetes management but data are limited on the frequency of hypoglycaemia incidents outside of clinical trials . the present study investigated the rates of self - reported non - severe hypoglycaemic events , hypoglycaemia awareness and physician discussion of events in people with type 1 diabetes mellitus or insulin - treated type 2 diabetes mellitus.methodspeople in seven european countries aged > 15 years with type 1 diabetes or insulin treated type 2 diabetes ( basal - only , basal - bolus and other insulin regimens ) were recruited via consumer panels , nurses , telephone recruitment and family referrals . respondents completed four online questionnaires . the first questionnaire collected background information on demographics and hypoglycaemia - related behaviour , whilst all four questionnaires collected data on non - severe hypoglycaemic events in the preceding 7 days.resultsanalysis was based on 11 440 respondent - weeks from 3827 respondents . all participants completed the first questionnaire and 57% completed all four . the mean number of events / respondent week was 1.8 ( type 1 diabetes ) and 0.40.7 ( type 2 diabetes , with different insulin treatments ) corresponding to annual event rates of 94 and 2136 , respectively . a total of 63% of respondents with type 1 diabetes and 4964% of respondents with type 2 diabetes , treated with different insulin regimens , who experienced hypoglycaemic events , reported impaired hypoglycaemia awareness or unawareness . a high proportion of respondents rarely or never informed their general practitioner / specialist about hypoglycaemia : 65% ( type 1 diabetes ) and 5059% ( type 2 diabetes ) . overall , 16% of respondents with type 1 diabetes and 26% of respondents with type 2 diabetes reported not being asked about hypoglycaemia during routine appointments.conclusionnon-severe hypoglycaemic events are common amongst people with type 1 diabetes and insulin treated type 2 diabetes in real - world settings . many rarely or never inform their general practitioner / specialist about their hypoglycaemia and the real burden of hypoglycaemia may be underestimated .

Whats new? Introduction Subjects and methods Results Discussion and conclusions Funding sources Competing interests

limited data exist on the frequency of non - severe hypoglycaemic events in people with type 1 or type 2 diabetes in real - world practice , as non - severe hypoglycaemic events , by definition , do not require healthcare professional interactions ( are not routinely registered ) . the frequency of non - severe hypoglycaemic events in real - world practice may differ from that observed in clinical trials because of the characteristics of clinical trial designs . to our knowledge , this is the first study reporting the frequency of non - severe hypoglycaemic events in real - world practice in the seven countries involved in our study . non - severe hypoglycaemic events are common amongst people with type 1 or insulin - treated type 2 diabetes . many people with diabetes rarely or never inform their general practitioner / specialist about their hypoglycaemia and the real burden may be underestimated . the goal of diabetes management for people with type 1 or type 2 diabetes mellitus is to maintain normoglycaemia so as to reduce diabetic complications and the risk of mortality ; however , the intensification of therapy to achieve this goal may increase the incidence of hypoglycaemic episodes . furthermore , hypoglycaemia presents a significant barrier to optimum diabetes management , as fear of hypoglycaemic events may cause exaggerated avoidance behaviour and consequently suboptimum insulin therapy and poor glycaemic control 9,10 . whilst the importance of education about the recognition and treatment of hypoglycaemia is acknowledged in the current european association for the study of diabetes and american diabetes association consensus statement 11 , the real - world levels of communication between healthcare professionals and people with diabetes regarding hypoglycaemia are not fully understood . data on the frequency of hypoglycaemia , specifically non - severe hypoglycaemic events , outside of clinical trial settings are limited and varied 1,5,6,8 . the variability of data is probably attributable to differing study populations ( degree of selection , type 1 diabetes and/or insulin - treated type 2 diabetes ) , targets for glycaemic control , duration of treatment , methods of data collection and country coverage within these studies . our aim was to investigate the real - world frequency of self - reported non - severe hypoglycaemic events , levels of impaired hypoglycaemia awareness and discussion of hypoglycaemic events within physician consultations . we used a multi - country questionnaire - based survey in a large non - interventional cohort of people with type 1 diabetes or insulin - treated type 2 diabetes . where sufficient numbers of respondents could not be identified via consumer panels , other methods of recruitment were initiated , including the use of advertisements on diabetes - related websites and patient association websites ( with a link to the screener for inclusion in the survey ) , face - to - face recruitment , telephone recruitment and subsequent referrals from friends / family . the inclusion criteria were a diagnosis of either type 1 diabetes or type 2 diabetes from a healthcare professional , current insulin treatment and age > 15 years . data collected in the first questionnaire included respondent demographics , previous experience with and awareness of hypoglycaemia , the impact of hypoglycaemia and the number of non - severe hypoglycaemic events and severe hypoglycaemic events . respondents were also asked about hypoglycaemia - related discussions during general practitioner / specialist consultations . respondents who had experienced a non - severe hypoglycaemic event were asked whether they normally informed their general practitioner / specialist after they had had a hypoglycaemic event . the subsequent questionnaires focused only on the number of non - severe hypoglycaemic events and the impact of these events . completion of the survey in four waves provided data for the number of non - severe hypoglycaemic events occurring over the past 4 weeks , whilst minimizing the recall period ( i.e. the first questionnaire collected data for non - severe hypoglycaemic events in the last 4 weeks and the last 7 days . all subsequent waves reported only the number of non - severe hypoglycaemic events in the last 7 days , so the estimated weekly rates from the 4-week rate provided in wave one could be matched with the weekly rates reported by the four times 7-day rates across waves one , two , three and four . standard descriptive methods ( means / percentage and standard deviations ) were used to report results for respondents in the following four groups : people with type 1 diabetes , people with type 2 diabetes receiving basal - only / long - acting insulin - only therapy , people with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy , or people with type 2 diabetes receiving another form of insulin therapy . the remaining 3827 respondents completed the initial survey , with 76 , 66 and 57% completing waves two , three and four , respectively , resulting in a total of 11 440 respondent - week records . the demographics for respondents with type 1 diabetes and those with type 2 diabetes are shown in table1 and were similar across countries ( data not shown ) . differences between respondents with type 1 diabetes and respondents with type 2 diabetes were consistent with those expected ( age , diabetes duration etc . ) respondent - related characteristics significant negative correlation with yearly number of non - severe hypoglycaemic events ( for both type 1 diabetes and type 2 diabetes , according to regression analysis ; p < 0.05 ) . significant positive correlation with yearly number of non - severe hypoglycaemic events ( for both type 1 diabetes and type 2 diabetes , according to regression analysis ; p < 0.05 ) . significant ( p < 0.05 ) negative correlation with yearly number of non - severe hypoglycaemic events ( type 2 diabetes only ) . a significant positive correlation was found between duration of treatment and yearly number of non - severe hypoglycaemic events ( for both type 1 diabetes and type 2 diabetes ; p < 0.05 ) . the mean self - reported non - severe hypoglycaemic event rate was 1.8 per respondent - week for respondents with type 1 diabetes and 0.5 for respondents with type 2 diabetes ( table2 ) . rates for respondents with type 2 diabetes were 0.4 ( respondents receiving basal - only / long - acting insulin - only therapy ) , 0.7 ( respondents receiving basal - bolus / both short- and long - acting insulin therapy ) and 0.5 ( respondents receiving another form of insulin therapy ; table2 ) . the proportion of nocturnal non - severe hypoglycaemic events were slightly greater in respondents with type 2 diabetes than in respondents with type 1 diabetes : 22% ( type 1 diabetes ) , 32% ( respondents with type 2 diabetes receiving basal - only / long - acting insulin - only therapy ) , 22% ( respondents with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy ) and 27% ( respondents with type 2 diabetes receiving another form of insulin therapy ; table2 ) . self - reported , recalled rates of hypoglycaemic events rw , respondent week ; nshe , non - severe hypoglycaemic event ; bot , basal - only / long - acting insulin - only therapy ; bb , basal bolus / short- and long - acting insulin therapy ; she , severe hypoglycaemic event ; other , e.g. the mean number of self - reported severe hypoglycaemic events experienced in the last year was 0.7 for respondents with type 1 diabetes , 0.1 for respondents with type 2 diabetes receiving basal - only / long - acting insulin - only therapy , 0.2 for respondents with type 2 diabetes receiving basal - bolus /both short- and long - acting insulin therapy and 0.2 for respondents with type 2 diabetes receiving another form of insulin therapy ( table2 ) . overall , 76% of study respondents ( 87% of respondents with type 1 and 5978% of respondents with type 2 diabetes ) had previously experienced a hypoglycaemic event at any point ( i.e. in respondents who had previous experience of hypoglycaemic events , impaired awareness was reported by 53% of respondents with type 1 diabetes , 45% of respondents with type 2 diabetes receiving basal - only / long - acting insulin therapy only , 43% of respondents with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy and 43% of respondents with type 2 diabetes receiving another form of insulin therapy ( table3 ) . respondents with type 1 diabetes who were unaware had significantly higher rates of non - severe hypoglycaemic events than those who were always aware ( p < 0.05 ; table3 ) . among respondents with type 2 diabetes receiving basal - only / long - acting insulin - only therapy and respondents with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy , respondents with impaired awareness had significantly higher non - severe hypoglycaemic event rates than those who were aware ( p < 0.05 ) . in respondents with type 2 diabetes receiving another form of insulin therapy , significantly lower non - severe hypoglycaemic event rates were observed in unaware respondents than in respondents who were always aware ( p < 0.05 ; table3 ) . significantly higher rates of severe hypoglycaemic events were reported by respondents with type 1 diabetes classified either as unaware or as having impaired awareness , compared with aware respondents ( p < 0.05 ) . self - reported respondent awareness of hypoglycaemia and corresponding event rates in respondents who had previously experienced a hypoglycaemic event bot , basal - only / long - acting insulin - only therapy ; bb , basal - bolus / short- and long - acting insulin therapy ; other , e.g. a high proportion of respondents who had experienced a non - severe hypoglycaemic event stated that they never informed their general practitioner / specialist about their hypoglycaemia : 65% ( respondents with type 1 diabetes ) , 50% ( respondents with type 2 diabetes receiving basal - only / long - acting insulin - only therapy ) , 59% ( respondents with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy ) and 53% ( respondents with type 2 diabetes receiving another form of insulin therapy ; table4 ) . the proportion of respondents in the netherlands who rarely or never informed their general practitioner / specialist about their hypoglycaemic events was 86% ( respondents with type 1 diabetes ) , 64% ( respondents with type 2 diabetes receiving basal - only therapy / long - acting insulin - only therapy ) , 77% ( respondents with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy ) and 79% ( respondents with type 2 diabetes receiving another form of insulin therapy ) . event rates for non - severe hypoglycaemic events were significantly higher for respondents with type 1 diabetes or type 2 diabetes who rarely or never informed a physician about their non - severe hypoglycaemic events ( p < 0.05 ) . when respondents were asked about topics discussed during general practitioner / specialist consultations , 17% ( type 1 diabetes ) , 28% ( type 2 diabetes receiving basal - only / long - acting insulin - only therapy ) , 26% ( type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy ) and 21% ( type 2 diabetes receiving another form of insulin therapy ) of respondents stated that their general practitioner / specialist did not ask about hypoglycaemia during routine appointments ( table4 ) . this study captures the self - reported , recalled rates of non - severe hypoglycaemic events and severe hypoglycaemic events in both people with type 1 diabetes and those with insulin - treated type 2 diabetes , and shows that hypoglycaemic events remain a common adverse event of insulin therapy in both groups . the majority of the published literature on hypoglycaemic event rates includes only people with type 1 diabetes , or is focused on reporting severe hypoglycaemic events only , and may not adequately reflect the frequency of hypoglycaemic events ( especially non - severe hypoglycaemic events ) across the insulin - treated diabetes population . in contrast , the present study explored the frequency of non - severe hypoglycaemic events and severe hypoglycaemic events in people with type 1 diabetes and people with insulin - treated type 2 diabetes across seven european countries . the recalled rates of non - severe hypoglycaemic events for respondents with type 1 diabetes ( 1.8 per respondent , per week ) in this study are comparable with results from three previously conducted studies in northern europe , which reported non - severe hypoglycaemic event rates of 1.8 , 2.0 and 2.2 per respondent , per week 6,16,17 . rates of non - severe hypoglycaemic events for respondents with type 2 diabetes in the current study are higher than those reported in a prospective single - centre study in scotland , uk ( 0.40.7 vs 0.3 per respondent , per week ) but this variation may be attributable to differences in the geographical region , type 2 diabetes treatment regimen , and study sample size , or the way in which hypoglycaemic events had been defined 5 . hypoglycaemic events occurred less frequently in respondents with insulin - treated type 2 diabetes compared with respondents with type 1 diabetes , and previous studies suggest the frequency of severe hypoglycaemic events in type 2 diabetes to be approximately one - third of that experienced by people with type 1 diabetes 5,18 . the results reported in the present study for severe hypoglycaemic events are consistent with this trend ( type 1 diabetes 0.7 ; type 2 0.10.2 ) and suggest a similar ratio for non - severe hypoglycaemic events ( type 1 diabetes 1.8 ; type 2 0.40.7 ) . it should be noted that the frequency of hypoglycaemic events in respondents with type 2 diabetes varies according to the treatment regimen ( basal - only / long - acting insulin - only therapy , basal - bolus / both short- and long - acting insulin therapy , or another form of insulin therapy ) ; however , this was to be expected given the different insulin coverage they provide 11 . in the present study , the proportion of overall non - severe hypoglycaemic events occurring at night was 22% ( respondents with type 1 diabetes ) , 32% ( respondents with type 2 diabetes receiving basal - only / long acting insulin - only therapy ) , 22% ( respondents with type 2 diabetes receiving basal - bolus / both short- and long - acting insulin therapy ) , and 27% ( respondents with type 2 diabetes receiving another form of insulin therapy ) . in the present study , we investigated levels of hypoglycaemia awareness and reported 10% ( type 1 diabetes ) and 619% ( type 2 diabetes ) of respondents to be classified as unaware and 53% ( type 1 diabetes ) and 4345% ( type 2 diabetes ) to have impaired awareness of hypoglycaemia ( based on respondents with experience of hypoglycaemic events ) . a comparable proportion of respondents with type 1 diabetes were found to have impaired awareness ( 47% ) or be classified as unaware ( 13% ) in a 1-year prospective study that used the validated question , do you recognise symptoms when you have a hypo? furthermore , a cross - sectional study in a cohort of 401 people with type 2 diabetes , also using this question , reported a similar proportion of respondents with impaired awareness ( 46% ) to that in the current study ( 4345% ) 18 . results of the current study show that respondents with type 1 diabetes classified as unaware or as having impaired awareness of hypoglycaemia reported significantly higher rates ( p < 0.05 ) of severe hypoglycaemic events than respondents who were always aware . unaware respondents with type 1 diabetes also reported significantly higher rates of non - severe hypoglycaemic events compared with aware respondents ( p < 0.05 ) . an important finding of the current study was the high proportion of respondents with type 1 diabetes ( 65% ) and type 2 diabetes ( 5059% ) who rarely or never informed their general practitioner / specialist about their hypoglycaemic events . despite these results , only 17% of respondents with type 1 diabetes and 2128% of respondents with type 2 diabetes said that their general practitioner / specialist did not ask them about hypoglycaemia during routine appointments , suggesting some level of communication regarding hypoglycaemic events is taking place . the reluctance of people with diabetes to discuss their hypoglycaemia may be caused by wider factors such as concerns regarding driving privileges 9 , implications for employment , or fear that they may be perceived by their general practitioner / specialist to have poor control of their diabetes . along with discussions on the frequency of non - severe hypoglycaemic events and severe hypoglycaemic events , other important aspects such as impaired hypoglycaemia awareness 18 and fear of hypoglycaemia 9,10 should be addressed , given that these are associated with an increased risk of severe hypoglycaemic events 18 and a risk of suboptimum glycaemic control 9,10 , respectively . the frequency of hypoglycaemic events reported during randomized trials , such as the diabetes control and complications trial 22 , and the united kingdom prospective diabetes study 23 , may not be reflective of the incidence in real - world practice because of trial inclusion and exclusion criteria and because observational studies have reported a higher incidence of hypoglycaemic events in unselected populations 1 . in addition , there are key benefits to obtaining data directly from people with diabetes , particularly since a high number of them are not reporting non - severe hypoglycaemic events to their doctor . respondent demographics show that 8% of respondents with type 1 diabetes receive long - acting insulin - only therapy . as a result , given that respondents with type 2 diabetes have fewer hypoglycaemic events , our study may underestimate the frequency of events for respondents with type 1 diabetes . also , a previous study has shown that people with type 1 diabetes and people with type 2 diabetes are able to accurately recall severe hypoglycaemic events within a 1-year period ( corresponding to the recall period in the current study ) 15 . there are therefore no reasons to suggest any selection bias towards people struggling with hypoglycaemia in the first wave of the study ; however , since the response rates for subsequent waves diminished ( 76 , 66 and 57% of respondents completed waves two , three and four , respectively ) we can not exclude the possibility that later waves were completed by respondents who had more experience of hypoglycaemic events . some respondents did not complete all four waves , but only small changes in the non - severe hypoglycaemic event rates ( 1.09 in wave 1 to 0.93 in wave 4 ) were seen when comparing data across waves . this was a descriptive study , therefore , few comparisons were explored and no adjustments were made for multiple cross - country comparisons ; however , variations in non - severe hypoglycaemic event rates across countries were 1.32.0 per respondent , per week in type 1 diabetes , and 0.21.0 per respondent , per week in type 2 diabetes ( 0.20.5 in type 2 diabetes respondents receiving basal - only / long - acting insulin - only therapy , 0.51.0 in type 2 diabetes respondents receiving basal - bolus / both short- and long - acting insulin therapy and 0.20.9 in type 2 diabetes respondents receiving another form of insulin therapy ) . despite these limitations , the present study reports the real - world rates of hypoglycaemic events in a large number of people with type 1 diabetes and people with type 2 diabetes across seven european countries and provides evidence for a need to minimize the frequency of hypoglycaemia . it is acknowledged that both severe and non - severe hypoglycaemic events are more frequent in people with type 1 diabetes , but the associated social and economic burden of events in people with type 2 diabetes is likely to be substantial given the global epidemic of type 2 diabetes 25 . hypoglycaemia presents a barrier to optimum glycaemic control , increasing the risk of diabetic complications and mortality ; therefore , discussion during physician consultations and education on the recognition and treatment of hypoglycaemic events for people with diabetes are imperative to encourage greater communication with physicians .

genetic code expansion has allowed the site - specific incorporation of more than 100 unnatural amino acids into proteins . however , the utility of these approaches may be limited by the efficiency with which unnatural amino acids are incorporated into proteins . the efficient , co - translational , site - specific incorporation of unnatural amino acids into proteins will enable emerging approaches for creating site - specifically modified recombinant proteins , as well as strategies to precisely control and image protein function in vivo , and many other approaches in which designer unnatural amino acids are used to control or report on protein function . orthogonal aminoacyl - trna synthetase / trna pairs direct the incorporation of unnatural amino acids , most commonly in response to the amber stop codon ( uag ) . the efficiency of unnatural amino acid incorporation is defined both by ( i ) the intrinsic efficiency with which the orthogonal synthetase / trna pair enables aminoacylation and translational elongation in response to a uag codon in the a site of the ribosome , and ( ii ) the efficiency with which release factors compete with the aminoacylated orthogonal trnacua to terminate protein synthesis . the pyrrolysyl - trna synthetase(pylrs)/trnacua pair is arguably the most useful pair to be developed for genetic code expansion because ( i ) it is orthogonal in a range of hosts including escherichia coli , yeast , mammalian cells , caenorhabditis elegans , and drosophila melanogaster ; ( ii ) pylrs does not recognize the common 20 amino acids ; ( iii ) pylrs does not recognize the anticodon of its cognate trnacua ; ( iv ) the active site of pylrs accommodates a range of unnatural amino acids bearing useful functional groups without the need for directed evolution ; ( v ) the active site of pylrs can be evolved to recognize structurally diverse unnatural amino acids bearing a range of useful functional groups in e. coli ; and ( vi ) the synthetase variants discovered in e. coli may be used in diverse eukaryotic hosts , where directed evolution of synthetases is challenging to implement . while unnatural amino acid incorporation is currently less efficient in mammalian cells than in e. coli , a systematic study of the effects of synthetase and trna promoter and copy number on unnatural amino acid incorporation efficiency has not been performed . moreover , a direct comparison of the efficiency of natural translation to the efficiency of unnatural amino acid incorporation into proteins in mammalian cells has not been performed . the incorporation of unnatural amino acids by the pylrs / trnacua pair in response to the amber stop codon may be limited by competition with the eukaryotic release factor 1 ( erf1)/eukaryotic release factor 3 ( erf3 ) complex that mediates translational termination in mammalian cells . while in prokaryotes , the two erf1 orthologs rf1 and rf2 selectively terminate protein synthesis at uag / uaa and uga / uaa codons , respectively , erf1 recognizes all three stop codons . thus , strategies developed in e. coli to enhance unnatural amino acid incorporation in response to the amber codon through selective disruption of rf1 function can not be extended to the mammalian system . indeed , there are no reports of engineering the eukaryotic translational machinery to enhance the efficiency with which unnatural amino acids are site - specifically incorporated into proteins in mammalian cells using orthogonal synthetase / trnacua pairs . here we define the efficiency of unnatural amino acid incorporation in mammalian cells relative to a natural translation control , allowing us to quantitatively benchmark improvements in unnatural amino acid incorporation efficiency . we demonstrate that expression of pylt ( encoding pyl trnacua ) is limiting for unnatural amino acid incorporation in mammalian cells , and we optimize the pylrs / trnacua expression system by increasing the levels of pyl trnacua expressed in mammalian cells . we engineer erf1 to enhance unnatural amino acid incorporation in response to the amber stop codon without increasing readthrough of opal and ochre stops . we demonstrate that the optimized system , which combines the best pylrs / trnacua expression system and an engineered erf1 , provides a 17- to 20-fold improvement in the yield of protein containing an unnatural amino acid incorporated in response to the amber stop codon , with optimized yields of protein approaching those for a no stop codon control . moreover , the optimized system increases the yield of proteins incorporating unnatural amino acids at three sites from unmeasurably low levels to up to 43% of a no stop codon control . we optimized the expression levels of trnacua to increase the efficiency of unnatural amino acid incorporation in mammalian cells . investigators have used different pylrs and trna plasmids that vary the copy number of pylrs , trnacua and the choice of promoters . however , there are no reports that quantify the yields of proteins bearing unnatural amino acids incorporated with the pylrs / trnacua pair in mammalian cells , nor are there reports that quantify the efficiencies of unnatural amino acid incorporation relative to the expression of a control protein expressed from a gene that does not contain an amber stop codon . these experiments are crucial for understanding how well unnatural amino acid incorporation in mammalian cells compares to natural protein synthesis . we first tested the efficiency of unnatural amino acid incorporation using plasmids b and c bearing a single copy of pylrs on a cmv promoter and four copies of trnacua each driven by a u6 promoter with a cmv enhancer ( construct schematics are shown in chart 1a ) . this system directed the incorporation of 1 ( n-[(tert - butoxy)carbonyl]-l - lysine ) , or 2 ( n-[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-l - lysine ) ( chart 1b ) , both known and efficient substrates for the pylrs / trnacua pair ) in response to an amber codon at position 150 in sfgfp ( cmv - sfgfp(tag ) ) with efficiencies of 5% and 7% ( figure 1a , b ) ; all incorporation efficiencies are reported as a percentage of sfgfp levels produced from an otherwise identical control construct bearing a leucine codon in place of the amber codon at position 150 ( plasmid a , chart 1 ) . next we replaced the four copies of trnacua with a single copy of trnacua on an optimized u6 promoter , leading to a small decrease in unnatural amino acid incorporation efficiency ( plasmids b and d ) . pylt is the gene encoding pyl trnacua and pylt * encodes the u25c variant . u6 indicates the u6 promoter , cmv is the cmv promoter , cmv enh is the 5 enhancer fragment of cmv promoter , ef1 prom is the ef1 promoter . ( b ) chemical structure of 1 ( n-[(tert - butoxy)carbonyl]-l - lysine ) and 2 ( n-[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-l - lysine ) . pylt is the gene encoding pyl trnacua and pylt * encodes the u25c variant . u6 indicates the u6 promoter , cmv is the cmv promoter , cmv enh is the 5 enhancer fragment of cmv promoter , ef1 prom is the ef1 promoter . ( b ) chemical structure of 1 ( n-[(tert - butoxy)carbonyl]-l - lysine ) and 2 ( n-[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-l - lysine ) . optimizing pylrs / trnacua expression vectors for the incorporation of unnatural amino acids in response to the tag codon in mammalian cells . ( a ) quantification of incorporation of 1 ( 2 mm ) and 2 ( 0.5 mm ) into superfolder green fluorescent protein ( sfgfp ) measured in a fluorescence assay . the indicated constructs were expressed transiently in hek293 t cells and sfgfp quantified in lysates by fluorescence at 520 nm , following excitation at 485 nm . a no amino acid control the data are plotted as a percentage of the fluorescence exhibited by an equivalent sfgfp control plasmid with a leucine codon in place of a stop codon ( construct a in chart 1a ) . equal amounts of cell lysate from cells transfected with the indicated vectors and grown in the presence of the indicated amino acid , or no amino acid , were immunoblotted with -gfp , -actin and -flag antibodies . ( c ) northern blot analysis of relative pylt / pylt * expression from constructs b + c , b + d , b + e , g + h , in the absence of amino acid . unlike the original four - copy cassette ( c ) , the new u6 trnacua cassette ( d ) does not contain the cmv enhancer , and produces a precise 5 end for the trna that does not require nuclease processing . northern blots ( figure 1c ) demonstrate that the levels of pyl trnacua produced from d are comparable to the levels produced from c. this indicates that the altered trna expression construct provides more copies of the trna per copy of the trna gene . replacing trnacua with a u25c variant increased the incorporation efficiency slightly from 2.73.5% to 4.75.1% ( plasmids b and e , chart 1 ) and increased the trnacua level . creating tandem arrays , each containing four copies of u6 pyl trnacua ( bearing u25c ) and switching the promoter for the protein coding genes from cmv to ef1 ( plasmids g and h , chart 1 ) led to a substantial increase in sfgfp bearing 1 or 2 . in this system , amino acid 1 was incorporated in response to the amber codon in sfgfp with an efficiency of 62% , while 2 was incorporated with an efficiency of approximately 129% . western blots demonstrate that changing the promoter of the protein coding genes to ef1 does not change the levels of pylrs ( anti - flag b + e vs g + h , figure 1b ) or wt sfgfp ( a vs f figure 1b ) , demonstrating that neither pylrs levels nor maximal levels of sfgfp expression are substantially altered by changing to the ef1 promoter . however , northern blots demonstrate that trna levels are much higher in this system than in all other systems tested , indicating that the large increases in unnatural amino acid incorporation efficiency we observe are correlated with an increase in trna level . next , we asked if we could further enhance unnatural amino acid incorporation efficiency , without increasing readthrough of other stop codons , by engineering erf1 . while the efficiency of unnatural amino acid incorporation was much improved with the optimized synthetase and trna system , we envisioned that erf1 engineering might further improve this efficiency and allow us to efficiently incorporate unnatural amino acids at multiple sites in a protein . we first identified amino acid positions in erf1 that are reported to have an effect on termination at amber codons from genetic or biochemical studies . these mutations are in the n - terminal domain of erf1 ( figure 2a ) that interacts with the stop codon on the mrna within the ribosome . to assess the effect of the erf1 mutants on translational termination in mammalian cells , we quantified suppressor trna independent readthrough at the amber , opal and ochre stop codon in hek 293 t cells and in hek 293 t cells bearing added , overexpressed human erf1 and erf1 mutants ( figure 2b ) . erf1 forms a complex with erf3 , primarily mediated through the c - terminal domain on erf1 that mediates translational termination . erf3 is present in cells at levels comparable to endogenous erf1 , and therefore , erf3 limits the number of termination complexes that may form . overexpression of erf1 n - terminal domain mutants may bias ( by mass action ) these complexes toward containing the erf1 mutants , thereby revealing the phenotype of the erf1 mutations . effect of mutations in erf1 on stop codon readthrough , and incorporation of 1 ( 2 mm ) using the pylrs / trnacua pair . structure of the n - terminal domain from erf1 ( pdb id:3e1y ) ; the residues mutated in this study are in red . ( b ) human erf1 variants are expressed following transient transfection of hek 293 t cells with perf1 ( x ) , where x designates the mutations introduced , and cmv - pylrs / cmv - dlr(tag ) . the negative control ( ) detects endogenous erf1 ; shrna is a knockdown of endogenous erf1 . ( c ) readthrough of all three stop codons is determined by the expression of a renilla - tag - firefly luciferase reporter and erf1 variants in hek293 t cells in the absence of a suppressor trna . cmv - pylrs / cmv - dlr(tag ) ( or the corresponding taa , tga or serine codon variant ) was transiently transfected into cells , expression levels determined after 20 h. tag , taa , or tga readthrough was normalized against data from the serine codon ( tcc ) containing construct . the negative control ( ) detects endogenous erf1 , shrna is a knockdown of endogenous erf1 . data for the 100 mutant are off scale ; the values are 1.6% ( taa ) , 2% ( tag ) and 15% ( tga ) . ( d ) transient transfection of hek 293 t cells with perf1 ( x ) , where x designates the mutations introduced , plasmid c expressing pylt from a u6 promoter ( chart 1a ) and cmv - pylrs / cmv - renilla - tag - firefly , a version of plasmid a ( chart 1a ) in which sfgfp is replaced by renilla - tag - firefly . the negative control ( ) detects endogenous erf1 ; shrna is a knockdown of endogenous erf1 . ( e ) erf1 ( x ) variants increase unnatural amino acid incorporation in response to an amber stop codon using the pyrolysyl trna / synthetase pair . t cells were transfected as described for panel d , and grown in the presence of 1 mm amino acid 1 , and measurements made after 20 h. percent readthrough was measured relative to a renilla - tcc - firefly reporter bearing a serine codon in place of the amber stop codon . we introduced each erf1 variant into cells ( figure 2b ) , and measured basal readthrough of stop codons , using three dual luciferase reporters ( figure 2c ) . each reporter contained an n - terminal renilla luciferase followed by a stop codon ( amber , opal or ochre ) and a c - terminal firefly luciferase . the readthrough of the stop codons was between 0.08 and 0.12% ( tag 0.09% , tga 0.12% , taa 0.08% ) , providing a benchmark for further experiments . ectopic overexpression of erf1 led to a decrease in readthrough of all three stop codons ( tag 0.03% , tga 0.07% , taa 0.04% ) , consistent with the increased level of erf1 in cells . this decrease in readthrough is small , consistent with the levels of erf3 being comparable to the levels of endogenous erf1 , and erf3 levels limiting the number of functional termination complexes that can be formed . introduction of erf1 variants increased stop codon readthrough with respect to the introduction of wild - type erf1 . however , for all erf1 mutants tested , except two , readthrough of all three stop codons was not increased above the levels found in the absence of ectopically expressed erf1 . we conclude that ectopic expression of most of the erf1 variants tested does not increase readthrough of stop codons above basal levels . the two erf1 mutants , which increase readthrough of stop codons above levels normally found in cells , are erf1 100 , a mutant that increases readthrough to 1.6% ( taa ) , 2% ( tag ) and 15% ( tga ) , and the t122q , s123f mutant that selectively increases readthrough at tga codons 2-fold . reduction of endogenous erf1 levels by shrna increased basal readthrough for all three stop codons 2- to 3-fold . the effect of the erf1 100 mutant on readthrough of all stop codons is expected , as the n - terminal domain , from which the residues are deleted , mediates recognition of all three stop codons in mrna , but does not mediate interactions with erf3 . the mutant is therefore predicted to form inactive complexes with erf3 , decreasing the number of functional erf1/erf3 complexes that can mediate termination . similarly , the effects of shrna against erf1 on all stop codons are expected since a decrease in erf1 should lead to a decrease in termination on all stop codons . to investigate the effects of erf1 , erf1 mutants and shrna on unnatural amino acid incorporation , we transfected cells with the relevant erf1 mutant ( figure 2d ) . each sample was also provided with the dual luciferase reporter of amber suppression , a single copy of the orthogonal pyrrolysyl trna- synthetase ( pylrs)/trnacua pair ( the arrangement shown as b + d in chart 1 , but with a dual luciferase reporter replacing sfgfp ) . we used this system to maximize the dynamic range with which we could measure the enhancement provided by erf1 variants . the amino acid 1 was added to all cells . in one case , an shrna construct targeting endogenous erf1 was added allowing us to compare the effects of ectopically expressed erf1 mutants to knocking down endogenous erf1 . the dual luciferase assay was used to determine the effects of erf1 on unnatural amino acid incorporation efficiency ( figure 2e ) . in the absence of ectopically expressed release factor , the efficiency of unnatural amino acid incorporation was approximately 5.3% in this assay . the incorporation efficiency was decreased slightly upon ectopic expression of wild - type release factor , and increased to 13% upon shrna knockdown of endogenous erf1 . the efficiency of incorporation for 1 increased in the presence of all mutant release factors except the s70a , g73s mutant . two erf1 mutants led to the most efficient unnatural amino acid incorporation : erf1 ( e55d ) , 27% ; and erf1 ( 100 ) , 36% . the incorporation efficiencies with the 100 mutant and the e55d mutant are 5- to 7-fold greater than the incorporation efficiency in cells that do not contain ectopically expressed release factor . interestingly , while strongly enhancing amber readthrough in the presence and absence of the pylrs / trnacua pair , the erf1100 mutant significantly reduced the total amount of luciferase produced in both situations , consistent with a drastic disruption of termination at all three stop codons having global effects on translation efficiency ( supporting information figure 2 ) in addition , the 100 mutant leads to readthrough of all three stop codons in the absence of suppressor trnas ; therefore , we did not investigate this release factor mutant further . this release factor mutant was previously identified in an in vitro assay in rabbit reticulocyte extract ( based on its ability to efficiently remove formyl - methionine from the initiator trna in the p site of the ribosome in response to an ochre or opal codon , but not an amber codon , in the a site ) . next , we combined the optimized synthetase and trna system and the e55d mutant of erf1 ( figure 3 ) . we find that the addition of erf1 ( e55d ) to cells containing the pylrs / trnacua pair , grown in the presence of 1 , increases the incorporation of 1 into sfgfp(tag ) from 62% to 85% ( figure 3a ) . similarly , the addition of the erf1 ( e55d ) increases the efficiency with which 1 is incorporated into sfgfp(tag)3 , that contains amber stop codons at positions 101 , 133 and 150 of gfp , from 5% to 12% ( figure 3a , b ) . the yield of sfgfp-1 from sfgfp(tag ) was 0.65 g from 10 cells , while the yield of sfgfp-(1)3 from sfgfp(tag)3 was 0.1 g per 10 cells ( supporting information figure 3 ; all yields are quoted per number of cells seeded , and were measured 48 h after transfection ) . combining erf1 e55d with an optimized pylrs / trnacua pair expression system enables efficient incorporation of multiple unnatural amino acids into recombinant proteins in mammalian cells . ( a ) plasmids g , h ( or i , chart 1a ) and erf1 e55d were transiently transfected into hek293 t cells , and grown in the presence or absence of 2 mm amino acid 1 for 48 h. full - length sfgfp was quantified in cell lysate at 520 nm , following excitation at 485 nm . ( b ) western blots from lysates . ( c ) as in panel a , but using 0.5 mm amino acid 2 . we find that the addition of the erf1 ( e55d ) to cells containing the pylrs / trnacua pair , grown in the presence of 2 , increases the incorporation of 2 into sfgfp(tag ) from 129% to 157% , and that the addition of the erf1 ( e55d ) quadruples the efficiency of producing of sfgfp-(2)3 from sfgfp(tag)3 from 11% to 43% ( figure 3c , d ) . the yield of sfgfp-2 from sfgfp(tag ) was 1.76 g per 10 cells , while the yield of sfgfp-(2)3 from sfgfp(tag)3 was 0.49 g per 10 cells ( supporting information figure 3 ) . full - length sfgfp was purified from cell lysates containing the optimized system ( figure 4a ) . electrospray ionization mass spectrometry demonstrated the site - specific incorporation of one and three molecules of 1 and 2 into sfgfp from sfgfp(tag ) and sfgfp(tag)3 respectively ( figure 4b , supporting information figure 4 ) . this data , in combination with the no amino acid controls in figures 4a demonstrate the high fidelity incorporation of unnatural amino acids in the presence of erf1 ( e55d ) . expression , purification and characterization of recombinant sfgfp incorporating one or three unnatural amino acids ( a ) . plasmids g , h ( or i , chart 1a ) and erf1 e55d were transiently transfected into hek293 t cells , and grown in the presence or absence of 2 mm amino acid 1 or 0.5 mm amino acid 2 for 48 h. full - length sfgfp was purified using anti - gfp resin . ( b ) electrospray ionization mass spectrometry confirms the quantitative incorporation of unnatural amino acids 1 and 2 , at one or three sites in sfgfp ( see also supporting information figure 4 ) . we have defined the efficiency of unnatural amino acid incorporation relative to a natural translation control , allowing us to quantitatively benchmark improvements in unnatural amino acid incorporation efficiency . the optimized system we have created provides a 17- to 20-fold improvement in unnatural amino acid incorporation efficiency with amino acids 1 and 2 in sfgfp . for amino acid 1 , the incorporation efficiency is increased from 5% to 85% , while for amino acid 2 , the incorporation efficiency is increased from 7% to 157% of a no stop codon control . moreover , the optimized system increases the yield of proteins incorporating 1 and 2 at three positions from unmeasurably low levels to 12% and 43% of a no stop control , respectively . two factors contribute to the dramatic improvement in unnatural amino acid incorporation : the optimization of trnacua levels to optimize pylrs / trnacua expression ; and the development and use of engineered erf1 variants . while the incorporation of unnatural amino acids is quite efficient in response to a single amber codon using the optimized pylrs / trnacua system alone , the efficiency is further improved by the addition of erf1 ( e55d ) . the effect of the erf1 mutant on unnatural amino acid incorporation is more dramatic when incorporating unnatural amino acids at multiple sites , increasing the yield of protein containing amino acid 1 at three sites , 2- to 3-fold and the yield of protein containing 2 at three sites , 4-fold . our data demonstrate that unnatural amino acid incorporation in mammalian cells can be very efficient . moreover , our data provide the first demonstration that , despite erf1 recognizing all three stop codons , it is possible to engineer erf1 to selectively enhance the efficiency of unnatural amino acid incorporation in eukaryotic cells in response to the amber stop codon , without increasing readthrough of opal or ochre stop codons . future work will further investigate the generality of the approaches we report for the efficient incorporation of diverse unnatural amino acids at diverse sites in diverse proteins in eukaryotic cells . we anticipate that our approach may enable the efficient production of site - specifically modified therapeutic proteins , as well as the quantitative replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function .

the efficient , site - specific introduction of unnatural amino acids into proteins in mammalian cells is an outstanding challenge in realizing the potential of genetic code expansion approaches . addressing this challenge will allow the synthesis of modified recombinant proteins and augment emerging strategies that introduce new chemical functionalities into proteins to control and image their function with high spatial and temporal precision in cells . the efficiency of unnatural amino acid incorporation in response to the amber stop codon ( uag ) in mammalian cells is commonly considered to be low . here we demonstrate that trna levels can be limiting for unnatural amino acid incorporation efficiency , and we develop an optimized pyrrolysyl - trna synthetase / trnacua expression system , with optimized trna expression for mammalian cells . in addition , we engineer erf1 , that normally terminates translation on all three stop codons , to provide a substantial increase in unnatural amino acid incorporation in response to the uag codon without increasing readthrough of other stop codons . by combining the optimized pyrrolysyl - trna synthetase / trnacua expression system and an engineered erf1 , we increase the yield of protein bearing unnatural amino acids at a single site 17- to 20-fold . using the optimized system , we produce proteins containing unnatural amino acids with comparable yields to a protein produced from a gene that does not contain a uag stop codon . moreover , the optimized system increases the yield of protein , incorporating an unnatural amino acid at three sites , from unmeasurably low levels up to 43% of a no amber stop control . our approach may enable the efficient production of site - specifically modified therapeutic proteins , and the quantitative replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function .

Introduction Results Discussion

genetic code expansion has allowed the site - specific incorporation of more than 100 unnatural amino acids into proteins . however , the utility of these approaches may be limited by the efficiency with which unnatural amino acids are incorporated into proteins . the efficient , co - translational , site - specific incorporation of unnatural amino acids into proteins will enable emerging approaches for creating site - specifically modified recombinant proteins , as well as strategies to precisely control and image protein function in vivo , and many other approaches in which designer unnatural amino acids are used to control or report on protein function . orthogonal aminoacyl - trna synthetase / trna pairs direct the incorporation of unnatural amino acids , most commonly in response to the amber stop codon ( uag ) . the efficiency of unnatural amino acid incorporation is defined both by ( i ) the intrinsic efficiency with which the orthogonal synthetase / trna pair enables aminoacylation and translational elongation in response to a uag codon in the a site of the ribosome , and ( ii ) the efficiency with which release factors compete with the aminoacylated orthogonal trnacua to terminate protein synthesis . the pyrrolysyl - trna synthetase(pylrs)/trnacua pair is arguably the most useful pair to be developed for genetic code expansion because ( i ) it is orthogonal in a range of hosts including escherichia coli , yeast , mammalian cells , caenorhabditis elegans , and drosophila melanogaster ; ( ii ) pylrs does not recognize the common 20 amino acids ; ( iii ) pylrs does not recognize the anticodon of its cognate trnacua ; ( iv ) the active site of pylrs accommodates a range of unnatural amino acids bearing useful functional groups without the need for directed evolution ; ( v ) the active site of pylrs can be evolved to recognize structurally diverse unnatural amino acids bearing a range of useful functional groups in e. coli ; and ( vi ) the synthetase variants discovered in e. coli may be used in diverse eukaryotic hosts , where directed evolution of synthetases is challenging to implement . while unnatural amino acid incorporation is currently less efficient in mammalian cells than in e. coli , a systematic study of the effects of synthetase and trna promoter and copy number on unnatural amino acid incorporation efficiency has not been performed . moreover , a direct comparison of the efficiency of natural translation to the efficiency of unnatural amino acid incorporation into proteins in mammalian cells has not been performed . the incorporation of unnatural amino acids by the pylrs / trnacua pair in response to the amber stop codon may be limited by competition with the eukaryotic release factor 1 ( erf1)/eukaryotic release factor 3 ( erf3 ) complex that mediates translational termination in mammalian cells . while in prokaryotes , the two erf1 orthologs rf1 and rf2 selectively terminate protein synthesis at uag / uaa and uga / uaa codons , respectively , erf1 recognizes all three stop codons . thus , strategies developed in e. coli to enhance unnatural amino acid incorporation in response to the amber codon through selective disruption of rf1 function can not be extended to the mammalian system . indeed , there are no reports of engineering the eukaryotic translational machinery to enhance the efficiency with which unnatural amino acids are site - specifically incorporated into proteins in mammalian cells using orthogonal synthetase / trnacua pairs . here we define the efficiency of unnatural amino acid incorporation in mammalian cells relative to a natural translation control , allowing us to quantitatively benchmark improvements in unnatural amino acid incorporation efficiency . we demonstrate that expression of pylt ( encoding pyl trnacua ) is limiting for unnatural amino acid incorporation in mammalian cells , and we optimize the pylrs / trnacua expression system by increasing the levels of pyl trnacua expressed in mammalian cells . we engineer erf1 to enhance unnatural amino acid incorporation in response to the amber stop codon without increasing readthrough of opal and ochre stops . we demonstrate that the optimized system , which combines the best pylrs / trnacua expression system and an engineered erf1 , provides a 17- to 20-fold improvement in the yield of protein containing an unnatural amino acid incorporated in response to the amber stop codon , with optimized yields of protein approaching those for a no stop codon control . moreover , the optimized system increases the yield of proteins incorporating unnatural amino acids at three sites from unmeasurably low levels to up to 43% of a no stop codon control . we optimized the expression levels of trnacua to increase the efficiency of unnatural amino acid incorporation in mammalian cells . however , there are no reports that quantify the yields of proteins bearing unnatural amino acids incorporated with the pylrs / trnacua pair in mammalian cells , nor are there reports that quantify the efficiencies of unnatural amino acid incorporation relative to the expression of a control protein expressed from a gene that does not contain an amber stop codon . these experiments are crucial for understanding how well unnatural amino acid incorporation in mammalian cells compares to natural protein synthesis . we first tested the efficiency of unnatural amino acid incorporation using plasmids b and c bearing a single copy of pylrs on a cmv promoter and four copies of trnacua each driven by a u6 promoter with a cmv enhancer ( construct schematics are shown in chart 1a ) . this system directed the incorporation of 1 ( n-[(tert - butoxy)carbonyl]-l - lysine ) , or 2 ( n-[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-l - lysine ) ( chart 1b ) , both known and efficient substrates for the pylrs / trnacua pair ) in response to an amber codon at position 150 in sfgfp ( cmv - sfgfp(tag ) ) with efficiencies of 5% and 7% ( figure 1a , b ) ; all incorporation efficiencies are reported as a percentage of sfgfp levels produced from an otherwise identical control construct bearing a leucine codon in place of the amber codon at position 150 ( plasmid a , chart 1 ) . next we replaced the four copies of trnacua with a single copy of trnacua on an optimized u6 promoter , leading to a small decrease in unnatural amino acid incorporation efficiency ( plasmids b and d ) . optimizing pylrs / trnacua expression vectors for the incorporation of unnatural amino acids in response to the tag codon in mammalian cells . a no amino acid control the data are plotted as a percentage of the fluorescence exhibited by an equivalent sfgfp control plasmid with a leucine codon in place of a stop codon ( construct a in chart 1a ) . unlike the original four - copy cassette ( c ) , the new u6 trnacua cassette ( d ) does not contain the cmv enhancer , and produces a precise 5 end for the trna that does not require nuclease processing . northern blots ( figure 1c ) demonstrate that the levels of pyl trnacua produced from d are comparable to the levels produced from c. this indicates that the altered trna expression construct provides more copies of the trna per copy of the trna gene . creating tandem arrays , each containing four copies of u6 pyl trnacua ( bearing u25c ) and switching the promoter for the protein coding genes from cmv to ef1 ( plasmids g and h , chart 1 ) led to a substantial increase in sfgfp bearing 1 or 2 . in this system , amino acid 1 was incorporated in response to the amber codon in sfgfp with an efficiency of 62% , while 2 was incorporated with an efficiency of approximately 129% . however , northern blots demonstrate that trna levels are much higher in this system than in all other systems tested , indicating that the large increases in unnatural amino acid incorporation efficiency we observe are correlated with an increase in trna level . next , we asked if we could further enhance unnatural amino acid incorporation efficiency , without increasing readthrough of other stop codons , by engineering erf1 . while the efficiency of unnatural amino acid incorporation was much improved with the optimized synthetase and trna system , we envisioned that erf1 engineering might further improve this efficiency and allow us to efficiently incorporate unnatural amino acids at multiple sites in a protein . to assess the effect of the erf1 mutants on translational termination in mammalian cells , we quantified suppressor trna independent readthrough at the amber , opal and ochre stop codon in hek 293 t cells and in hek 293 t cells bearing added , overexpressed human erf1 and erf1 mutants ( figure 2b ) . erf3 is present in cells at levels comparable to endogenous erf1 , and therefore , erf3 limits the number of termination complexes that may form . effect of mutations in erf1 on stop codon readthrough , and incorporation of 1 ( 2 mm ) using the pylrs / trnacua pair . ( c ) readthrough of all three stop codons is determined by the expression of a renilla - tag - firefly luciferase reporter and erf1 variants in hek293 t cells in the absence of a suppressor trna . ( e ) erf1 ( x ) variants increase unnatural amino acid incorporation in response to an amber stop codon using the pyrolysyl trna / synthetase pair . t cells were transfected as described for panel d , and grown in the presence of 1 mm amino acid 1 , and measurements made after 20 h. percent readthrough was measured relative to a renilla - tcc - firefly reporter bearing a serine codon in place of the amber stop codon . we introduced each erf1 variant into cells ( figure 2b ) , and measured basal readthrough of stop codons , using three dual luciferase reporters ( figure 2c ) . ectopic overexpression of erf1 led to a decrease in readthrough of all three stop codons ( tag 0.03% , tga 0.07% , taa 0.04% ) , consistent with the increased level of erf1 in cells . this decrease in readthrough is small , consistent with the levels of erf3 being comparable to the levels of endogenous erf1 , and erf3 levels limiting the number of functional termination complexes that can be formed . introduction of erf1 variants increased stop codon readthrough with respect to the introduction of wild - type erf1 . however , for all erf1 mutants tested , except two , readthrough of all three stop codons was not increased above the levels found in the absence of ectopically expressed erf1 . we conclude that ectopic expression of most of the erf1 variants tested does not increase readthrough of stop codons above basal levels . the two erf1 mutants , which increase readthrough of stop codons above levels normally found in cells , are erf1 100 , a mutant that increases readthrough to 1.6% ( taa ) , 2% ( tag ) and 15% ( tga ) , and the t122q , s123f mutant that selectively increases readthrough at tga codons 2-fold . reduction of endogenous erf1 levels by shrna increased basal readthrough for all three stop codons 2- to 3-fold . the effect of the erf1 100 mutant on readthrough of all stop codons is expected , as the n - terminal domain , from which the residues are deleted , mediates recognition of all three stop codons in mrna , but does not mediate interactions with erf3 . similarly , the effects of shrna against erf1 on all stop codons are expected since a decrease in erf1 should lead to a decrease in termination on all stop codons . to investigate the effects of erf1 , erf1 mutants and shrna on unnatural amino acid incorporation , we transfected cells with the relevant erf1 mutant ( figure 2d ) . the dual luciferase assay was used to determine the effects of erf1 on unnatural amino acid incorporation efficiency ( figure 2e ) . in the absence of ectopically expressed release factor , the efficiency of unnatural amino acid incorporation was approximately 5.3% in this assay . two erf1 mutants led to the most efficient unnatural amino acid incorporation : erf1 ( e55d ) , 27% ; and erf1 ( 100 ) , 36% . the incorporation efficiencies with the 100 mutant and the e55d mutant are 5- to 7-fold greater than the incorporation efficiency in cells that do not contain ectopically expressed release factor . interestingly , while strongly enhancing amber readthrough in the presence and absence of the pylrs / trnacua pair , the erf1100 mutant significantly reduced the total amount of luciferase produced in both situations , consistent with a drastic disruption of termination at all three stop codons having global effects on translation efficiency ( supporting information figure 2 ) in addition , the 100 mutant leads to readthrough of all three stop codons in the absence of suppressor trnas ; therefore , we did not investigate this release factor mutant further . next , we combined the optimized synthetase and trna system and the e55d mutant of erf1 ( figure 3 ) . similarly , the addition of the erf1 ( e55d ) increases the efficiency with which 1 is incorporated into sfgfp(tag)3 , that contains amber stop codons at positions 101 , 133 and 150 of gfp , from 5% to 12% ( figure 3a , b ) . the yield of sfgfp-1 from sfgfp(tag ) was 0.65 g from 10 cells , while the yield of sfgfp-(1)3 from sfgfp(tag)3 was 0.1 g per 10 cells ( supporting information figure 3 ; all yields are quoted per number of cells seeded , and were measured 48 h after transfection ) . combining erf1 e55d with an optimized pylrs / trnacua pair expression system enables efficient incorporation of multiple unnatural amino acids into recombinant proteins in mammalian cells . we find that the addition of the erf1 ( e55d ) to cells containing the pylrs / trnacua pair , grown in the presence of 2 , increases the incorporation of 2 into sfgfp(tag ) from 129% to 157% , and that the addition of the erf1 ( e55d ) quadruples the efficiency of producing of sfgfp-(2)3 from sfgfp(tag)3 from 11% to 43% ( figure 3c , d ) . this data , in combination with the no amino acid controls in figures 4a demonstrate the high fidelity incorporation of unnatural amino acids in the presence of erf1 ( e55d ) . ( b ) electrospray ionization mass spectrometry confirms the quantitative incorporation of unnatural amino acids 1 and 2 , at one or three sites in sfgfp ( see also supporting information figure 4 ) . we have defined the efficiency of unnatural amino acid incorporation relative to a natural translation control , allowing us to quantitatively benchmark improvements in unnatural amino acid incorporation efficiency . the optimized system we have created provides a 17- to 20-fold improvement in unnatural amino acid incorporation efficiency with amino acids 1 and 2 in sfgfp . for amino acid 1 , the incorporation efficiency is increased from 5% to 85% , while for amino acid 2 , the incorporation efficiency is increased from 7% to 157% of a no stop codon control . moreover , the optimized system increases the yield of proteins incorporating 1 and 2 at three positions from unmeasurably low levels to 12% and 43% of a no stop control , respectively . two factors contribute to the dramatic improvement in unnatural amino acid incorporation : the optimization of trnacua levels to optimize pylrs / trnacua expression ; and the development and use of engineered erf1 variants . while the incorporation of unnatural amino acids is quite efficient in response to a single amber codon using the optimized pylrs / trnacua system alone , the efficiency is further improved by the addition of erf1 ( e55d ) . the effect of the erf1 mutant on unnatural amino acid incorporation is more dramatic when incorporating unnatural amino acids at multiple sites , increasing the yield of protein containing amino acid 1 at three sites , 2- to 3-fold and the yield of protein containing 2 at three sites , 4-fold . our data demonstrate that unnatural amino acid incorporation in mammalian cells can be very efficient . moreover , our data provide the first demonstration that , despite erf1 recognizing all three stop codons , it is possible to engineer erf1 to selectively enhance the efficiency of unnatural amino acid incorporation in eukaryotic cells in response to the amber stop codon , without increasing readthrough of opal or ochre stop codons . future work will further investigate the generality of the approaches we report for the efficient incorporation of diverse unnatural amino acids at diverse sites in diverse proteins in eukaryotic cells . we anticipate that our approach may enable the efficient production of site - specifically modified therapeutic proteins , as well as the quantitative replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function .

anorexia nervosa ( an ) is a serious psychiatric disorder affecting mainly young females and it is associated with poor long - term outcome.1 according to the current version of the diagnostic and statistical manual of mental disorders , the essential features of an are a significantly low body weight for the developmental stage of the subject , an intense fear of gaining weight or persistent behavior that interferes with weight gain , and disturbance in experiencing body weight and shape.2 two different subtypes of the disorders are specified , the restricting type ( an - r ) , in which weight loss is accomplished exclusively through a massive reduction in food intake and excessive exercise over the previous 3 months , and the binge - eating / purging type ( an - be ) , in which binge eating is usually associated with purging through self - induced vomiting or misuse of laxatives and diuretics or enemas during the previous 3 months.2 despite neither diagnostic criteria nor reports on common associated psychopathology describe empathic alterations in an , clinical observation as well as some research data suggests that empathic difficulty could be part of the an phenotype.3 empathy is a complex multidimensional construct and a core component of social cognition . a schematic distinction of empathy includes : a ) sharing the emotional states of others ( affective empathy [ ae ] ) , and b ) understanding the perspective of another person ( cognitive empathy [ ce]).4 a phenomenon partially overlapping with empathy is theory of mind ( tom ) . tom consists of the ability to make inferences regarding others emotions ( affective or emotional tom ) or beliefs and motivations ( cognitive tom ) ; while the latter is strongly similar to ce , the former may be related to both affective and cognitive empathy.5 a considerable subgroup of an subjects with associated empathy disorders was revealed in a gillberg et al study:6 the authors described these patients as impaired in understanding the cognitive and emotional perspectives of other people and with a clinical picture suggestive of an autism spectrum disorder ( asd ) . subsequent reports confirmed autistic traits as predictive1 or comorbid factors in an patients:7 this has led some authors to consider an as a version of asd.8 however , an impaired empathic profile in an is not a consistent finding ; for example , a self - report evaluation of empathic abilities failed to detect statistically significant differences between an patients and healthy controls.9 on the other hand , tchan - turia et al10 first explored cognitive tom in women with an , revealing a clear impairment in a subgroup of these patients of which the study lacked the power to demonstrate statistically . later reports showed abnormalities in both affective and cognitive tom,11 in emotional tom only,12 or no relevant differences between adult patients with an and controls in emotional tom.13 finally , a recent investigation that evaluated , among other things , the social perception domain through a multidomain neuropsychological battery designed specifically for children and adolescents , reported an absence of deficit in this area in an - r patients.14 the assessment of empathic response can be performed through physiological measurements ( eg , heart rate or skin conductance registration ) , indirect indices ( facial , gestural , bodily , and vocal expression recognition ) , and psychological evaluation ( test administration , reports from significant others , and self - reported questionnaires ) . among self - reported psychological tests aimed at evaluating empathic functioning , the interpersonal reactivity index ( iri ) represents one of the most widely used and comprehensive instruments.4 in fact , different abnormalities in ce and ae levels , assessed through the iri , have been reported in a variety of psychiatric conditions , including psychopathy,15 borderline personality disorder,16 asperger syndrome,17 schizophrenia,18 major depressive disorder,19 bipolar disorder,20 and patients with alcohol dependence.21 to our knowledge , no study has evaluated empathy abnormalities in adolescents with an - r . in this study , we analyzed the empathic abilities of a homogeneous group of adolescents with an - r . as an - r subjects are characterized by starvation and associated internalizing psychopathology that can impact cognitive abilities,22 we specifically investigated the empathic profile controlling for disease severity and associated psychopathology . thirty - two adolescent females ( mean age standard deviation [ sd ] = 14.78 1.75 years ; range = 11.0017.33 years ) with an - r were consecutively recruited from the inpatient eating disorders unit at the irccs stella maris foundation . these adolescents ( all between 11 and 18 years ) were characterized by restrictive behaviors and weight loss purely by dieting without binge eating or purging behaviors . they fulfilled the criteria for the diagnosis of an - r according to the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv).23 patients with psychotic symptoms , intelligence quotient [ iq ] below 80 , medical pathology not correlated with the eating disorder , current or history of substance abuse , and significant intrinsic instability requiring constant medical care supervision ( such as severe bradycardia , dehydration , or electrolyte imbalance ) were excluded . the mean body mass index was 15.07 1.54 kg / m and the mean duration of illness was 19.56 18.38 months . twenty - seven patients ( 84.38% ) fulfilled the criteria for an axis i anxiety and/or mood disorder ( assessed through the italian version of the kiddie - sads - present and lifetime version [ k - sads - pl])24 and 3 of these 27 patients also met the criteria for an axis ii obsessive - compulsive personality disorder ( evaluated by the structured clinical interview for dsm - iv axis ii personality disorders).25 twelve patients ( 37.5% ) received psychopharmacological treatment with selective serotonin reuptake inhibitors and/or atypical antipsychotics and/or mood modulators , while the remaining 20 subjects were medication - nave . the an - r clinical sample was compared to a healthy control group ( hc ) composed of 41 healthy female adolescents recruited from students ( mean age = 14.02 1.69 years ; range = 11.0817.16 years ) of a middle and a high school in the metropolitan area of pisa . the following established measures , which were part of a larger suite of tests , were administered for this study . the iri is a 28-item self - reported questionnaire that allows a multi - dimensional assessment of empathy , which can be measured by two cognitive subscales ( perspective taking [ pt ] ; fantasy [ fs ] ) and two affective subscales ( empathic concern [ ec ] ; personal distress [ pd ] ) . participants respond to each item using a 5-point likert scale ranging from 2 does not describe me well to + 2 does describe me well . therefore the scores of each subscale range between 14 and + 14 points where higher scores indicate more empathic abilities . to address eating disorder severity , the following measures were considered : 1 ) the body mass index ( bmi ) calculated by dividing body weight in kilograms by height in meters squared ( kg / m ) ; 2 ) the eating attitudes test ( eat-26),27 a 26-item self - reported questionnaire of disordered eating patterns . the total score ranges from 0 to 78 ; a higher score reflects a greater degree of eating pathology , with a score over 20 indicating a possible eating problem ; 3 ) disease duration , defined as the time interval between the onset of the first eating disorder symptoms and the administration of the tests . to address psychopathological traits associated with the eating disorder , two questionnaires were used : 1 ) the child behavior checklist ( cbcl 618),28 a 118 item parent - reported measure designed to record the problem behaviors as well as the competencies of children or adolescents . each item describes a specific behavior and the parent is asked to rate its frequency on a 3-point likert scale . the scoring gives a summary profile including internalizing ( int ) , external - izing ( ext ) , and total problems ( tp ) , and a syndrome profile ( withdrawn , anxious / depressed , somatic complaints , social problems , thought problems , attention problems , delinquent behavior , and aggressive behavior ) ; 2 ) the youth self report ( ysr 1118 ) , an118 items self - reported questionnaire for adolescents from 11 to 18 years old.28 the ysr provides a summary profile and a syndrome profile paralleling those of the cbcl 618 . the study was carried out according to the standards for good ethical practice of the irccs stella maris foundation , and was approved by the educational board of each school of the hc group . patients and their parents completed all the tests ( iri , eat-26 , cbcl 618 , and ysr 1118 ) during the first days of the clinical assessment . hc , in addition to the iri , completed the eat-26 and the ysr 1118 , in order to rule out subjects with eating problems and/or axis i psychopathology , respectively . in fact , the ysr has been found to show a significant correlation with clinical dsm - iv diagnoses.2830 thus , subjects with an eat-26 score > 20 , and/or a score > 60 on int and/or ext and/or tp at the ysr , were excluded from the study . hc data were collected by a physician resident in child and adolescent psychiatry ( pf ) during school hours and informed consent was obtained from participants and their parents through the head teacher s office at each school . a student s t - test was used to compare differences in eat-26 , ysr , and iri mean scores between an - r and hc groups ; multivariate analysis of variance ( anova ) with eat-26 and with int , ext , and tp mean scores of the ysr as a covariate was used to identify iri scales discriminating between an - r and hc groups ; spearman correlations were adopted to assess the relationship between iri scales and 1 ) associated psychopathology ( cbcl ) , and 2 ) eating disorder severity ( bmi and disease duration ) , respectively . all statistical analyses were performed using spss 18.0 for windows ( ibm corporation , armonk , ny , usa ) . thirty - two adolescent females ( mean age standard deviation [ sd ] = 14.78 1.75 years ; range = 11.0017.33 years ) with an - r were consecutively recruited from the inpatient eating disorders unit at the irccs stella maris foundation . these adolescents ( all between 11 and 18 years ) were characterized by restrictive behaviors and weight loss purely by dieting without binge eating or purging behaviors . they fulfilled the criteria for the diagnosis of an - r according to the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv).23 patients with psychotic symptoms , intelligence quotient [ iq ] below 80 , medical pathology not correlated with the eating disorder , current or history of substance abuse , and significant intrinsic instability requiring constant medical care supervision ( such as severe bradycardia , dehydration , or electrolyte imbalance ) were excluded . the mean body mass index was 15.07 1.54 kg / m and the mean duration of illness was 19.56 18.38 months . twenty - seven patients ( 84.38% ) fulfilled the criteria for an axis i anxiety and/or mood disorder ( assessed through the italian version of the kiddie - sads - present and lifetime version [ k - sads - pl])24 and 3 of these 27 patients also met the criteria for an axis ii obsessive - compulsive personality disorder ( evaluated by the structured clinical interview for dsm - iv axis ii personality disorders).25 twelve patients ( 37.5% ) received psychopharmacological treatment with selective serotonin reuptake inhibitors and/or atypical antipsychotics and/or mood modulators , while the remaining 20 subjects were medication - nave . the an - r clinical sample was compared to a healthy control group ( hc ) composed of 41 healthy female adolescents recruited from students ( mean age = 14.02 1.69 years ; range = 11.0817.16 years ) of a middle and a high school in the metropolitan area of pisa . the following established measures , which were part of a larger suite of tests , were administered for this study . the iri is a 28-item self - reported questionnaire that allows a multi - dimensional assessment of empathy , which can be measured by two cognitive subscales ( perspective taking [ pt ] ; fantasy [ fs ] ) and two affective subscales ( empathic concern [ ec ] ; personal distress [ pd ] ) . participants respond to each item using a 5-point likert scale ranging from 2 does not describe me well to + 2 does describe me well . therefore the scores of each subscale range between 14 and + 14 points where higher scores indicate more empathic abilities . to address eating disorder severity , the following measures were considered : 1 ) the body mass index ( bmi ) calculated by dividing body weight in kilograms by height in meters squared ( kg / m ) ; 2 ) the eating attitudes test ( eat-26),27 a 26-item self - reported questionnaire of disordered eating patterns . the total score ranges from 0 to 78 ; a higher score reflects a greater degree of eating pathology , with a score over 20 indicating a possible eating problem ; 3 ) disease duration , defined as the time interval between the onset of the first eating disorder symptoms and the administration of the tests . to address psychopathological traits associated with the eating disorder , two questionnaires were used : 1 ) the child behavior checklist ( cbcl 618),28 a 118 item parent - reported measure designed to record the problem behaviors as well as the competencies of children or adolescents . each item describes a specific behavior and the parent is asked to rate its frequency on a 3-point likert scale . the scoring gives a summary profile including internalizing ( int ) , external - izing ( ext ) , and total problems ( tp ) , and a syndrome profile ( withdrawn , anxious / depressed , somatic complaints , social problems , thought problems , attention problems , delinquent behavior , and aggressive behavior ) ; 2 ) the youth self report ( ysr 1118 ) , an118 items self - reported questionnaire for adolescents from 11 to 18 years old.28 the ysr provides a summary profile and a syndrome profile paralleling those of the cbcl 618 . the study was carried out according to the standards for good ethical practice of the irccs stella maris foundation , and was approved by the educational board of each school of the hc group . written informed consent from a parent or guardian of each patient was obtained . patients and their parents completed all the tests ( iri , eat-26 , cbcl 618 , and ysr 1118 ) during the first days of the clinical assessment . hc , in addition to the iri , completed the eat-26 and the ysr 1118 , in order to rule out subjects with eating problems and/or axis i psychopathology , respectively . in fact , the ysr has been found to show a significant correlation with clinical dsm - iv diagnoses.2830 thus , subjects with an eat-26 score > 20 , and/or a score > 60 on int and/or ext and/or tp at the ysr , were excluded from the study . hc data were collected by a physician resident in child and adolescent psychiatry ( pf ) during school hours and informed consent was obtained from participants and their parents through the head teacher s office at each school . the iri is a 28-item self - reported questionnaire that allows a multi - dimensional assessment of empathy , which can be measured by two cognitive subscales ( perspective taking [ pt ] ; fantasy [ fs ] ) and two affective subscales ( empathic concern [ ec ] ; personal distress [ pd ] ) . participants respond to each item using a 5-point likert scale ranging from 2 does not describe me well to + 2 does describe me well . therefore the scores of each subscale range between 14 and + 14 points where higher scores indicate more empathic abilities . to address eating disorder severity , the following measures were considered : 1 ) the body mass index ( bmi ) calculated by dividing body weight in kilograms by height in meters squared ( kg / m ) ; 2 ) the eating attitudes test ( eat-26),27 a 26-item self - reported questionnaire of disordered eating patterns . the total score ranges from 0 to 78 ; a higher score reflects a greater degree of eating pathology , with a score over 20 indicating a possible eating problem ; 3 ) disease duration , defined as the time interval between the onset of the first eating disorder symptoms and the administration of the tests . to address psychopathological traits associated with the eating disorder , two questionnaires were used : 1 ) the child behavior checklist ( cbcl 618),28 a 118 item parent - reported measure designed to record the problem behaviors as well as the competencies of children or adolescents . each item describes a specific behavior and the parent is asked to rate its frequency on a 3-point likert scale . the scoring gives a summary profile including internalizing ( int ) , external - izing ( ext ) , and total problems ( tp ) , and a syndrome profile ( withdrawn , anxious / depressed , somatic complaints , social problems , thought problems , attention problems , delinquent behavior , and aggressive behavior ) ; 2 ) the youth self report ( ysr 1118 ) , an118 items self - reported questionnaire for adolescents from 11 to 18 years old.28 the ysr provides a summary profile and a syndrome profile paralleling those of the cbcl 618 . the study was carried out according to the standards for good ethical practice of the irccs stella maris foundation , and was approved by the educational board of each school of the hc group . patients and their parents completed all the tests ( iri , eat-26 , cbcl 618 , and ysr 1118 ) during the first days of the clinical assessment . hc , in addition to the iri , completed the eat-26 and the ysr 1118 , in order to rule out subjects with eating problems and/or axis i psychopathology , respectively . in fact , the ysr has been found to show a significant correlation with clinical dsm - iv diagnoses.2830 thus , subjects with an eat-26 score > 20 , and/or a score > 60 on int and/or ext and/or tp at the ysr , were excluded from the study . hc data were collected by a physician resident in child and adolescent psychiatry ( pf ) during school hours and informed consent was obtained from participants and their parents through the head teacher s office at each school . a student s t - test was used to compare differences in eat-26 , ysr , and iri mean scores between an - r and hc groups ; multivariate analysis of variance ( anova ) with eat-26 and with int , ext , and tp mean scores of the ysr as a covariate was used to identify iri scales discriminating between an - r and hc groups ; spearman correlations were adopted to assess the relationship between iri scales and 1 ) associated psychopathology ( cbcl ) , and 2 ) eating disorder severity ( bmi and disease duration ) , respectively . all statistical analyses were performed using spss 18.0 for windows ( ibm corporation , armonk , ny , usa ) . independent t - test analysis indicated no significant differences between the an - r and the hc groups in age ( t=1.88 ; p=0.065 ) . the two groups showed significant differences between eat-26 ( t=8.47 ; p<0.001 ) and ysr ( int : t=4.96 ; p<0.001 ; ext : t=2.68 ; p=0.009 ; tp : t=3.78 ; p<0.001 ) scores . to examine the cognitive versus affective aspects of empathy , the total scores for the cognitive ( pt + fs ) scales and the affective ( ec + pd ) scales were calculated . as observed in figure 1 , an - r had a significantly lower ce mean score than hc ( an - r mean score : 0.44 6.87 ; hc mean score : 5.24 6.45 ; t = 3.0 ; p=0.003 ) . the an - r group scored significantly lower on both pt ( t = 2.36 ; p=0.021 ) and fs ( t = 2.18 ; p=0.032 ) scales . the two groups did nt show significant differences on ae mean score ( an - r mean score : 4.69 7.08 ; hc mean score : 4.20 4.75 ; t = 0.35 ; p=0.725 ) and the two related subscales ( see table 1 ) . significant differences between groups with respect to ce ( f=5.19 ; p=0.008 ) and the pt subscale ( f=3.70 ; p=0.03 ) when eat-26 mean scores were used as a covariate ; no significant differences on the fs subscale ( f=2.61 ; p=0.081 ) were obtained . significant differences between groups in iri when ysr mean scores were used as a covariate were noted . specifically , when int was used as a covariate : ce ( f=5.15 ; p=0.008 ) , pt ( f=2.41 ; p=0.098 ) , fs ( f=3.52 ; p=0.035 ) ; when ext was used as a covariate : ce ( f=5.08 ; p=0.009 ) , pt ( f=5.24 ; p=0.008 ) , fs ( f=2.29 ; p=0.109 ) ; when tp was used as a covariate : ce ( f=4.46 ; p=0.015 ) , pt ( f=2.92 ; p=0.061 ) , fs ( f=3.25 ; p=0.045 ) . no statistically significant associations between cognitive empathy and : 1 ) bmi ( rho=0.32 ; p=0.069 ) ; 2 ) disease duration ( rho=0.07 ; p=0.69 ) ; or 3 ) cbcl 618 ( int : rho=0.23 ; p=0.213 ; ext = 0.05 ; p=0.806 ; tp = 0.16 ; p=0.401 ) were detected . to examine the cognitive versus affective aspects of empathy , the total scores for the cognitive ( pt + fs ) scales and the affective ( ec + pd ) scales were calculated . as observed in figure 1 , an - r had a significantly lower ce mean score than hc ( an - r mean score : 0.44 6.87 ; hc mean score : 5.24 6.45 ; t = 3.0 ; p=0.003 ) . the an - r group scored significantly lower on both pt ( t = 2.36 ; p=0.021 ) and fs ( t = 2.18 ; p=0.032 ) scales . the two groups did nt show significant differences on ae mean score ( an - r mean score : 4.69 7.08 ; hc mean score : 4.20 4.75 ; t = 0.35 ; p=0.725 ) and the two related subscales ( see table 1 ) . significant differences between groups with respect to ce ( f=5.19 ; p=0.008 ) and the pt subscale ( f=3.70 ; p=0.03 ) when eat-26 mean scores were used as a covariate ; no significant differences on the fs subscale ( f=2.61 ; p=0.081 ) were obtained . significant differences between groups in iri when ysr mean scores were used as a covariate were noted . specifically , when int was used as a covariate : ce ( f=5.15 ; p=0.008 ) , pt ( f=2.41 ; p=0.098 ) , fs ( f=3.52 ; p=0.035 ) ; when ext was used as a covariate : ce ( f=5.08 ; p=0.009 ) , pt ( f=5.24 ; p=0.008 ) , fs ( f=2.29 ; p=0.109 ) ; when tp was used as a covariate : ce ( f=4.46 ; p=0.015 ) , pt ( f=2.92 ; p=0.061 ) , fs ( f=3.25 ; p=0.045 ) . no statistically significant associations between cognitive empathy and : 1 ) bmi ( rho=0.32 ; p=0.069 ) ; 2 ) disease duration ( rho=0.07 ; p=0.69 ) ; or 3 ) cbcl 618 ( int : rho=0.23 ; p=0.213 ; ext = 0.05 ; p=0.806 ; tp = 0.16 ; p=0.401 ) were detected . this study adds to the limited literature regarding empathy in an - r and the results show statistically significant differences between patients with an - r and the hc with respect to empathic profile assessed using the iri.4 the main finding arising from the current investigation , performed on a carefully selected population of adolescent females with an - r , is the impairment in empathic abilities limited to the cognitive domain , while sparing the affective component . cognitive capacity is the ability to take the perspective of the other person in real ( perspective taking scale ) and in fictional ( fantasy scale ) situations . in particular , a low score on the pt scale is indicative of an impairment in interpersonal functioning,4 and converges with the repeated findings of altered social skills in anorectic patients.6 moreover , since executive functions contribute to the effective development of ce31 and , at the same time , are consistently reported as impaired in an subjects,32 further research is warranted to clarify the possibility that executive functions deficits interfere with the reduced ce abilities of an patients . only two other studies assessed empathic abilities in an - r through the iri.33,34 in contrast with our results , these investigations reported no significant differences between patients and controls33 and higher ratings for emotional empathy in an subjects than hc participants.34 moreover , a mixed sample of both an - r and an - be patients were enrolled in this latter study , whereas in the current investigation subjects who engaged in binging / purging behaviors were excluded . hence , it is difficult to compare our work with the other two , as they do present substantial differences not only with respect to sample age ( adolescents in our report versus adults in guttman and laporte , and beadle et al)33,34 and clinical severity ( inpatients versus outpatients ) , but also as to the methods of score attribution . a lower ce and similar ae scores , compared to controls , is instead a finding that our an patients have commonalities with asperger subjects,17 providing further support for the similarities between the two disorders.7,8 results point out specific ce impairment in adolescents with an - r . in particular , this finding suggests three key issues : first , on the basis of its independence from associated psychopathology assessed through the ysr , a ce deficit could be considered a specific marker of the adolescent an - r , not mediated by comorbid symptoms ; second , the independence of a ce deficit from the severity of illness suggests that it is not directly attributable to the profound effects of starvation on neuropsychological functioning.35 in the future , longitudinal studies comparing ill and recovered an - r patients may clarify whether the reduction in ce is a state - correlated characteristic or if it also persists after recovery . third , the absence of correlation between ce scores and disease duration indicates the possible presence of a deficit in ce from the beginning of the disorder or even before an - r onset . the hypothesis of ce impairment as a precursor of an - r is consistent with other studies on the personality traits involved in creating a predisposition to developing this disorder.1,36 in this view , low ce could become one of the risk factors that precede the onset of a restrictive eating disorder . however , a prospective exploration of empathic skills in a non - clinical population is required to sustain the hypothesis of an association with the subsequent development of an . finally , empathic impairment limited to the cognitive domain supports its functional independence from the affective one ; neuroimaging ( for a review , see decety and meyer)37 as well as lesion studies5 sustain this hypothesis . in particular , two distinct brain systems for cognitive and ae have been described : the ventromedial prefrontal cortex is involved in ce , whereas the inferior frontal gyrus is a part of the ae network . interestingly , ventromedial prefrontal cortex anomalies have been frequently reported in neuroimaging studies conducted in an patients ( eg , uher et al);38,39 future functional mri research is needed to investigate the response to empathy - eliciting stimuli in an - r . the main result of this study is that adolescents with an - r type do not show global empathy abilities deficit , but rather a specific ce impairment . specifically , self - reported measurements lack of objectivity , can be biased by denial or exaggeration of symptoms , as well as social desirability , and only reflect the self - perception of empathic characteristics . however , this issue was mitigated in the present study by having a clinical psychologist remain with the subject in order to clarify possible misunderstandings during the administration of the iri scale . secondly , the control subjects were screened for possible axis i psychopathology through the ysr . however , besides the possible false positive and false negative results , the ysr does not evaluate personality disorders that could be present in the subjects enrolled in the study as controls . the vast majority ( 84.4% ) of an - r patients involved in this study also meet diagnostic criteria for an additional internalizing diagnosis assessed through the k - sads - pl.24 since internalizing disorders could impact empathic abilities,19 we can not rule out that the low levels of ce may be due to the influence of comorbid psychiatric symptoms rather than to an - r itself . also , while subjects with major depressive disorders assessed through the iri reported significantly lower levels of both cognitive and affective empathy,19 our an - r patients showed a selective impairment of ce . however , in order to investigate the specific role of internalizing symptoms on empathic abilities , future studies should include an additional control sample composed of depressed / anxious subjects matched for demographic characteristics . further , the selective inclusion criteria of the patient group imply a relatively small sample size that limits the statistical power of results . notwithstanding the problematic issues raised above , these data , if supported by a larger replication study , could have a 3-fold implications for an - r : a ) clinical , since the overall management of these patients should take into account the compromised empathic skills ; b ) therapeutic , with the development of specific intervention goals focused on ce enhancement ; and c ) preventive , with the aim of early individuation of subjects with ce deficits , a marker that , in addition to other susceptibility factors , may play an important role in the development and maintenance of the anorexic disorder .

backgrounda growing , but conflicting body of literature suggests altered empathic abilities in subjects with anorexia nervosa - restricting type ( an - r ) . this study aims to characterize the cognitive and affective empathic profiles of adolescents with purely an-r.methodsas part of a standardized clinical and research protocol , the interpersonal reactivity index ( iri ) , a valid and reliable self - reported instrument to measure empathy , was administered to 32 female adolescents with an - r and in 41 healthy controls ( hc ) comparisons , matched for age and gender . correlational analyses were performed to evaluate the links between empathy scores and psychopathological measures.resultspatients scored significantly lower than hc on cognitive empathy ( ce ) , while they did not differ from controls on affective empathy ( ae ) . the deficit in ce was not related to either disease severity nor was it related to associated psychopathology.conclusionthese results , albeit preliminary , suggest that a dysfunctional pattern of ce capacity may be a stable trait of an - r that should be taken into account not only for the clinical management , but also in preventive and therapeutic intervention .

Introduction Materials and methods Participants Measures Assessment of empathic abilities Assessment of eating disorder severity Assessment of possible psychopathological traits associated to the eating disorder Procedure Data analysis Results Cognitive and affective empathy Cognitive empathy, disease severity, and associated psychopathology Discussion Conclusion

anorexia nervosa ( an ) is a serious psychiatric disorder affecting mainly young females and it is associated with poor long - term outcome.1 according to the current version of the diagnostic and statistical manual of mental disorders , the essential features of an are a significantly low body weight for the developmental stage of the subject , an intense fear of gaining weight or persistent behavior that interferes with weight gain , and disturbance in experiencing body weight and shape.2 two different subtypes of the disorders are specified , the restricting type ( an - r ) , in which weight loss is accomplished exclusively through a massive reduction in food intake and excessive exercise over the previous 3 months , and the binge - eating / purging type ( an - be ) , in which binge eating is usually associated with purging through self - induced vomiting or misuse of laxatives and diuretics or enemas during the previous 3 months.2 despite neither diagnostic criteria nor reports on common associated psychopathology describe empathic alterations in an , clinical observation as well as some research data suggests that empathic difficulty could be part of the an phenotype.3 empathy is a complex multidimensional construct and a core component of social cognition . a schematic distinction of empathy includes : a ) sharing the emotional states of others ( affective empathy [ ae ] ) , and b ) understanding the perspective of another person ( cognitive empathy [ ce]).4 a phenomenon partially overlapping with empathy is theory of mind ( tom ) . tom consists of the ability to make inferences regarding others emotions ( affective or emotional tom ) or beliefs and motivations ( cognitive tom ) ; while the latter is strongly similar to ce , the former may be related to both affective and cognitive empathy.5 a considerable subgroup of an subjects with associated empathy disorders was revealed in a gillberg et al study:6 the authors described these patients as impaired in understanding the cognitive and emotional perspectives of other people and with a clinical picture suggestive of an autism spectrum disorder ( asd ) . subsequent reports confirmed autistic traits as predictive1 or comorbid factors in an patients:7 this has led some authors to consider an as a version of asd.8 however , an impaired empathic profile in an is not a consistent finding ; for example , a self - report evaluation of empathic abilities failed to detect statistically significant differences between an patients and healthy controls.9 on the other hand , tchan - turia et al10 first explored cognitive tom in women with an , revealing a clear impairment in a subgroup of these patients of which the study lacked the power to demonstrate statistically . later reports showed abnormalities in both affective and cognitive tom,11 in emotional tom only,12 or no relevant differences between adult patients with an and controls in emotional tom.13 finally , a recent investigation that evaluated , among other things , the social perception domain through a multidomain neuropsychological battery designed specifically for children and adolescents , reported an absence of deficit in this area in an - r patients.14 the assessment of empathic response can be performed through physiological measurements ( eg , heart rate or skin conductance registration ) , indirect indices ( facial , gestural , bodily , and vocal expression recognition ) , and psychological evaluation ( test administration , reports from significant others , and self - reported questionnaires ) . among self - reported psychological tests aimed at evaluating empathic functioning , the interpersonal reactivity index ( iri ) represents one of the most widely used and comprehensive instruments.4 in fact , different abnormalities in ce and ae levels , assessed through the iri , have been reported in a variety of psychiatric conditions , including psychopathy,15 borderline personality disorder,16 asperger syndrome,17 schizophrenia,18 major depressive disorder,19 bipolar disorder,20 and patients with alcohol dependence.21 to our knowledge , no study has evaluated empathy abnormalities in adolescents with an - r . in this study , we analyzed the empathic abilities of a homogeneous group of adolescents with an - r . as an - r subjects are characterized by starvation and associated internalizing psychopathology that can impact cognitive abilities,22 we specifically investigated the empathic profile controlling for disease severity and associated psychopathology . thirty - two adolescent females ( mean age standard deviation [ sd ] = 14.78 1.75 years ; range = 11.0017.33 years ) with an - r were consecutively recruited from the inpatient eating disorders unit at the irccs stella maris foundation . they fulfilled the criteria for the diagnosis of an - r according to the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv).23 patients with psychotic symptoms , intelligence quotient [ iq ] below 80 , medical pathology not correlated with the eating disorder , current or history of substance abuse , and significant intrinsic instability requiring constant medical care supervision ( such as severe bradycardia , dehydration , or electrolyte imbalance ) were excluded . the an - r clinical sample was compared to a healthy control group ( hc ) composed of 41 healthy female adolescents recruited from students ( mean age = 14.02 1.69 years ; range = 11.0817.16 years ) of a middle and a high school in the metropolitan area of pisa . the following established measures , which were part of a larger suite of tests , were administered for this study . the iri is a 28-item self - reported questionnaire that allows a multi - dimensional assessment of empathy , which can be measured by two cognitive subscales ( perspective taking [ pt ] ; fantasy [ fs ] ) and two affective subscales ( empathic concern [ ec ] ; personal distress [ pd ] ) . to address eating disorder severity , the following measures were considered : 1 ) the body mass index ( bmi ) calculated by dividing body weight in kilograms by height in meters squared ( kg / m ) ; 2 ) the eating attitudes test ( eat-26),27 a 26-item self - reported questionnaire of disordered eating patterns . to address psychopathological traits associated with the eating disorder , two questionnaires were used : 1 ) the child behavior checklist ( cbcl 618),28 a 118 item parent - reported measure designed to record the problem behaviors as well as the competencies of children or adolescents . the scoring gives a summary profile including internalizing ( int ) , external - izing ( ext ) , and total problems ( tp ) , and a syndrome profile ( withdrawn , anxious / depressed , somatic complaints , social problems , thought problems , attention problems , delinquent behavior , and aggressive behavior ) ; 2 ) the youth self report ( ysr 1118 ) , an118 items self - reported questionnaire for adolescents from 11 to 18 years old.28 the ysr provides a summary profile and a syndrome profile paralleling those of the cbcl 618 . patients and their parents completed all the tests ( iri , eat-26 , cbcl 618 , and ysr 1118 ) during the first days of the clinical assessment . in fact , the ysr has been found to show a significant correlation with clinical dsm - iv diagnoses.2830 thus , subjects with an eat-26 score > 20 , and/or a score > 60 on int and/or ext and/or tp at the ysr , were excluded from the study . a student s t - test was used to compare differences in eat-26 , ysr , and iri mean scores between an - r and hc groups ; multivariate analysis of variance ( anova ) with eat-26 and with int , ext , and tp mean scores of the ysr as a covariate was used to identify iri scales discriminating between an - r and hc groups ; spearman correlations were adopted to assess the relationship between iri scales and 1 ) associated psychopathology ( cbcl ) , and 2 ) eating disorder severity ( bmi and disease duration ) , respectively . thirty - two adolescent females ( mean age standard deviation [ sd ] = 14.78 1.75 years ; range = 11.0017.33 years ) with an - r were consecutively recruited from the inpatient eating disorders unit at the irccs stella maris foundation . they fulfilled the criteria for the diagnosis of an - r according to the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv).23 patients with psychotic symptoms , intelligence quotient [ iq ] below 80 , medical pathology not correlated with the eating disorder , current or history of substance abuse , and significant intrinsic instability requiring constant medical care supervision ( such as severe bradycardia , dehydration , or electrolyte imbalance ) were excluded . the an - r clinical sample was compared to a healthy control group ( hc ) composed of 41 healthy female adolescents recruited from students ( mean age = 14.02 1.69 years ; range = 11.0817.16 years ) of a middle and a high school in the metropolitan area of pisa . the following established measures , which were part of a larger suite of tests , were administered for this study . the iri is a 28-item self - reported questionnaire that allows a multi - dimensional assessment of empathy , which can be measured by two cognitive subscales ( perspective taking [ pt ] ; fantasy [ fs ] ) and two affective subscales ( empathic concern [ ec ] ; personal distress [ pd ] ) . to address eating disorder severity , the following measures were considered : 1 ) the body mass index ( bmi ) calculated by dividing body weight in kilograms by height in meters squared ( kg / m ) ; 2 ) the eating attitudes test ( eat-26),27 a 26-item self - reported questionnaire of disordered eating patterns . the scoring gives a summary profile including internalizing ( int ) , external - izing ( ext ) , and total problems ( tp ) , and a syndrome profile ( withdrawn , anxious / depressed , somatic complaints , social problems , thought problems , attention problems , delinquent behavior , and aggressive behavior ) ; 2 ) the youth self report ( ysr 1118 ) , an118 items self - reported questionnaire for adolescents from 11 to 18 years old.28 the ysr provides a summary profile and a syndrome profile paralleling those of the cbcl 618 . patients and their parents completed all the tests ( iri , eat-26 , cbcl 618 , and ysr 1118 ) during the first days of the clinical assessment . in fact , the ysr has been found to show a significant correlation with clinical dsm - iv diagnoses.2830 thus , subjects with an eat-26 score > 20 , and/or a score > 60 on int and/or ext and/or tp at the ysr , were excluded from the study . the iri is a 28-item self - reported questionnaire that allows a multi - dimensional assessment of empathy , which can be measured by two cognitive subscales ( perspective taking [ pt ] ; fantasy [ fs ] ) and two affective subscales ( empathic concern [ ec ] ; personal distress [ pd ] ) . to address eating disorder severity , the following measures were considered : 1 ) the body mass index ( bmi ) calculated by dividing body weight in kilograms by height in meters squared ( kg / m ) ; 2 ) the eating attitudes test ( eat-26),27 a 26-item self - reported questionnaire of disordered eating patterns . the scoring gives a summary profile including internalizing ( int ) , external - izing ( ext ) , and total problems ( tp ) , and a syndrome profile ( withdrawn , anxious / depressed , somatic complaints , social problems , thought problems , attention problems , delinquent behavior , and aggressive behavior ) ; 2 ) the youth self report ( ysr 1118 ) , an118 items self - reported questionnaire for adolescents from 11 to 18 years old.28 the ysr provides a summary profile and a syndrome profile paralleling those of the cbcl 618 . in fact , the ysr has been found to show a significant correlation with clinical dsm - iv diagnoses.2830 thus , subjects with an eat-26 score > 20 , and/or a score > 60 on int and/or ext and/or tp at the ysr , were excluded from the study . a student s t - test was used to compare differences in eat-26 , ysr , and iri mean scores between an - r and hc groups ; multivariate analysis of variance ( anova ) with eat-26 and with int , ext , and tp mean scores of the ysr as a covariate was used to identify iri scales discriminating between an - r and hc groups ; spearman correlations were adopted to assess the relationship between iri scales and 1 ) associated psychopathology ( cbcl ) , and 2 ) eating disorder severity ( bmi and disease duration ) , respectively . independent t - test analysis indicated no significant differences between the an - r and the hc groups in age ( t=1.88 ; p=0.065 ) . to examine the cognitive versus affective aspects of empathy , the total scores for the cognitive ( pt + fs ) scales and the affective ( ec + pd ) scales were calculated . as observed in figure 1 , an - r had a significantly lower ce mean score than hc ( an - r mean score : 0.44 6.87 ; hc mean score : 5.24 6.45 ; t = 3.0 ; p=0.003 ) . the an - r group scored significantly lower on both pt ( t = 2.36 ; p=0.021 ) and fs ( t = 2.18 ; p=0.032 ) scales . the two groups did nt show significant differences on ae mean score ( an - r mean score : 4.69 7.08 ; hc mean score : 4.20 4.75 ; t = 0.35 ; p=0.725 ) and the two related subscales ( see table 1 ) . to examine the cognitive versus affective aspects of empathy , the total scores for the cognitive ( pt + fs ) scales and the affective ( ec + pd ) scales were calculated . as observed in figure 1 , an - r had a significantly lower ce mean score than hc ( an - r mean score : 0.44 6.87 ; hc mean score : 5.24 6.45 ; t = 3.0 ; p=0.003 ) . the an - r group scored significantly lower on both pt ( t = 2.36 ; p=0.021 ) and fs ( t = 2.18 ; p=0.032 ) scales . the two groups did nt show significant differences on ae mean score ( an - r mean score : 4.69 7.08 ; hc mean score : 4.20 4.75 ; t = 0.35 ; p=0.725 ) and the two related subscales ( see table 1 ) . this study adds to the limited literature regarding empathy in an - r and the results show statistically significant differences between patients with an - r and the hc with respect to empathic profile assessed using the iri.4 the main finding arising from the current investigation , performed on a carefully selected population of adolescent females with an - r , is the impairment in empathic abilities limited to the cognitive domain , while sparing the affective component . in particular , a low score on the pt scale is indicative of an impairment in interpersonal functioning,4 and converges with the repeated findings of altered social skills in anorectic patients.6 moreover , since executive functions contribute to the effective development of ce31 and , at the same time , are consistently reported as impaired in an subjects,32 further research is warranted to clarify the possibility that executive functions deficits interfere with the reduced ce abilities of an patients . only two other studies assessed empathic abilities in an - r through the iri.33,34 in contrast with our results , these investigations reported no significant differences between patients and controls33 and higher ratings for emotional empathy in an subjects than hc participants.34 moreover , a mixed sample of both an - r and an - be patients were enrolled in this latter study , whereas in the current investigation subjects who engaged in binging / purging behaviors were excluded . hence , it is difficult to compare our work with the other two , as they do present substantial differences not only with respect to sample age ( adolescents in our report versus adults in guttman and laporte , and beadle et al)33,34 and clinical severity ( inpatients versus outpatients ) , but also as to the methods of score attribution . a lower ce and similar ae scores , compared to controls , is instead a finding that our an patients have commonalities with asperger subjects,17 providing further support for the similarities between the two disorders.7,8 results point out specific ce impairment in adolescents with an - r . in particular , this finding suggests three key issues : first , on the basis of its independence from associated psychopathology assessed through the ysr , a ce deficit could be considered a specific marker of the adolescent an - r , not mediated by comorbid symptoms ; second , the independence of a ce deficit from the severity of illness suggests that it is not directly attributable to the profound effects of starvation on neuropsychological functioning.35 in the future , longitudinal studies comparing ill and recovered an - r patients may clarify whether the reduction in ce is a state - correlated characteristic or if it also persists after recovery . third , the absence of correlation between ce scores and disease duration indicates the possible presence of a deficit in ce from the beginning of the disorder or even before an - r onset . the hypothesis of ce impairment as a precursor of an - r is consistent with other studies on the personality traits involved in creating a predisposition to developing this disorder.1,36 in this view , low ce could become one of the risk factors that precede the onset of a restrictive eating disorder . however , a prospective exploration of empathic skills in a non - clinical population is required to sustain the hypothesis of an association with the subsequent development of an . in particular , two distinct brain systems for cognitive and ae have been described : the ventromedial prefrontal cortex is involved in ce , whereas the inferior frontal gyrus is a part of the ae network . the main result of this study is that adolescents with an - r type do not show global empathy abilities deficit , but rather a specific ce impairment . specifically , self - reported measurements lack of objectivity , can be biased by denial or exaggeration of symptoms , as well as social desirability , and only reflect the self - perception of empathic characteristics . the vast majority ( 84.4% ) of an - r patients involved in this study also meet diagnostic criteria for an additional internalizing diagnosis assessed through the k - sads - pl.24 since internalizing disorders could impact empathic abilities,19 we can not rule out that the low levels of ce may be due to the influence of comorbid psychiatric symptoms rather than to an - r itself . also , while subjects with major depressive disorders assessed through the iri reported significantly lower levels of both cognitive and affective empathy,19 our an - r patients showed a selective impairment of ce . however , in order to investigate the specific role of internalizing symptoms on empathic abilities , future studies should include an additional control sample composed of depressed / anxious subjects matched for demographic characteristics . notwithstanding the problematic issues raised above , these data , if supported by a larger replication study , could have a 3-fold implications for an - r : a ) clinical , since the overall management of these patients should take into account the compromised empathic skills ; b ) therapeutic , with the development of specific intervention goals focused on ce enhancement ; and c ) preventive , with the aim of early individuation of subjects with ce deficits , a marker that , in addition to other susceptibility factors , may play an important role in the development and maintenance of the anorexic disorder .

an aneurysm arising from the internal carotid artery ( ica ) at the origin of the anterior choroidal artery ( acha ) is one of the most easily accessible surgical lesions that can be reached via a pterional or even a supraorbital keyhole approach without much difficulty . notwithstanding , the surgical results can still be unsatisfactory due to inadvertent occlusion of the acha , leading to a permanent acha syndrome with an incidence of 5 to 16%1,5,6,10,17,20 ) . furthermore , the low incidence of acha aneurysms has hampered the accumulation of surgical experience with infrequent publications on the surgical outcomes of acha aneurysms . however , recent advancements in surgical techniques and intraoperative monitoring to preserve the acha have decreased ischemic complications and improved surgical outcomes . accordingly , the authors reviewed a single - institution surgical experience based on 62 patients who underwent surgical clipping for an acha aneurysm to document the ischemic complications and surgical outcomes . the design of this study was a retrospective review of data from a series of 62 consecutive patients who underwent surgical clipping of an acha aneurysm by a single surgeon ( j.p . ) between november 2004 and june 2012 . meanwhile , during this same period , other 12 patients with an acha aneurysm were treated using endovascular coiling . information was obtained from the patient charts , surgical reports , video records , reviews of radiological investigations , and notes from follow - up visits to investigate the ischemic complications and surgical outcomes . a detailed description of the supraorbital keyhole approach has already been published elsewhere by the authors12,13 ) . frontal lobe retraction after dissecting the most proximal part of the sylvian fissure allows visualization up to the carotid bifurcation . the arachnoid is dissected along the superior wall of the ica and rolled laterally to expose the aneurysm base and origins of the posterior communicating artery ( pcoa ) and acha . a few small vessels arising from the ica can be observed and should be preserved as they can also supply the acha territory . determining the exact origin of the acha is crucial and can only be confirmed by rigorous visual scrutiny under high microscopic magnification . the origin of the acha is most commonly located at the inferior or posterior aspect of the aneurysm . when the acha origin is posterior to the aneurysm obscuring the artery from view , the aneurysm can be compressed and manipulated using a blunt dissector to reveal the hidden acha origin after softening the aneurysm by temporary ica clipping . in cases of a ruptured aneurysm , tentative clipping at the rupture point or trapping the aneurysm using proximal and distal ica clips can facilitate dissection and manipulation of the aneurysm . when placing the aneurysm clips , utmost effort must be made to avoid compromising the acha origin . if the acha arises inferior to the aneurysm , a clip is applied perpendicular to the axis of the ica . the proximal blade of the clip is positioned a small distance away from the origin of the acha so as not to constrict the acha origin , while the distal blade is positioned nuzzled close to the ica ( fig . meanwhile , when the acha arises posterior to the aneurysm base , the acha origin is visualized by gentle compression of the aneurysm , while avoiding any inadvertent injury to the neck of the aneurysm . a straight clip is then applied across the neck of the aneurysm perpendicular to the axis of the ica , being tilted laterally away from the origin of the acha ( fig . 2 ) . when manipulation of the aneurysm is limited by a thick wall or large size of the aneurysm furthermore , even when the acha origin is not visible behind the aneurysm , its presence should still be assumed present until proven otherwise and the clip only placed in a safe area . intraoperative monitoring , such as a motor evoked potentials ( meps ) , is crucial in this situation . after placing the clip , the clip blades are inspected to ensure complete occlusion of the aneurysm , and that the acha is not compromised and the oculomotor nerve is free . additional verification of the acha blood flow can be obtained using microvascular doppler sonography , indocyanine green near - infrared angiography , and meps2,3,7 ) . if the dome of the aneurysm is adherent to the oculomotor nerve , it should be divided or punctured with microscissors rather than dissected from the nerve in order to avoid nerve injury . the statistical analyses were performed with the aid of an spss package ( version 18 for windows ; spss , inc . , chicago , il , usa ) . a -test was used to evaluate the affects of the preoperative world federation of neurologic surgeons ( wfns ) grade on the clinical outcomes ( mrs ) . the design of this study was a retrospective review of data from a series of 62 consecutive patients who underwent surgical clipping of an acha aneurysm by a single surgeon ( j.p . ) between november 2004 and june 2012 . meanwhile , during this same period , other 12 patients with an acha aneurysm were treated using endovascular coiling . information was obtained from the patient charts , surgical reports , video records , reviews of radiological investigations , and notes from follow - up visits to investigate the ischemic complications and surgical outcomes . the acha aneurysms were approached using a pterional or supraorbital keyhole approach . a detailed description of the supraorbital keyhole approach has already been published elsewhere by the authors12,13 ) . frontal lobe retraction after dissecting the most proximal part of the sylvian fissure allows visualization up to the carotid bifurcation . the arachnoid is dissected along the superior wall of the ica and rolled laterally to expose the aneurysm base and origins of the posterior communicating artery ( pcoa ) and acha . a few small vessels arising from the ica can be observed and should be preserved as they can also supply the acha territory . determining the exact origin of the acha is crucial and can only be confirmed by rigorous visual scrutiny under high microscopic magnification . the origin of the acha is most commonly located at the inferior or posterior aspect of the aneurysm . when the acha origin is posterior to the aneurysm obscuring the artery from view , the aneurysm can be compressed and manipulated using a blunt dissector to reveal the hidden acha origin after softening the aneurysm by temporary ica clipping . in cases of a ruptured aneurysm , tentative clipping at the rupture point or trapping the aneurysm using proximal and distal ica clips can facilitate dissection and manipulation of the aneurysm . when placing the aneurysm clips , utmost effort must be made to avoid compromising the acha origin . if the acha arises inferior to the aneurysm , a clip is applied perpendicular to the axis of the ica . the proximal blade of the clip is positioned a small distance away from the origin of the acha so as not to constrict the acha origin , while the distal blade is positioned nuzzled close to the ica ( fig . meanwhile , when the acha arises posterior to the aneurysm base , the acha origin is visualized by gentle compression of the aneurysm , while avoiding any inadvertent injury to the neck of the aneurysm . a straight clip is then applied across the neck of the aneurysm perpendicular to the axis of the ica , being tilted laterally away from the origin of the acha ( fig . 2 ) . when manipulation of the aneurysm is limited by a thick wall or large size of the aneurysm , the use of an endoscope can be helpful . furthermore , even when the acha origin is not visible behind the aneurysm , its presence should still be assumed present until proven otherwise and the clip only placed in a safe area . intraoperative monitoring , such as a motor evoked potentials ( meps ) , is crucial in this situation . after placing the clip , the clip blades are inspected to ensure complete occlusion of the aneurysm , and that the acha is not compromised and the oculomotor nerve is free . additional verification of the acha blood flow can be obtained using microvascular doppler sonography , indocyanine green near - infrared angiography , and meps2,3,7 ) . if the dome of the aneurysm is adherent to the oculomotor nerve , it should be divided or punctured with microscissors rather than dissected from the nerve in order to avoid nerve injury . the statistical analyses were performed with the aid of an spss package ( version 18 for windows ; spss , inc . , chicago , il , usa ) . a -test was used to evaluate the affects of the preoperative world federation of neurologic surgeons ( wfns ) grade on the clinical outcomes ( mrs ) . the clinical and angiographic characteristics of the 62 patients with an acha aneurysm are summarized in table 1 . the patient ages ranged from 32 to 75 years [ meanstandard deviation ( sd ) : 54.99.7 yr ] . thirty - six ( 58.1% ) patients presented with an acute subarachnoid hemorrhage related to a ruptured acha aneurysm ( n=21 ) or other ruptured aneurysm ( n=15 ) , while 26 patients had an unruptured acha aneurysm that was asymptomatic ( n=24 ) or symptomatic ( n=2 ) with oculomotor nerve palsy . thirty - two ( 51.6% ) patients presented with wfns grade 1 to 3 , while 4 ( 6.5% ) patients presented with wfns grade 4 to 5 . in the preoperative angiograms , the aneurysm height , i.e. the maximal perpendicular distance from the neck plane to the top of the aneurysm dome , ranged from 3 to 13 mm ( meansd : 6.02.2 mm ) , while the maximum transverse diameter of the aneurysm neck ranged from 2 to 7 mm ( meansd : 3.90.8 mm ) and the maximum aneurysm diameter , which is the largest of all cross - sections along the height of the aneurysm , ranged from 3 to 12 mm ( meansd : 4.51.4 mm ) . the origins of the acha in relation to the aneurysm were identified in the preoperative angiograms , and emitted from the base of the aneurysm in 30 ( 48.4% ) patients and from the junction of the ica and the aneurysm in 32 ( 51.6% ) patients . successful aneurysm clipping was achieved in all patients . for the patients ( n=20 ) with a ruptured acha aneurysm and who presented with wfns grade 1 to 4 , the operation was performed within a few hours after admission . however , for the patient who presented with wfns grade 5 , consent for surgery was only obtained when the patient 's level of consciousness improved to glasgow coma scale ( gcs ) score 7 ( wfns grade 4 ) . the operation was then performed the day after admission . a pterional approach was performed for most patients with a subarachnoid hemorrhage ( pterional approach in 33 cases ; supraorbital keyhole approach in 4 cases ) , whereas a supraorbital keyhole approach was applied for most of the unruptured cases ( supraorbital keyhole approach in 18 cases ; pterional approach in 7 cases ) . digital subtraction angiography was performed in 18 cases at the surgeon 's discretion on the basis of surgical findings suggesting the possibility of incomplete aneurysm obliteration . fifty - seven ( 91.9% ) patients exhibited complete aneurysm clipping , while five ( 8.1% ) patients showed a small residual neck , where the incomplete clipping of the aneurysms was caused by intentional placement of the clip beyond the aneurysm neck to avoid acha insufficiency . in these cases , an atherosclerotic aneurysm base , the acha densely adherent to the aneurysm base , and complex aneurysm shapes led to the incomplete obliteration . in the present series , there was no case of permanent acha syndrome or oculomotor nerve palsy induced by the surgical procedures . however , three ( 4.8% ) patients suffered transient acha syndrome lasting less than a week postoperatively . for a 63-year - old woman harboring a small unruptured acha aneurysm with a daughter sac , the location of the acha origin behind the aneurysm was ascertained by gentle compression of the aneurysm . a side - curved clip was then placed nuzzled close to the origin of the acha , yet leading to inadvertent constriction of the acha ( fig . however , this was not discovered as meps were not routinely determined in the early cases of the present series and the microvascular doppler was out of order . when the patient awoke from surgery in the operating room , she exhibited contralateral hemiplegia , so a re - operation was performed immediately without radiological verification of an acha occlusion and the curved clip replaced with a straight one placed further away from the acha origin ( fig . the duration of the acha occlusion , from the initial clip placement to the clip repositioning , was only 70 minutes . the patient then awoke with an improved hemiparesis ( grade 3/5 ) , and recovered completely after 6 hours . the clinical outcomes of the patients in this series were found to be related to their preoperative wfns grade ( fig . 4 ) . as regards the 3-month mrs , significant differences were found between the patients with an unruptured aneurysm ( wfns grade 0 ; n=26 ) , good - grade subarachnoid hemorrhage ( wfns grade 1 to 3 ; n=32 ) , and poor - grade subarachnoid hemorrhage ( wfns grade 4 ; n=4 ) ( p=0.000 ) . the patients ( n=26 ) without an acute subarachnoid hemorrhage remained neurologically intact or improved from their preoperative clinical status . two patients with an acha aneurysm inducing oculomotor nerve palsy recovered completely after 3 months , while the other 24 patients with an asymptomatic unruptured aneurysm remained neurologically intact postoperatively . among the patients ( n=36 ) with an acute subarachnoid hemorrhage , 32 were wfns grade 1 to 3 , while 4 were grade 4 to 5 . for the wfns grade 1 to 3 patients , the 3-month mrs score was 0 ( n=26 , 81.3% ) or 1 ( n=6 , 18.8% ) . meanwhile , for the 4 wfns grade 4 to 5 patients , the 3-month mrs score was 1 ( n=1 , 25.0% ) or 2 ( n=3 , 75.0% ) , where their symptoms and disabilities were related to a cognitive dysfunction . for the patients who presented with a subarachnoid hemorrhage , the acha aneurysm was ruptured in 21 patients , while other anterior circulation aneurysms arising at the pcoa origin ( n=7 ) , ica bifurcation ( n=1 ) , anterior communicating artery ( n=5 ) , and middle cerebral artery ( n=2 ) were ruptured in 15 patients . four ( 19.0% ) of the 21 patients with a ruptured acha aneurysm presented with contralateral hemiparesis or hemiplegia on admission ( fig . 5 ) . the ct scans of 3 hemiplegic patients revealed a caudoputaminal hemorrhage , massive intraventricular hemorrhage , and considerable amount of cisternal bleeding on the insular cortex , respectively , but no direct disruption of the corticospinal tract was observed . the clinical outcomes were favorable with 3-month mrs scores of 1 or 2 and complete recovery from hemiplegia within 6 months . oculomotor nerve palsy in relation to ruptured ( n=2 ) and unruptured ( n=2 ) acha aneurysms was observed in four patients on admission . however , total recovery from the complete ( n=2 ) and incomplete ( n=2 ) palsy was experienced within 3 months . no recurrence or rebleeding of the clipped aneurysms occurred during the follow - up period , ranging from 3 months to 7 years ( meansd : 3.22.2 years ) the clinical and angiographic characteristics of the 62 patients with an acha aneurysm are summarized in table 1 . the patient ages ranged from 32 to 75 years [ meanstandard deviation ( sd ) : 54.99.7 yr ] . thirty - six ( 58.1% ) patients presented with an acute subarachnoid hemorrhage related to a ruptured acha aneurysm ( n=21 ) or other ruptured aneurysm ( n=15 ) , while 26 patients had an unruptured acha aneurysm that was asymptomatic ( n=24 ) or symptomatic ( n=2 ) with oculomotor nerve palsy . thirty - two ( 51.6% ) patients presented with wfns grade 1 to 3 , while 4 ( 6.5% ) patients presented with wfns grade 4 to 5 . in the preoperative angiograms , the aneurysm height , i.e. the maximal perpendicular distance from the neck plane to the top of the aneurysm dome , ranged from 3 to 13 mm ( meansd : 6.02.2 mm ) , while the maximum transverse diameter of the aneurysm neck ranged from 2 to 7 mm ( meansd : 3.90.8 mm ) and the maximum aneurysm diameter , which is the largest of all cross - sections along the height of the aneurysm , ranged from 3 to 12 mm ( meansd : 4.51.4 mm ) . the origins of the acha in relation to the aneurysm were identified in the preoperative angiograms , and emitted from the base of the aneurysm in 30 ( 48.4% ) patients and from the junction of the ica and the aneurysm in 32 ( 51.6% ) patients . successful aneurysm clipping was achieved in all patients . for the patients ( n=20 ) with a ruptured acha aneurysm and who presented with wfns grade 1 to 4 , the operation was performed within a few hours after admission . however , for the patient who presented with wfns grade 5 , consent for surgery was only obtained when the patient 's level of consciousness improved to glasgow coma scale ( gcs ) score 7 ( wfns grade 4 ) . the operation a pterional approach was performed for most patients with a subarachnoid hemorrhage ( pterional approach in 33 cases ; supraorbital keyhole approach in 4 cases ) , whereas a supraorbital keyhole approach was applied for most of the unruptured cases ( supraorbital keyhole approach in 18 cases ; pterional approach in 7 cases ) . digital subtraction angiography was performed in 18 cases at the surgeon 's discretion on the basis of surgical findings suggesting the possibility of incomplete aneurysm obliteration . fifty - seven ( 91.9% ) patients exhibited complete aneurysm clipping , while five ( 8.1% ) patients showed a small residual neck , where the incomplete clipping of the aneurysms was caused by intentional placement of the clip beyond the aneurysm neck to avoid acha insufficiency . in these cases , an atherosclerotic aneurysm base , the acha densely adherent to the aneurysm base , and complex aneurysm shapes led to the incomplete obliteration . in the present series , there was no case of permanent acha syndrome or oculomotor nerve palsy induced by the surgical procedures . however , three ( 4.8% ) patients suffered transient acha syndrome lasting less than a week postoperatively . for a 63-year - old woman harboring a small unruptured acha aneurysm with a daughter sac , the location of the acha origin behind the aneurysm was ascertained by gentle compression of the aneurysm . a side - curved clip was then placed nuzzled close to the origin of the acha , yet leading to inadvertent constriction of the acha ( fig . however , this was not discovered as meps were not routinely determined in the early cases of the present series and the microvascular doppler was out of order . when the patient awoke from surgery in the operating room , she exhibited contralateral hemiplegia , so a re - operation was performed immediately without radiological verification of an acha occlusion and the curved clip replaced with a straight one placed further away from the acha origin ( fig . the duration of the acha occlusion , from the initial clip placement to the clip repositioning , was only 70 minutes . the patient then awoke with an improved hemiparesis ( grade 3/5 ) , and recovered completely after 6 hours . the clinical outcomes of the patients in this series were found to be related to their preoperative wfns grade ( fig . 4 ) . as regards the 3-month mrs , significant differences were found between the patients with an unruptured aneurysm ( wfns grade 0 ; n=26 ) , good - grade subarachnoid hemorrhage ( wfns grade 1 to 3 ; n=32 ) , and poor - grade subarachnoid hemorrhage ( wfns grade 4 ; n=4 ) ( p=0.000 ) . the patients ( n=26 ) without an acute subarachnoid hemorrhage remained neurologically intact or improved from their preoperative clinical status . two patients with an acha aneurysm inducing oculomotor nerve palsy recovered completely after 3 months , while the other 24 patients with an asymptomatic unruptured aneurysm remained neurologically intact postoperatively . among the patients ( n=36 ) with an acute subarachnoid hemorrhage , 32 were wfns grade 1 to 3 , while 4 were grade 4 to 5 . for the wfns grade 1 to 3 patients , the 3-month mrs score was 0 ( n=26 , 81.3% ) or 1 ( n=6 , 18.8% ) . meanwhile , for the 4 wfns grade 4 to 5 patients , the 3-month mrs score was 1 ( n=1 , 25.0% ) or 2 ( n=3 , 75.0% ) , where their symptoms and disabilities were related to a cognitive dysfunction . for the patients who presented with a subarachnoid hemorrhage , the acha aneurysm was ruptured in 21 patients , while other anterior circulation aneurysms arising at the pcoa origin ( n=7 ) , ica bifurcation ( n=1 ) , anterior communicating artery ( n=5 ) , and middle cerebral artery ( n=2 ) were ruptured in 15 patients . four ( 19.0% ) of the 21 patients with a ruptured acha aneurysm presented with contralateral hemiparesis or hemiplegia on admission ( fig . 5 ) . the ct scans of 3 hemiplegic patients revealed a caudoputaminal hemorrhage , massive intraventricular hemorrhage , and considerable amount of cisternal bleeding on the insular cortex , respectively , but no direct disruption of the corticospinal tract was observed . the clinical outcomes were favorable with 3-month mrs scores of 1 or 2 and complete recovery from hemiplegia within 6 months . oculomotor nerve palsy in relation to ruptured ( n=2 ) and unruptured ( n=2 ) acha aneurysms was observed in four patients on admission . however , total recovery from the complete ( n=2 ) and incomplete ( n=2 ) palsy was experienced within 3 months . no recurrence or rebleeding of the clipped aneurysms occurred during the follow - up period , ranging from 3 months to 7 years ( meansd : 3.22.2 years ) due to the low incidence of acha aneurysms , only 2 to 5% of intracranial aneurysms4,19 ) , there are relatively few reports on the related outcomes of surgical and endovascular treatments , as summarized in table 2 and 31,5,6,8 - 11,15 - 17,20 ) , which only include studies of more than 10 individuals with a non - giant aneurysm published in a peer - reviewed journal . acha insufficiency as an ischemic complication of aneurysm clipping develops into transient or permanent acha syndrome , which is typified by contralateral hemiplegia / hemiparesis with less consistent contralateral hemianesthesia or hemianopsia . the incidence of permanent acha syndrome after surgical clipping of an acha aneurysm has consistently been reported to range from 5.3% to 15.7%1,5,6,10,11,17,20 ) . when compared with surgical series , endovascular series have a much lower incidence of acha insufficiency , although only a few series have been reported8 - 10,15,16 ) . however , the favorable surgical outcomes of the current series , no occurrence of permanent acha syndrome in the 62 patients , would seem to suggest that the risk of acha insufficiency can be reduced with appropriate surgical techniques and intraoperative monitoring18 ) . while the surgical access to an acha aneurysm after dural opening in a craniotomy is technically simple and straightforward , the surgical results are actually determined by the ensuing maneuvers , including identification of the acha at its origin and around the aneurysm , careful microdissection separating the acha from the aneurysm , and appropriate clip application . preserving the patency of the acha is sometimes demanding and requires careful attention to avoid a devastating postoperative acha infarction . regarding the endovascular coiling of acha aneurysms , the previous reports show that endovascular treatment is effective for preventing aneurysmal ruptures and recurrent bleeding with low morbidity and mortality rates8 - 10,15,16 ) . however , there are significant concerns with an endovascular procedure , including thrombotic occlusion of the acha , a fatal procedural aneurysm rupture , and high recurrence rate of occluded aneurysms over a long - term period8 - 10,14 - 16 ) . the case of an acha aneurysm where the acha arises from the aneurysm base is a more challenging situation to preserve the acha patency for both surgical and endovascular treatment . in the current and six other series that reported on the origin of the acha in association with the aneurysm ( table 2 , 3 ) , a total of 172 ( 45.5% ) out of 378 aneurysms had the acha arising from the aneurysm base rather than the junction of the ica and the aneurysm1,8,9,11,15,16 ) . in yasargil 's report on 21 patients with a ruptured acha aneurysm19 ) , four patients presented with contralateral hemiparesis on admission . these patients showed an incomplete recovery of hemiparesis ( n=2 ) , aggravated hemiparesis due to acha occlusion after aneurysm clipping ( n=1 ) , and early death related to severe brain swelling and a large hemispheric infarct ( n=1 ) . the reasons for the 2 cases of incomplete hemiparesis recovery , in contrast to the 4 cases of complete recovery of initial hemiparesis in the present series , can not be explained as no ct scans were available at that time . although the current study is limited based on a retrospective review of a case series from a single institution , it may help reappraise the surgical outcomes of acha aneurysms and contribute to the limited available literature . surgical treatment of acha aneurysms involves a significant risk of acha insufficiency , yet this risk can be minimized by careful effort to preserve the acha patency based on acha - avoiding clip placement and intraoperative monitoring . in addition , the surgical outcome is primarily determined by the preoperative clinical state ( wfns grade ) .

objectivesurgical results for anterior choroidal artery ( acha ) aneurysms have previously been reported as unsatisfactory due to inadvertent occlusion of the acha , while the low incidence of acha aneurysms hampers the accumulation of surgical experience . the authors reviewed their related surgical experience to document the ischemic complications and surgical outcomes.methodsidentification of the acha at its origin by rigorous visual scrutiny , careful microdissection , and meticulous clip placement to avoid the acha origin are all crucial surgical maneuvers . a retrospective review of a surgical series of 62 consecutive cases of an acha aneurysm between 2004 and 2012 was performed.resultsall patients , except for five ( 8.1% ) with a small residual neck , showed complete aneurysm obliteration in postoperative angiographic evaluations . there was no incidence of procedure - related permanent acha syndrome or oculomotor nerve palsy , while three ( 4.8% ) patients suffered from transient acha syndrome . the clinical outcomes [ the 3-month modified rankin scale ( mrs ) ] of the patients were related to their preoperative world federation of neurologic surgeons ( wfns ) grade . as regards the 3-month mrs , significant differences were found between patients with an unruptured aneurysm ( wfns grade 0 ; n=20 ) , good - grade subarachnoid hemorrhage ( wfns grade 1 - 3 ; n=30 ) , and poor - grade subarachnoid hemorrhage ( wfns grade 4 - 5 ; n=4).conclusionin surgical treatment of acha aneurysms , a risk of acha insufficiency can be minimized by taking every precaution to preserve the acha patency and intraoperative monitoring . in addition , the surgical outcome is primarily determined by the preoperative clinical state .

INTRODUCTION MATERIALS AND METHODS Patients Operative technique Statistical analysis RESULTS Clinical and angiographic characteristics Surgery Angiographic outcomes Surgical complications Clinical outcomes DISCUSSION CONCLUSION

an aneurysm arising from the internal carotid artery ( ica ) at the origin of the anterior choroidal artery ( acha ) is one of the most easily accessible surgical lesions that can be reached via a pterional or even a supraorbital keyhole approach without much difficulty . notwithstanding , the surgical results can still be unsatisfactory due to inadvertent occlusion of the acha , leading to a permanent acha syndrome with an incidence of 5 to 16%1,5,6,10,17,20 ) . furthermore , the low incidence of acha aneurysms has hampered the accumulation of surgical experience with infrequent publications on the surgical outcomes of acha aneurysms . however , recent advancements in surgical techniques and intraoperative monitoring to preserve the acha have decreased ischemic complications and improved surgical outcomes . accordingly , the authors reviewed a single - institution surgical experience based on 62 patients who underwent surgical clipping for an acha aneurysm to document the ischemic complications and surgical outcomes . the design of this study was a retrospective review of data from a series of 62 consecutive patients who underwent surgical clipping of an acha aneurysm by a single surgeon ( j.p . ) information was obtained from the patient charts , surgical reports , video records , reviews of radiological investigations , and notes from follow - up visits to investigate the ischemic complications and surgical outcomes . determining the exact origin of the acha is crucial and can only be confirmed by rigorous visual scrutiny under high microscopic magnification . when the acha origin is posterior to the aneurysm obscuring the artery from view , the aneurysm can be compressed and manipulated using a blunt dissector to reveal the hidden acha origin after softening the aneurysm by temporary ica clipping . the proximal blade of the clip is positioned a small distance away from the origin of the acha so as not to constrict the acha origin , while the distal blade is positioned nuzzled close to the ica ( fig . meanwhile , when the acha arises posterior to the aneurysm base , the acha origin is visualized by gentle compression of the aneurysm , while avoiding any inadvertent injury to the neck of the aneurysm . after placing the clip , the clip blades are inspected to ensure complete occlusion of the aneurysm , and that the acha is not compromised and the oculomotor nerve is free . additional verification of the acha blood flow can be obtained using microvascular doppler sonography , indocyanine green near - infrared angiography , and meps2,3,7 ) . a -test was used to evaluate the affects of the preoperative world federation of neurologic surgeons ( wfns ) grade on the clinical outcomes ( mrs ) . the design of this study was a retrospective review of data from a series of 62 consecutive patients who underwent surgical clipping of an acha aneurysm by a single surgeon ( j.p . ) meanwhile , during this same period , other 12 patients with an acha aneurysm were treated using endovascular coiling . information was obtained from the patient charts , surgical reports , video records , reviews of radiological investigations , and notes from follow - up visits to investigate the ischemic complications and surgical outcomes . determining the exact origin of the acha is crucial and can only be confirmed by rigorous visual scrutiny under high microscopic magnification . when the acha origin is posterior to the aneurysm obscuring the artery from view , the aneurysm can be compressed and manipulated using a blunt dissector to reveal the hidden acha origin after softening the aneurysm by temporary ica clipping . the proximal blade of the clip is positioned a small distance away from the origin of the acha so as not to constrict the acha origin , while the distal blade is positioned nuzzled close to the ica ( fig . meanwhile , when the acha arises posterior to the aneurysm base , the acha origin is visualized by gentle compression of the aneurysm , while avoiding any inadvertent injury to the neck of the aneurysm . after placing the clip , the clip blades are inspected to ensure complete occlusion of the aneurysm , and that the acha is not compromised and the oculomotor nerve is free . a -test was used to evaluate the affects of the preoperative world federation of neurologic surgeons ( wfns ) grade on the clinical outcomes ( mrs ) . the clinical and angiographic characteristics of the 62 patients with an acha aneurysm are summarized in table 1 . thirty - six ( 58.1% ) patients presented with an acute subarachnoid hemorrhage related to a ruptured acha aneurysm ( n=21 ) or other ruptured aneurysm ( n=15 ) , while 26 patients had an unruptured acha aneurysm that was asymptomatic ( n=24 ) or symptomatic ( n=2 ) with oculomotor nerve palsy . thirty - two ( 51.6% ) patients presented with wfns grade 1 to 3 , while 4 ( 6.5% ) patients presented with wfns grade 4 to 5 . the maximal perpendicular distance from the neck plane to the top of the aneurysm dome , ranged from 3 to 13 mm ( meansd : 6.02.2 mm ) , while the maximum transverse diameter of the aneurysm neck ranged from 2 to 7 mm ( meansd : 3.90.8 mm ) and the maximum aneurysm diameter , which is the largest of all cross - sections along the height of the aneurysm , ranged from 3 to 12 mm ( meansd : 4.51.4 mm ) . the origins of the acha in relation to the aneurysm were identified in the preoperative angiograms , and emitted from the base of the aneurysm in 30 ( 48.4% ) patients and from the junction of the ica and the aneurysm in 32 ( 51.6% ) patients . for the patients ( n=20 ) with a ruptured acha aneurysm and who presented with wfns grade 1 to 4 , the operation was performed within a few hours after admission . however , for the patient who presented with wfns grade 5 , consent for surgery was only obtained when the patient 's level of consciousness improved to glasgow coma scale ( gcs ) score 7 ( wfns grade 4 ) . a pterional approach was performed for most patients with a subarachnoid hemorrhage ( pterional approach in 33 cases ; supraorbital keyhole approach in 4 cases ) , whereas a supraorbital keyhole approach was applied for most of the unruptured cases ( supraorbital keyhole approach in 18 cases ; pterional approach in 7 cases ) . fifty - seven ( 91.9% ) patients exhibited complete aneurysm clipping , while five ( 8.1% ) patients showed a small residual neck , where the incomplete clipping of the aneurysms was caused by intentional placement of the clip beyond the aneurysm neck to avoid acha insufficiency . in the present series , there was no case of permanent acha syndrome or oculomotor nerve palsy induced by the surgical procedures . however , three ( 4.8% ) patients suffered transient acha syndrome lasting less than a week postoperatively . for a 63-year - old woman harboring a small unruptured acha aneurysm with a daughter sac , the location of the acha origin behind the aneurysm was ascertained by gentle compression of the aneurysm . a side - curved clip was then placed nuzzled close to the origin of the acha , yet leading to inadvertent constriction of the acha ( fig . when the patient awoke from surgery in the operating room , she exhibited contralateral hemiplegia , so a re - operation was performed immediately without radiological verification of an acha occlusion and the curved clip replaced with a straight one placed further away from the acha origin ( fig . the clinical outcomes of the patients in this series were found to be related to their preoperative wfns grade ( fig . as regards the 3-month mrs , significant differences were found between the patients with an unruptured aneurysm ( wfns grade 0 ; n=26 ) , good - grade subarachnoid hemorrhage ( wfns grade 1 to 3 ; n=32 ) , and poor - grade subarachnoid hemorrhage ( wfns grade 4 ; n=4 ) ( p=0.000 ) . two patients with an acha aneurysm inducing oculomotor nerve palsy recovered completely after 3 months , while the other 24 patients with an asymptomatic unruptured aneurysm remained neurologically intact postoperatively . among the patients ( n=36 ) with an acute subarachnoid hemorrhage , 32 were wfns grade 1 to 3 , while 4 were grade 4 to 5 . for the wfns grade 1 to 3 patients , the 3-month mrs score was 0 ( n=26 , 81.3% ) or 1 ( n=6 , 18.8% ) . meanwhile , for the 4 wfns grade 4 to 5 patients , the 3-month mrs score was 1 ( n=1 , 25.0% ) or 2 ( n=3 , 75.0% ) , where their symptoms and disabilities were related to a cognitive dysfunction . for the patients who presented with a subarachnoid hemorrhage , the acha aneurysm was ruptured in 21 patients , while other anterior circulation aneurysms arising at the pcoa origin ( n=7 ) , ica bifurcation ( n=1 ) , anterior communicating artery ( n=5 ) , and middle cerebral artery ( n=2 ) were ruptured in 15 patients . four ( 19.0% ) of the 21 patients with a ruptured acha aneurysm presented with contralateral hemiparesis or hemiplegia on admission ( fig . no recurrence or rebleeding of the clipped aneurysms occurred during the follow - up period , ranging from 3 months to 7 years ( meansd : 3.22.2 years ) the clinical and angiographic characteristics of the 62 patients with an acha aneurysm are summarized in table 1 . thirty - six ( 58.1% ) patients presented with an acute subarachnoid hemorrhage related to a ruptured acha aneurysm ( n=21 ) or other ruptured aneurysm ( n=15 ) , while 26 patients had an unruptured acha aneurysm that was asymptomatic ( n=24 ) or symptomatic ( n=2 ) with oculomotor nerve palsy . thirty - two ( 51.6% ) patients presented with wfns grade 1 to 3 , while 4 ( 6.5% ) patients presented with wfns grade 4 to 5 . the maximal perpendicular distance from the neck plane to the top of the aneurysm dome , ranged from 3 to 13 mm ( meansd : 6.02.2 mm ) , while the maximum transverse diameter of the aneurysm neck ranged from 2 to 7 mm ( meansd : 3.90.8 mm ) and the maximum aneurysm diameter , which is the largest of all cross - sections along the height of the aneurysm , ranged from 3 to 12 mm ( meansd : 4.51.4 mm ) . the origins of the acha in relation to the aneurysm were identified in the preoperative angiograms , and emitted from the base of the aneurysm in 30 ( 48.4% ) patients and from the junction of the ica and the aneurysm in 32 ( 51.6% ) patients . for the patients ( n=20 ) with a ruptured acha aneurysm and who presented with wfns grade 1 to 4 , the operation was performed within a few hours after admission . however , for the patient who presented with wfns grade 5 , consent for surgery was only obtained when the patient 's level of consciousness improved to glasgow coma scale ( gcs ) score 7 ( wfns grade 4 ) . the operation a pterional approach was performed for most patients with a subarachnoid hemorrhage ( pterional approach in 33 cases ; supraorbital keyhole approach in 4 cases ) , whereas a supraorbital keyhole approach was applied for most of the unruptured cases ( supraorbital keyhole approach in 18 cases ; pterional approach in 7 cases ) . fifty - seven ( 91.9% ) patients exhibited complete aneurysm clipping , while five ( 8.1% ) patients showed a small residual neck , where the incomplete clipping of the aneurysms was caused by intentional placement of the clip beyond the aneurysm neck to avoid acha insufficiency . in the present series , there was no case of permanent acha syndrome or oculomotor nerve palsy induced by the surgical procedures . however , three ( 4.8% ) patients suffered transient acha syndrome lasting less than a week postoperatively . for a 63-year - old woman harboring a small unruptured acha aneurysm with a daughter sac , the location of the acha origin behind the aneurysm was ascertained by gentle compression of the aneurysm . when the patient awoke from surgery in the operating room , she exhibited contralateral hemiplegia , so a re - operation was performed immediately without radiological verification of an acha occlusion and the curved clip replaced with a straight one placed further away from the acha origin ( fig . the duration of the acha occlusion , from the initial clip placement to the clip repositioning , was only 70 minutes . the clinical outcomes of the patients in this series were found to be related to their preoperative wfns grade ( fig . as regards the 3-month mrs , significant differences were found between the patients with an unruptured aneurysm ( wfns grade 0 ; n=26 ) , good - grade subarachnoid hemorrhage ( wfns grade 1 to 3 ; n=32 ) , and poor - grade subarachnoid hemorrhage ( wfns grade 4 ; n=4 ) ( p=0.000 ) . the patients ( n=26 ) without an acute subarachnoid hemorrhage remained neurologically intact or improved from their preoperative clinical status . two patients with an acha aneurysm inducing oculomotor nerve palsy recovered completely after 3 months , while the other 24 patients with an asymptomatic unruptured aneurysm remained neurologically intact postoperatively . among the patients ( n=36 ) with an acute subarachnoid hemorrhage , 32 were wfns grade 1 to 3 , while 4 were grade 4 to 5 . for the wfns grade 1 to 3 patients , the 3-month mrs score was 0 ( n=26 , 81.3% ) or 1 ( n=6 , 18.8% ) . meanwhile , for the 4 wfns grade 4 to 5 patients , the 3-month mrs score was 1 ( n=1 , 25.0% ) or 2 ( n=3 , 75.0% ) , where their symptoms and disabilities were related to a cognitive dysfunction . for the patients who presented with a subarachnoid hemorrhage , the acha aneurysm was ruptured in 21 patients , while other anterior circulation aneurysms arising at the pcoa origin ( n=7 ) , ica bifurcation ( n=1 ) , anterior communicating artery ( n=5 ) , and middle cerebral artery ( n=2 ) were ruptured in 15 patients . no recurrence or rebleeding of the clipped aneurysms occurred during the follow - up period , ranging from 3 months to 7 years ( meansd : 3.22.2 years ) due to the low incidence of acha aneurysms , only 2 to 5% of intracranial aneurysms4,19 ) , there are relatively few reports on the related outcomes of surgical and endovascular treatments , as summarized in table 2 and 31,5,6,8 - 11,15 - 17,20 ) , which only include studies of more than 10 individuals with a non - giant aneurysm published in a peer - reviewed journal . the incidence of permanent acha syndrome after surgical clipping of an acha aneurysm has consistently been reported to range from 5.3% to 15.7%1,5,6,10,11,17,20 ) . when compared with surgical series , endovascular series have a much lower incidence of acha insufficiency , although only a few series have been reported8 - 10,15,16 ) . however , the favorable surgical outcomes of the current series , no occurrence of permanent acha syndrome in the 62 patients , would seem to suggest that the risk of acha insufficiency can be reduced with appropriate surgical techniques and intraoperative monitoring18 ) . while the surgical access to an acha aneurysm after dural opening in a craniotomy is technically simple and straightforward , the surgical results are actually determined by the ensuing maneuvers , including identification of the acha at its origin and around the aneurysm , careful microdissection separating the acha from the aneurysm , and appropriate clip application . regarding the endovascular coiling of acha aneurysms , the previous reports show that endovascular treatment is effective for preventing aneurysmal ruptures and recurrent bleeding with low morbidity and mortality rates8 - 10,15,16 ) . however , there are significant concerns with an endovascular procedure , including thrombotic occlusion of the acha , a fatal procedural aneurysm rupture , and high recurrence rate of occluded aneurysms over a long - term period8 - 10,14 - 16 ) . the case of an acha aneurysm where the acha arises from the aneurysm base is a more challenging situation to preserve the acha patency for both surgical and endovascular treatment . in the current and six other series that reported on the origin of the acha in association with the aneurysm ( table 2 , 3 ) , a total of 172 ( 45.5% ) out of 378 aneurysms had the acha arising from the aneurysm base rather than the junction of the ica and the aneurysm1,8,9,11,15,16 ) . although the current study is limited based on a retrospective review of a case series from a single institution , it may help reappraise the surgical outcomes of acha aneurysms and contribute to the limited available literature . surgical treatment of acha aneurysms involves a significant risk of acha insufficiency , yet this risk can be minimized by careful effort to preserve the acha patency based on acha - avoiding clip placement and intraoperative monitoring . in addition , the surgical outcome is primarily determined by the preoperative clinical state ( wfns grade ) .

in current therapeutic strategies , large bone defects caused by trauma , tumors , or infections are filled by bone auto- or allografts [ 1 , 2 ] . these methods imply disadvantages such as limited availability , donor site morbidities , immunological reactions , or the risk of infections [ 35 ] . synthetic implants provide an alternative to the limited resources of autografts and the problems in the use of allogenic or xenogenic grafts . the success of such implants is determined by various factors : the materials used have to be biocompatible and corrosion - resistant , they must have the correct mechanical properties , and the architecture of the graft has to favor tissue ingrowth into the scaffold . commonly , synthetic three - dimensional ( 3d ) scaffolds were used , whose structures were phenomenologically optimized for cell seeding [ 68 ] . however , in vitro studies of bone cell ingrowth into scaffolds demonstrated an impaired cellular proliferation and reduced differentiation in the core region of scaffolds with increasing scaffold volume [ 9 , 10 ] . consequently , osteoblast growth into porous scaffolds with pore sizes between 400 m and 800 m in diameter [ 11 , 12 ] was observed , but migration to the interior of such scaffolds was limited for static in vitro cultures without nutrient flow . the results were interpreted by a concentration gradient from the surface to the core due to a restriction of medium diffusion in the scaffold , followed by insufficient nutrient and oxygen supply ( hypoxia ) and waste accumulation ( acidification ) for cells in the core region [ 9 , 14 , 15 ] . hypoxia influences osteogenic differentiation in cell cultures [ 1618 ] and may cause cell death inside the implant . therefore , cell nutrition in the core region of a scaffold is frequently supported by medium flow in vitro , with perfusion bioreactor systems yielding the best results [ 1923 ] . commercial perfusion systems for tissue culture , for example , minucells perfusion containers ( minucells and minutissue vertriebs gmbh , bad abbach , germany ) , the osteogen bioreactor ( instron , norwood , usa ) , the 3d bioreactor ( 3d biotek , sigma - aldrich , munich , germany ) , or the zrp bioreactor ( zellwerk , oberkrmer , germany ) as well as custom - made bioreactors , were used in published studies [ 1925 ] . however , the use of dynamic cell cultures reveals a dilemma : while sufficient cell nutrition requires relatively high medium flow rates , the faster movement of the liquid leads to increased shear stress and as a consequence to increased levels of cell death [ 2628 ] . the local supply rates as well as the local shear stress depend on the exact channel structure inside the scaffold and , consequently , an optimal medium flow rate has to be determined for each scaffold architecture . in turn , this means that not every channel structure allows an optimal supply for the cells . to gain a better understanding of the nutrient supply in the scaffold , the applied methods range from brownian dynamics simulations that explicitly calculate the trajectory of each nutrient particle to simulations on finite element basis . frequently , the diffusion and the directed flow of a particle species are taken into account and the local balance of consumed and freshly supplied molecules is calculated . in particular , many studies focus on the simulation of the oxygen concentration inside the scaffold and come to the result that the oxygen supply is a critical factor in determining cell fate [ 3234 ] . however , the calculated channel structures [ 31 , 32 ] are very simple and allow only limited conclusions for real systems . to overcome this limitation , an in vitro 3d cell culture module was developed that allows the cultivation of osteoblasts in a 3d porous structure at different nutrient flow rates . we compared the wet - lab data ( cell viability ) with those from computer simulations . these in silico data based on the finite element method ( fem ) predicted the local oxygen supply and shear stress inside the scaffold and let us draw conclusions for the optimization of perfusion flow rates and the channel design of the scaffold . ta scaffolds ( zimmer , freiburg , germany ) of 14 mm radius and 5 mm thickness were used ( figure 1 ) . this porous trabecular ta has a typical porosity of 80% and a pore size of around 550 m . the scaffolds were molded on one side at the edge to fit into the clamping ring ( figure 2 ) . the ta scaffolds and the clamping ring were sterilized by autoclaving ( 121c steam , superior , mammooth , italy ) . the clamping ring was made of titanium ( grade 2 ) with an inner radius of 12 mm , and a mounting thread ensured that the plane surfaces were situated on top of each other . in this way the in vitro 3d module simulated one scaffold ( total height : 10 mm ) , enabling nondestructive cell observation on four different levels without cutting the material : one apical ( level 1 ) , two medial ( levels 2 and 3 ) , and one basal ( level 4 ) surface . mg-63 osteoblastic cells ( osteosarcoma cell line , atcc , lgc promochem , wesel , germany ) were used as a well - established cell model for in vitro research in biomaterials science [ 3336 ] . cells were cultured in dulbecco 's modified eagle medium ( dmem ) ( invitrogen , darmstadt , germany ) supplemented with 10% fetal calf serum ( fcs ) ( paa gold , paa laboratories , clbe , germany ) and 1% gentamicin ( ratiopharm , ulm , germany ) at 37c in a humidified atmosphere with 5% co2 . near confluence , cells were detached with 0.05% trypsin/0.02% edta for 5 min . after stopping trypsinization by the addition of cell culture medium , an aliquot of 100 l was put into 10 ml of casy ton buffer solution ( roche innovatis , reutlingen , germany ) and the cell number was measured in the counter casy model dt ( schrfe system , reutlingen , germany ) . two porous 3d ta scaffolds with the molded side up were placed into a 6-well tissue culture plate ( greiner bio one , frickenhausen , germany ) and covered with 4 ml of dmem . 1 10 cells in 100 l dmem were seeded in a meandering pattern onto the surface of the medium - incubated ta scaffolds . after 15 min , when the cells had adhered to the materials , the 6-well plate was incubated at 37c in a humidified atmosphere with 5% co2 for another 6 h. then the ta scaffolds were inverted ( molded side down ) and the seeding procedure was repeated for the opposite sides . after a further incubation overnight at 37c in a humidified atmosphere with 5% co2 , the two porous 3d ta scaffolds were stacked horizontally and fixed within the clamping ring ( figure 2 ) , with the plane surfaces on each other . for 3d dynamic cell cultures in scaffolds , we developed not only the in vitro 3d module but also a perfusion cell culture reactor ( cellynyzer , institute for polymer technologies wismar , germany ) . this cell culture reactor was made by rapid prototyping on the basis of a biocompatible methacrylate resin ( fotomed led.a , innovation meditech gmbh , germany ) ( height of 60 mm and 25 mm in radius ) . the interior was cylindrical , designed to precisely fit the 3d module to guarantee perfusion , and completed by three luer cones for the connection to luer lock systems ( figure 3 ) . the reactor was conceived to be extendable in height by adding a spacer ring between the base and the upper section . more than two scaffolds or heightened scaffolds could thereby be easily incorporated into the system . the perfusion cell culture reactor cellynyzer was sterilized by autoclaving ( 121c steam , mammooth ) . gas permeable tubes ( fluidflex silicon hg , liquid - scan , germany ) were chosen for the supply of medium to the cell culture reactor due to their ph maintenance at 37c in a humidified atmosphere with 5% co2 . the tubes were sterilized by rinsing with ethanol ( 70% ) , sterile pbs , and cell culture medium before connecting them to the cell culture reactor . the cell reactor was connected to a peristaltic pump ( ipc - n 4 , ismatec , germany ) , a medium reservoir , and a waste container . the pump and the medium reservoir were placed outside the incubator : the pump at room temperature and the medium in the refrigerator standing beside it . the medium was heated to 37c by elongating the tube inside the incubator up to 50 cm before reaching the reactor . the pump was calibrated before the experiments and medium consumption was measured during the experiments . the seeded in vitro 3d module ( see figure 2 ) was integrated into the perfusion cell culture reactor . the cell culture medium was pumped through the 3d module with a flow direction being bottom - up within the reactor . dynamic culture experiments were done in complete dmem as described for 7 days under continuous flow with different flow rates ( 5 , 15 , 30 , 45 , and 60 l / min ) . control experiments were done under static culture conditions without flow in the same cell reactor . for this purpose , the culture medium was removed using a sterile syringe and carefully replaced by new medium every other day . after cell culturing for the appropriate time under the defined medium flow , cells on every level of the in vitro 3d module underwent a separate cell - biological analysis . this was easily realized by demounting the module and taking the two scaffolds apart ( see figure 2 ) . in this way , cells from the core of the in vitro 3d module could be analyzed without cutting any material and cell observation was therefore nondestructive . mg-63 cells were cultured in the in vitro 3d module system in dynamic as well as static mode ( without flow ) for 7 days as described . vitality was analyzed by live / dead staining ( l7013 live / dead cell viability kit , invitrogen , germany ) after demounting the ta scaffold , washing carefully with hank 's buffered salt solution ( hbss , paa laboratories gmbh , germany ) and staining the cells with a solution of syto 10 and dead red ( 8 l of each dye in 4 ml hbss ) for 15 min at 37c in the dark . then the scaffolds were inverted and incubated for another 15 min at 37c in the dark ; the scaffolds were washed with hbss and incubated with 4% glutaraldehyde in hbss for at least 1 h at 4c . cells were examined on the fluorescence microscope ( axio scope.a1 , 10x air lens , carl zeiss , germany ) using the standard fluorescein long pass filter . the nuclei of living cells appeared green and could thereby be distinguished from dead cells ( red nuclei ) . the porous structure of the ta scaffold material was examined by fesem ( supra 25 , zeiss , germany ) . cell density on the different levels of the in vitro 3d module was visualized after 7 days of culture under static conditions . for this purpose , the upper and lower ta scaffolds were separated ( see figure 2 ) , washed with pbs , fixed with 4% glutaraldehyde , and dehydrated using a graded series of acetone . after critical point drying ( k 850 , emitech , germany ) , the scaffolds were examined by fesem . the oxygen supply of the cell culture was analyzed by a computer model based on the finite element method ( fem ) . the simulation box consisted of a section through the structured scaffold as well as the reservoir regions below and above the ta scaffold . for reasons of computing time the simulation box was split into a lower region with the medium inlet and an upper part with the medium outlet ; both regions were linked together by the equating of the concentrations and pressures at their contact faces . the channel structure of the ta scaffold was modeled by double - walled connected spheres ( figures 4(b)4(d ) ) ; the narrow region between the outer and inner wall represents the volume in which the cells were located . periodic boundary conditions were assumed in the horizontal directions ( figure 4(d ) ) ; each end face was linked to the symmetry - equivalent end face on the opposite site of the simulation box leading to a total number of 94 periodic boundary conditions for each half of the simulation box . however , along the main flow direction of the medium ( bottom - up ) the full scaffold disc was calculated . the local oxygen concentration was evaluated for a dense cell layer on the channel walls of the scaffold . it was derived from the balance of consumed molecules by the cells and supplied molecules by diffusion and by inflowing medium . the external reservoir of fresh medium was assumed to be large compared to the volume of the perfusion reactor , and consequently the oxygen concentration was set to a constant bulk value of c0 outside the reactor as well as at the inlet-/outlet - interfaces . in the cell - occupied regions oxygen is consumed at a given consumption rate r. for the calculation of the oxygen concentration , the simulation procedures of coletti et al . and buchwald et al . the diffusion of oxygen was described by the general diffusion equation ( 1)ct+dc = ruc , where c is the local oxygen concentration , d is the diffusion constant of oxygen in the medium , is the nabla - operator , and u is the local velocity vector of the media . because the oxygen consumption of the cells depends on the locally available amount of oxygen , a michaelis - menten - type expression was used for r , similar to the calculations of buchwald et al . : ( 2)r=0,culture medium , rmaxcc+cmmccc , cell volume . in the equation rmax is the maximal oxygen consumption rate of the osteoblast cells , cmm , the michaelis - menten constant , that is , the oxygen concentration at which the cells consume only 50% of rmax . cc is a critical oxygen concentration at which the cells die ( and then consume no more oxygen ) . the delta function returns a value of 1 for c cc and a value of 0 otherwise . it ensures that oxygen consumption ceases ( r = 0 ) for regions with dead cells ( c < cc ) . the local velocity field u in ( 1 ) was calculated by solving the navier - stokes equation [ 33 , 34](3)ut+uu+p=2u+ffor an incompressible liquid medium ( u = 0 ) . here , is the density of the liquid medium , is the viscosity , and f is the vector of the body force density . the oxygen concentration c0 for a fresh medium at 37c was set to 0.42 mol / m ; the diffusion constant d of oxygen was set to 2.68 10 m / s . for reasons of comparability , the values for the critical concentration and the michaelis - menten constant were taken from the simulations of buchwald et al . and set to cc = 10 mol / m and cmm = 10 mol / m . . demonstrated that the consumption rates of osteoblasts vary between 5.56 10 mol/(cellmin ) for osteoblasts cultured in static t - flasks and 1.25 10 mol/(cellmin ) for encapsulated medium in vitro . considering the cell numbers and the cell volume given in , oxygen consumption rates between rmax = 2.7 10 mol/(ms ) and rmax = 1.23 10 mol/(ms ) arise . accordingly , an average oxygen consumption rate of rmax = 2.0 10 mol/(ms ) was used in this study . medium supply rates of 5 , 15 , 30 , 45 , and 60 l / min as well as the static cell culture were analyzed . please note that all specified flow rates refer to the entire scaffold ( such as in the experiments ) and are internally converted to the region of the simulation box by analyzing the ratio of the respective surfaces . m was used for the scaffold ( representing the inner region of the ta disk , which is not blocked by the clamping ring ) and 8.40 10 m for the simulation box . the porous channel structure of the scaffold was constructed by double - walled spheres with an outer radius of 250 m and an inner radius of 245 m ( thickness of the cell layer : 5 m ) . the connections were built up of double - walled cylinders with an outer radius of 80 m , an inner radius of 75 m , and a connection length of 2 25 m . the channel structure was meshed with a free tetrahedral mesh ( 1,244,382 elements ) and the equations were solved using the parallel direct sparse solver ( pardiso ) . the iteration was terminated when the relative error was less than 10 . a finer mesh or a larger number of iterations all calculations were performed with comsol multiphysics version 4.2a and carried out on an intel xeon x5680 processor ( 2 processors with 3.33 ghz and 48 gb of main memory ) . perfusion culture in a bioreactor with a pump system that perfuses nutrition media directly through the cell - seeded scaffold can obviously mitigate internal diffusional limitations which result in cell death [ 23 , 38 ] . different perfusion bioreactor systems used with different flow rates or velocities are mentioned in the literature [ 1923 ] . perfusion ensures an adequate supply to the cells but higher continuous flow rates led to increased levels of cell death due to shear stress [ 2628 ] . optimal flow velocities depend on the scaffold volume and the pore size and have to be determined separately for each perfusion system . in earlier studies we could demonstrate colonization of ta scaffolds by mg-63 osteoblastic cells in our in vitro 3d module [ 39 , 40 ] . to investigate cell survival inside scaffolds with dimensions suitable for large bone defects , the same module was used here . a setup in which all levels of the 3d module were seeded by osteoblast cells before assembling the module enabled the analysis of the cell survival in the core of the scaffold ( see figure 2 ) . to promote the sufficient supply of the cells in the core of the scaffold we complemented the in vitro 3d module with a perfusion bioreactor , thus creating a novel dynamic system . because the commercially available perfusion systems did not meet our demands for the size and special shape of our 3d module , we developed a custom - made direct perfusion bioreactor usable in a standard lab incubator with a humidified atmosphere and 5% co2 . to identify the optimal flow rate for our system , we started with a very low pump velocity ( 5 l / min ) . cell viability and cell density were analyzed on the different levels after 7 days of culture and demounting the scaffolds from the dynamic in vitro 3d module . as can be seen in figure 5 , insufficient cell survival occurred on all levels . therefore velocity was enhanced progressively for the next experiments taking into consideration enhanced shear stress caused by high medium flow . cell vitality on the different scaffold levels was correlated with the medium flow rate for every experiment . in this way we found that cell survival rose on level 4 with a higher medium flow up to 45 l / min . cell vitality and density in the core region ( levels 2 and 3 ) were very low with flow rates below 30 l / min but increased at rates up to 60 l / min . as the flow direction in the cell culture reactor was bottom - up , cells on level 4 were always supplied with fresh medium . if the flow was too low , for example , 5 l / min , cells on the upper levels were supplied with used medium which was depleted of oxygen and nutrients and enriched with metabolic products by the cells from the levels below . therefore it is not surprising that on levels 2 - 3 dead cells were detected at low flow rates . interestingly , cell viability also decreased with the flow rate 60 l / min . dead cells could be detected especially on level 4 and the cell density was reduced . this is an obvious contrast to the vitality on this level at 45 l / min . the higher continuous flow ( 60 l / min ) seems to induce cell death . on the other hand , cell survival improved in the core ( levels 2 and 3 ) , but on these levels dead cells could be detected too . therefore a flow rate of 60 l / min was considered as suboptimal . cells occupied the whole surface as could be seen in the fesem images and were almost all vital on these levels ( figure 6 ) . cell survival in the core region ( levels 2 and 3 ) was reduced as expected . to understand the nutrient supply in the scaffold , the experiments were supported by computer simulations . figure 7 displays the local oxygen concentration in the simulation box for different nutrient supply rates . the flow direction of the nutrient medium was from the bottom ( inlet ) to the top ( outlet ) . regions marked in red represent areas that are well supplied with oxygen , in yellow and green regions in which the oxygen concentration is significantly reduced , and in the dark blue areas a critical lack of oxygen . without flow the lowest oxygen concentrations were found in the middle of the scaffold , because new oxygen could diffuse from the inlet and from the outlet into the system but did not reach the inner regions before it was consumed . at low flow rates ( 5 l / min30 l / min ) , the oxygen supply did not significantly improve , but the undersupplied region shifted in the direction of the outlet . the reason for this shift was that on the one hand fresh medium flowed into the system and increased the oxygen concentration in the vicinity of the inlet . on the other hand , however , the flow counteracted the inward diffusion of oxygen at the outlet so that the equilibrium concentration was reduced there . the system was only sufficiently supplied with oxygen at high flow rates ( 45 l / min60 l / min ) , because incoming molecules were not completely consumed before they reached the region in the middle of the scaffold . figure 8 shows the oxygen concentration at the four analysis levels of the in vitro 3d module in the perfusion reactor for different flow rates . in the static cell culture , the oxygen concentration was reduced at level 1 ( outlet ) and level 4 ( inlet ) compared to fresh medium and levels 2 and 3 ( in the core of the scaffold ) were insufficiently supplied with oxygen . in comparison , the oxygen concentration decreased at level 1 and increased at level 4 for low flow rates ( 5 l / min30 l / min ) ; at level 2 and level 3 no significant change in the oxygen concentration was registered . only at high flow rates ( 45 l / min60 l / min ) all levels in addition to the described changes in the oxygen concentration , increased shear rates have to be considered if the medium flow is increased . figure 9 displays the calculated shear rates at the cell surface for the four analysis levels and the different flow rates . the higher the flow rate , the greater the shear rate . however , the difference between the outer levels ( 1 , 4 ) and the inner levels ( 2 , 3 ) also increased at higher flow rates . these different shear rates may have an influence on the growth rate of the cells . the comparison of results from the cell culture experiments in the dynamic 3d in vitro cell culture module and the computer simulation of oxygen supply and shear stress indicate a correlation between local population of vital cells and oxygen supply in the scaffold . by enhancing the medium flow insufficiently , unfortunately , medium flow rates for adequate oxygen supply in all levels result in high levels of shear stress in the outer levels 1 and 4 . therefore it can be deduced that if oxygen supply does not impede the cell population in the scaffold , shear stress will . for lower medium flow rates between 15 l / min and 30 l / min cells will occupy the outer levels 1 and 4 . with rising flow rates up to 45 l / min the cell vitality in the core region ( levels 2 and 3 ) will grow through sufficient oxygen supply to the cells . for further enhanced flows up to 60 l / min the cell population will remain viable in the interior only ( levels 2 and 3 ) . therefore a flow of 45 l / min can be assumed as the optimal medium flow rate for this dynamic 3d in vitro cell culture module . however , please note that only the local balance of oxygen was considered in this study due to the high computation time of the complex 3d scaffold . even if the presence of a sufficient amount of oxygen is believed to be a key factor for cell growth in tissue engineering , the presence of other molecules like nutrients and metabolic waste products may also influence cell fate . nevertheless , jonitz et al . were able to show a significantly reduced oxygen supply of cells in the core region of a similar 3d module already after one day of static culture , but no significant acidification by cell metabolites . these results indicate that the local oxygen concentration is affected more quickly by the cells than other parameters and therefore is probably the most critical parameter for cell growth . moreover , we would expect similar ( e.g. , for the nutrient molecules ) or inverted ( e.g. , for metabolic products ) concentration profiles as calculated for oxygen , because the diffusion and flow properties of these molecules do not fundamentally differ from oxygen . consequently , the consideration of further molecules would moderately intensify or reduce the effects observed here but would not fundamentally change our interpretation . in the present study , an in vitro 3d module with a perfusion cell culture reactor for dynamic cell cultures was developed and used to achieve enhanced cell viability in the core of scaffolds with dimensions suitable for large bone defects . the optimal flow rate for the survival of mg-63 cells was obtained at around 45 l / min in the dynamic mode , where fully cell - seeded scaffolds showed vital cells and cell death could not be detected on all four levels after 7 days of culture . reduced cell survival with higher continuous flow rates could be attributed to cell death induced by shear stress , as shown by our simulation and as other groups also observed [ 2628 ] . however , fluid shear stress was reported to induce osteoblast cell differentiation , whereby cell proliferation decreases [ 26 , 28 ] . sikavitsas et al . analyzed the influence of shear forces on rat bone marrow stromal cells with perfusing culture media of different viscosities and detected that mechanical stimulation enhanced the expression of the osteoblastic phenotype . investigated the influence of enhanced medium flow on the cell proliferation and differentiation of mc3t3-e1 osteoblast - like cells perfused for 1 week at flow rates of 10 , 100 , and 200 l / min and 1.0 ml / min . they showed enhanced cell proliferation at a flow rate of 10 l / min and induced cell differentiation at 200 l / min in bone constructs seeded with mouse osteoblast precursor cells . campos et al . also determined cell differentiation dependent on fluid flow rates in a perfusion culture . on hydroxyapatite / collagen scaffolds colonized with stro-1a cells they found stimulated proliferation at flow rates of 300 l / min , but osteogenic marker gene expression was enhanced with a low flow of only 30 l / min therefore , the results seem to be contradictory and it is difficult to deduce the optimal flow from these examples . taking into account that scaffold and pore size influence the distribution of the fluid and alter the shear forces generated in the scaffold , large scaffolds with higher pore size should require higher flow rates in perfusion culture than small ones . li et al . investigated the effects of flow shear stress on the construction of a large - scale tissue - engineered -tricalcium phosphate scaffold ( 14 mm in diameter , 30 mm in height , with a tunnel of 3.5 mm in diameter ) seeded with human bone marrow stromal cells . they used flow rates of 3.0 to 9.0 ml / min and altered shear forces by enhancing the viscosity of the medium . the results showed accelerated osteoblastic differentiation due to shear stress , while lower flow enhanced cell proliferation . a bidirectional continuous perfusion bioreactor for culturing constructs of significant dimensions was developed by gardel et al . . fiber mesh scaffolds made from starch and polycaprolactone ( 16 mm in diameter , 3 mm in height ) were seeded with goat marrow stromal cells and stacked , creating a 42 mm thick construct . the samples were cultured at a flow rate of 1.0 ml / min for up to 21 days . the number of cells in the constructs showed lower values compared to static culture , while dynamic conditions tended to enhance osteogenic differentiation . also used scaffolds with higher volumes ( 6.35 mm in diameter , 6.35 mm in height ) suitable for large bone defects . they were able to determine cell death in the constructs with an average pore size of 645 m at a flow rate of 1.0 ml / min and enhanced cell proliferation at 10 l / min . as insufficient cell supply in the core of a scaffold could result in impaired cell migration towards the center , peripherally seeded scaffolds should be cultured under optimal perfusion flow for cell survival in the core first , followed by induction of osteogenic differentiation through enhanced flow . in this context , the optimal flow rate of 45 l / min for osteoblast survival in the combined large scaffold with a height of 10 mm in our dynamic in vitro 3d module was within the assumed range . in this preliminary study , we observed acceptable cell survival on the different levels after a cultivation time of seven days by live / dead staining of the cells . these results were confirmed by a simulation model for oxygen supply and shear stress . using the optimal conditions for cell survival , our dynamic in vitro module can also be used for osteoblast migration studies by changing the cell seeding setup . for this purpose , cells could be seeded on only one outer level in the 3d module . in this way , active cell migration against gravity could be analyzed , for instance , dependent on the surface properties ( chemical gradients ) of the scaffolds . the optimal perfusion flow for osteogenic differentiation in our dynamic in vitro 3d module will be determined in further studies with mesenchymal stem cells or preosteoblasts . for optimal oxygen supply of all cells in the scaffold new architecture of scaffolds should be developed , where small cell - seeded channels are supplied with nutrients by diffusion from separate channels which are not seeded by cells . in these nonseeded channels higher medium flow rates the dynamic in vitro 3d module described here is a system well suited for the nondestructive investigation of cell behavior inside scaffolds with dimensions corresponding to large bone defects . for this 3d module , simulation of oxygen supply and shear stress combined with in vitro experiments result in an optimal flow rate near 45 l / min as a reasonable compromise . the 3d module combined with a dynamic cell culture in the perfusion reactor can be used for the observation of cell growth inside porous implant materials . scaffolds with pore sizes of around 500 m and a minimum height of 10 mm can be investigated with respect to material composition and chemical surface modifications under an optimal perfusion flow rate .

studies on bone cell ingrowth into synthetic , porous three - dimensional ( 3d ) implants showed difficulties arising from impaired cellular proliferation and differentiation in the core region of these scaffolds with increasing scaffold volume in vitro . therefore , we developed an in vitro perfusion cell culture module , which allows the analysis of cells in the interior of scaffolds under different medium flow rates . for each flow rate the cell viability was measured and compared with results from computer simulations that predict the local oxygen supply and shear stress inside the scaffold based on the finite element method . we found that the local cell viability correlates with the local oxygen concentration and the local shear stress . on the one hand the oxygen supply of the cells in the core becomes optimal with a higher perfusion flow . on the other hand shear stress caused by high flow rates impedes cell vitality , especially at the surface of the scaffold . our results demonstrate that both parameters must be considered to derive an optimal nutrient flow rate .

1. Introduction 2. Material and Methods 3. Results and Discussion 4. Conclusions

the success of such implants is determined by various factors : the materials used have to be biocompatible and corrosion - resistant , they must have the correct mechanical properties , and the architecture of the graft has to favor tissue ingrowth into the scaffold . commonly , synthetic three - dimensional ( 3d ) scaffolds were used , whose structures were phenomenologically optimized for cell seeding [ 68 ] . however , in vitro studies of bone cell ingrowth into scaffolds demonstrated an impaired cellular proliferation and reduced differentiation in the core region of scaffolds with increasing scaffold volume [ 9 , 10 ] . consequently , osteoblast growth into porous scaffolds with pore sizes between 400 m and 800 m in diameter [ 11 , 12 ] was observed , but migration to the interior of such scaffolds was limited for static in vitro cultures without nutrient flow . the results were interpreted by a concentration gradient from the surface to the core due to a restriction of medium diffusion in the scaffold , followed by insufficient nutrient and oxygen supply ( hypoxia ) and waste accumulation ( acidification ) for cells in the core region [ 9 , 14 , 15 ] . therefore , cell nutrition in the core region of a scaffold is frequently supported by medium flow in vitro , with perfusion bioreactor systems yielding the best results [ 1923 ] . however , the use of dynamic cell cultures reveals a dilemma : while sufficient cell nutrition requires relatively high medium flow rates , the faster movement of the liquid leads to increased shear stress and as a consequence to increased levels of cell death [ 2628 ] . the local supply rates as well as the local shear stress depend on the exact channel structure inside the scaffold and , consequently , an optimal medium flow rate has to be determined for each scaffold architecture . to gain a better understanding of the nutrient supply in the scaffold , the applied methods range from brownian dynamics simulations that explicitly calculate the trajectory of each nutrient particle to simulations on finite element basis . frequently , the diffusion and the directed flow of a particle species are taken into account and the local balance of consumed and freshly supplied molecules is calculated . in particular , many studies focus on the simulation of the oxygen concentration inside the scaffold and come to the result that the oxygen supply is a critical factor in determining cell fate [ 3234 ] . to overcome this limitation , an in vitro 3d cell culture module was developed that allows the cultivation of osteoblasts in a 3d porous structure at different nutrient flow rates . we compared the wet - lab data ( cell viability ) with those from computer simulations . these in silico data based on the finite element method ( fem ) predicted the local oxygen supply and shear stress inside the scaffold and let us draw conclusions for the optimization of perfusion flow rates and the channel design of the scaffold . after stopping trypsinization by the addition of cell culture medium , an aliquot of 100 l was put into 10 ml of casy ton buffer solution ( roche innovatis , reutlingen , germany ) and the cell number was measured in the counter casy model dt ( schrfe system , reutlingen , germany ) . two porous 3d ta scaffolds with the molded side up were placed into a 6-well tissue culture plate ( greiner bio one , frickenhausen , germany ) and covered with 4 ml of dmem . 1 10 cells in 100 l dmem were seeded in a meandering pattern onto the surface of the medium - incubated ta scaffolds . for 3d dynamic cell cultures in scaffolds , we developed not only the in vitro 3d module but also a perfusion cell culture reactor ( cellynyzer , institute for polymer technologies wismar , germany ) . this cell culture reactor was made by rapid prototyping on the basis of a biocompatible methacrylate resin ( fotomed led.a , innovation meditech gmbh , germany ) ( height of 60 mm and 25 mm in radius ) . the perfusion cell culture reactor cellynyzer was sterilized by autoclaving ( 121c steam , mammooth ) . gas permeable tubes ( fluidflex silicon hg , liquid - scan , germany ) were chosen for the supply of medium to the cell culture reactor due to their ph maintenance at 37c in a humidified atmosphere with 5% co2 . the seeded in vitro 3d module ( see figure 2 ) was integrated into the perfusion cell culture reactor . the cell culture medium was pumped through the 3d module with a flow direction being bottom - up within the reactor . after cell culturing for the appropriate time under the defined medium flow , cells on every level of the in vitro 3d module underwent a separate cell - biological analysis . in this way , cells from the core of the in vitro 3d module could be analyzed without cutting any material and cell observation was therefore nondestructive . mg-63 cells were cultured in the in vitro 3d module system in dynamic as well as static mode ( without flow ) for 7 days as described . vitality was analyzed by live / dead staining ( l7013 live / dead cell viability kit , invitrogen , germany ) after demounting the ta scaffold , washing carefully with hank 's buffered salt solution ( hbss , paa laboratories gmbh , germany ) and staining the cells with a solution of syto 10 and dead red ( 8 l of each dye in 4 ml hbss ) for 15 min at 37c in the dark . cell density on the different levels of the in vitro 3d module was visualized after 7 days of culture under static conditions . the oxygen supply of the cell culture was analyzed by a computer model based on the finite element method ( fem ) . the channel structure of the ta scaffold was modeled by double - walled connected spheres ( figures 4(b)4(d ) ) ; the narrow region between the outer and inner wall represents the volume in which the cells were located . periodic boundary conditions were assumed in the horizontal directions ( figure 4(d ) ) ; each end face was linked to the symmetry - equivalent end face on the opposite site of the simulation box leading to a total number of 94 periodic boundary conditions for each half of the simulation box . the local oxygen concentration was evaluated for a dense cell layer on the channel walls of the scaffold . the external reservoir of fresh medium was assumed to be large compared to the volume of the perfusion reactor , and consequently the oxygen concentration was set to a constant bulk value of c0 outside the reactor as well as at the inlet-/outlet - interfaces . in the cell - occupied regions oxygen is consumed at a given consumption rate r. for the calculation of the oxygen concentration , the simulation procedures of coletti et al . the diffusion of oxygen was described by the general diffusion equation ( 1)ct+dc = ruc , where c is the local oxygen concentration , d is the diffusion constant of oxygen in the medium , is the nabla - operator , and u is the local velocity vector of the media . because the oxygen consumption of the cells depends on the locally available amount of oxygen , a michaelis - menten - type expression was used for r , similar to the calculations of buchwald et al . in the equation rmax is the maximal oxygen consumption rate of the osteoblast cells , cmm , the michaelis - menten constant , that is , the oxygen concentration at which the cells consume only 50% of rmax . the oxygen concentration c0 for a fresh medium at 37c was set to 0.42 mol / m ; the diffusion constant d of oxygen was set to 2.68 10 m / s . for reasons of comparability , the values for the critical concentration and the michaelis - menten constant were taken from the simulations of buchwald et al . demonstrated that the consumption rates of osteoblasts vary between 5.56 10 mol/(cellmin ) for osteoblasts cultured in static t - flasks and 1.25 10 mol/(cellmin ) for encapsulated medium in vitro . please note that all specified flow rates refer to the entire scaffold ( such as in the experiments ) and are internally converted to the region of the simulation box by analyzing the ratio of the respective surfaces . m was used for the scaffold ( representing the inner region of the ta disk , which is not blocked by the clamping ring ) and 8.40 10 m for the simulation box . the porous channel structure of the scaffold was constructed by double - walled spheres with an outer radius of 250 m and an inner radius of 245 m ( thickness of the cell layer : 5 m ) . the channel structure was meshed with a free tetrahedral mesh ( 1,244,382 elements ) and the equations were solved using the parallel direct sparse solver ( pardiso ) . perfusion culture in a bioreactor with a pump system that perfuses nutrition media directly through the cell - seeded scaffold can obviously mitigate internal diffusional limitations which result in cell death [ 23 , 38 ] . different perfusion bioreactor systems used with different flow rates or velocities are mentioned in the literature [ 1923 ] . perfusion ensures an adequate supply to the cells but higher continuous flow rates led to increased levels of cell death due to shear stress [ 2628 ] . optimal flow velocities depend on the scaffold volume and the pore size and have to be determined separately for each perfusion system . a setup in which all levels of the 3d module were seeded by osteoblast cells before assembling the module enabled the analysis of the cell survival in the core of the scaffold ( see figure 2 ) . to promote the sufficient supply of the cells in the core of the scaffold we complemented the in vitro 3d module with a perfusion bioreactor , thus creating a novel dynamic system . because the commercially available perfusion systems did not meet our demands for the size and special shape of our 3d module , we developed a custom - made direct perfusion bioreactor usable in a standard lab incubator with a humidified atmosphere and 5% co2 . to identify the optimal flow rate for our system , we started with a very low pump velocity ( 5 l / min ) . cell viability and cell density were analyzed on the different levels after 7 days of culture and demounting the scaffolds from the dynamic in vitro 3d module . therefore velocity was enhanced progressively for the next experiments taking into consideration enhanced shear stress caused by high medium flow . cell vitality on the different scaffold levels was correlated with the medium flow rate for every experiment . in this way we found that cell survival rose on level 4 with a higher medium flow up to 45 l / min . cell vitality and density in the core region ( levels 2 and 3 ) were very low with flow rates below 30 l / min but increased at rates up to 60 l / min . as the flow direction in the cell culture reactor was bottom - up , cells on level 4 were always supplied with fresh medium . if the flow was too low , for example , 5 l / min , cells on the upper levels were supplied with used medium which was depleted of oxygen and nutrients and enriched with metabolic products by the cells from the levels below . interestingly , cell viability also decreased with the flow rate 60 l / min . dead cells could be detected especially on level 4 and the cell density was reduced . on the other hand , cell survival improved in the core ( levels 2 and 3 ) , but on these levels dead cells could be detected too . cell survival in the core region ( levels 2 and 3 ) was reduced as expected . to understand the nutrient supply in the scaffold , the experiments were supported by computer simulations . figure 7 displays the local oxygen concentration in the simulation box for different nutrient supply rates . regions marked in red represent areas that are well supplied with oxygen , in yellow and green regions in which the oxygen concentration is significantly reduced , and in the dark blue areas a critical lack of oxygen . without flow the lowest oxygen concentrations were found in the middle of the scaffold , because new oxygen could diffuse from the inlet and from the outlet into the system but did not reach the inner regions before it was consumed . at low flow rates ( 5 l / min30 l / min ) , the oxygen supply did not significantly improve , but the undersupplied region shifted in the direction of the outlet . the reason for this shift was that on the one hand fresh medium flowed into the system and increased the oxygen concentration in the vicinity of the inlet . on the other hand , however , the flow counteracted the inward diffusion of oxygen at the outlet so that the equilibrium concentration was reduced there . the system was only sufficiently supplied with oxygen at high flow rates ( 45 l / min60 l / min ) , because incoming molecules were not completely consumed before they reached the region in the middle of the scaffold . figure 8 shows the oxygen concentration at the four analysis levels of the in vitro 3d module in the perfusion reactor for different flow rates . in the static cell culture , the oxygen concentration was reduced at level 1 ( outlet ) and level 4 ( inlet ) compared to fresh medium and levels 2 and 3 ( in the core of the scaffold ) were insufficiently supplied with oxygen . in comparison , the oxygen concentration decreased at level 1 and increased at level 4 for low flow rates ( 5 l / min30 l / min ) ; at level 2 and level 3 no significant change in the oxygen concentration was registered . only at high flow rates ( 45 l / min60 l / min ) all levels in addition to the described changes in the oxygen concentration , increased shear rates have to be considered if the medium flow is increased . figure 9 displays the calculated shear rates at the cell surface for the four analysis levels and the different flow rates . however , the difference between the outer levels ( 1 , 4 ) and the inner levels ( 2 , 3 ) also increased at higher flow rates . these different shear rates may have an influence on the growth rate of the cells . the comparison of results from the cell culture experiments in the dynamic 3d in vitro cell culture module and the computer simulation of oxygen supply and shear stress indicate a correlation between local population of vital cells and oxygen supply in the scaffold . by enhancing the medium flow insufficiently , unfortunately , medium flow rates for adequate oxygen supply in all levels result in high levels of shear stress in the outer levels 1 and 4 . therefore it can be deduced that if oxygen supply does not impede the cell population in the scaffold , shear stress will . for lower medium flow rates between 15 l / min and 30 l / min cells will occupy the outer levels 1 and 4 . with rising flow rates up to 45 l / min the cell vitality in the core region ( levels 2 and 3 ) will grow through sufficient oxygen supply to the cells . for further enhanced flows up to 60 l / min the cell population will remain viable in the interior only ( levels 2 and 3 ) . therefore a flow of 45 l / min can be assumed as the optimal medium flow rate for this dynamic 3d in vitro cell culture module . were able to show a significantly reduced oxygen supply of cells in the core region of a similar 3d module already after one day of static culture , but no significant acidification by cell metabolites . these results indicate that the local oxygen concentration is affected more quickly by the cells than other parameters and therefore is probably the most critical parameter for cell growth . in the present study , an in vitro 3d module with a perfusion cell culture reactor for dynamic cell cultures was developed and used to achieve enhanced cell viability in the core of scaffolds with dimensions suitable for large bone defects . the optimal flow rate for the survival of mg-63 cells was obtained at around 45 l / min in the dynamic mode , where fully cell - seeded scaffolds showed vital cells and cell death could not be detected on all four levels after 7 days of culture . reduced cell survival with higher continuous flow rates could be attributed to cell death induced by shear stress , as shown by our simulation and as other groups also observed [ 2628 ] . investigated the influence of enhanced medium flow on the cell proliferation and differentiation of mc3t3-e1 osteoblast - like cells perfused for 1 week at flow rates of 10 , 100 , and 200 l / min and 1.0 ml / min . on hydroxyapatite / collagen scaffolds colonized with stro-1a cells they found stimulated proliferation at flow rates of 300 l / min , but osteogenic marker gene expression was enhanced with a low flow of only 30 l / min therefore , the results seem to be contradictory and it is difficult to deduce the optimal flow from these examples . taking into account that scaffold and pore size influence the distribution of the fluid and alter the shear forces generated in the scaffold , large scaffolds with higher pore size should require higher flow rates in perfusion culture than small ones . investigated the effects of flow shear stress on the construction of a large - scale tissue - engineered -tricalcium phosphate scaffold ( 14 mm in diameter , 30 mm in height , with a tunnel of 3.5 mm in diameter ) seeded with human bone marrow stromal cells . the number of cells in the constructs showed lower values compared to static culture , while dynamic conditions tended to enhance osteogenic differentiation . as insufficient cell supply in the core of a scaffold could result in impaired cell migration towards the center , peripherally seeded scaffolds should be cultured under optimal perfusion flow for cell survival in the core first , followed by induction of osteogenic differentiation through enhanced flow . in this context , the optimal flow rate of 45 l / min for osteoblast survival in the combined large scaffold with a height of 10 mm in our dynamic in vitro 3d module was within the assumed range . in this preliminary study , we observed acceptable cell survival on the different levels after a cultivation time of seven days by live / dead staining of the cells . these results were confirmed by a simulation model for oxygen supply and shear stress . using the optimal conditions for cell survival , our dynamic in vitro module can also be used for osteoblast migration studies by changing the cell seeding setup . in this way , active cell migration against gravity could be analyzed , for instance , dependent on the surface properties ( chemical gradients ) of the scaffolds . the optimal perfusion flow for osteogenic differentiation in our dynamic in vitro 3d module will be determined in further studies with mesenchymal stem cells or preosteoblasts . for optimal oxygen supply of all cells in the scaffold new architecture of scaffolds should be developed , where small cell - seeded channels are supplied with nutrients by diffusion from separate channels which are not seeded by cells . in these nonseeded channels higher medium flow rates the dynamic in vitro 3d module described here is a system well suited for the nondestructive investigation of cell behavior inside scaffolds with dimensions corresponding to large bone defects . for this 3d module , simulation of oxygen supply and shear stress combined with in vitro experiments result in an optimal flow rate near 45 l / min as a reasonable compromise . the 3d module combined with a dynamic cell culture in the perfusion reactor can be used for the observation of cell growth inside porous implant materials . scaffolds with pore sizes of around 500 m and a minimum height of 10 mm can be investigated with respect to material composition and chemical surface modifications under an optimal perfusion flow rate .

the neanderthals are a well - known middle pleistocene population , which was autochthonous in europe during mois 6 , 5 , and 4 . the european neanderthals are associated with mousterian assemblages . during the later part of mois3 , in the late pleistocene , europe was also populated by modern humans . the presence in europe of modern humans is inferred , according to some authors , in the oldest eastern european sites by association with protoaurignacian or aurignacian assemblages and , for later periods of mois3 , by association also with fossil remains . the dates available for the protoaurignacian , aurignacian , and late mousterian sites show that , after the arrival of modern humans , there was a period of coexistence between these two populations in europe for at least 15,000 years . this period is marked by an increase of aurignacian sites throughout europe , the appearance of so - called transitional assemblage sites ( chatelperronian , uluzzian , szeletian , lincombian , ranisian , jerzmanowician ) , and the decline of mousterian sites . after 25,000 years bp , the mousterian sites and the neanderthal population completely disappeared in europe , and only modern humans survived on this continent . despite numerous investigations , the debate concerning whether neanderthals became extinct because of climate change or competition with modern humans is still unresolved . some researchers argue that competition alone can not be the cause of neanderthal extinction [ 47 ] . by contrast , other authors support the existence of competitive exclusion for the same niche and argue that competition played a major role in the demise of the neanderthal population . some analyses , which are based on mathematical modeling , lack plausibility because they are too theoretical ; others , which are based on more integrative simulations or which take into account archeological and ethnologic examples , are more convincing . the modelling approach is used to understand complex systems by working on a simplified model of these systems . thus , this process involves the choice of certain parameters and variables which , if they are simplified , are nonetheless controlled in such a way that they are capable of representing the system as a whole . therefore , the model used in this paper does not attempt to determine the kind of food that neanderthals and modern humans consumed but to highlight the potential differences in dietary habits characteristic of these two populations . the aim of this paper is to test the hypothesis that resource competition , analysed through isotopic modelling , was strong between neanderthals and modern humans . it assumes that if a model shows similar dietary patterns for neanderthals and modern humans , then these two populations would be in competition for resources . by contrast , if the models show differences in dietary patterns , this would signify that resource competition would be less intense . for some years , isotopic biochemistry allowed us to improve our knowledge about past human diet using carbon and nitrogen isotopic ratios [ 1115 ] . since 1990 's these methods have been increasingly used to study paleontological populations such as neanderthals or early modern humans , in order to understand their relationship with the local environment ( e.g. , [ 11 , 1623 ] ) . therefore , the literature contains a substantial number of isotopic data , mainly on carbon and nitrogen isotopic values measured on bones and dental collagen . the previous studies suggested that prehistoric peoples had a carnivorous diet similar to that of contemporaneous predators , such as cave lions or cave hyenas [ 19 , 24 ] . these isotopic studies are consistent with zooarchaeological investigations which showed that neanderthals and modern humans were big game hunters ( hunting mainly big ungulates ) [ 2530 ] . the isotopic modelling used in this paper presents a new method of investigation that intends to contribute to the debate on resource competition between neanderthals and modern humans which has often been assumed but never really demonstrated . isotopic data from 51 major archaeological sites in europe ( figure 1 ) were compiled from 42 publications . of these archaeological sites , , isotopic data from 945 specimens ( faunal and human ) was assembled from the literature . this paper focuses on the transition between mois3 and mois2 ; as such , isotope data from species unavailable during these time periods was eliminated from the data set . furthermore , the models employed in this paper rely exclusively on three faunal types ( reindeer , horse , and bovid ) because these were the only remains present at all sites . as a result , only isotope data from 599 specimens were included in this analysis ( table 1 and tables si1 , si2 , si3 ) . as drucker has shown , local environmental context can influence isotopic signatures of plants and consequently those of consumers . as a result , the first step of our analysis was to verify isotopic modifications for each faunal type through time and space . thus , in addition to chronology ( e.g. , late mois3 versus mois2 ) , data relating to geography and environment were also considered ( table 2 ) . radiocarbon dates were updated by jris and street 2008 . in order to study the transition between late mois3 and mois2 , we created models by grouping faunal types and humans species into three chronological groups : ( 1 ) mois3 neanderthals , ( 2 ) mois3 modern humans , ( 3 ) mois2 modern humans . in this paper mois3 in order to nominate the coevolution period of neanderthals and modern humans in europe ; thus these groups were analysed in three different ways : ( 1 ) an absence of cluster ( global ) , ( 2 ) a geographical cluster , ( 3 ) an environmental cluster . due to limitations related to the faunal isotopic data available for each cluster and the fact that models have to be run with the same characteristics for diachronic comparison , models were limited to : ( 1 ) the whole dataset , ( 2 ) the data of south - western area , ( 3 ) the data relating to cold environments of tundra - steppe and open boreal woodland ( table 3 ) . patterns in human and animal food consumption since collagen is protein , the stable isotope ratio of this tissue provides information on the protein component of the diet over approximately the last 10 years of an individual 's life [ 63 , 64 ] . because plants and animals differ in their carbon and nitrogen isotope ratios it is possible to use their ratio to infer past dietary patterns . carbon isotope ratios are typically used to differentiate between the consumption of c3 versus c4 plants or marine fish versus fresh water fish [ 11 , 14 , 63 , 65 , 66 ] . in contrast , nitrogen isotope ratios are indicative of trophic level ( i.e. , an individual position in the food web ) [ 65 , 67 ] . stable isotope ratios reflect the type of primary protein sources and are successively enriched in the heavy isotope ( c , n ) with each step up the food web [ 65 , 68 ] . thus , the relative isotopic variability between different organisms of a terrestrial and aquatic trophic web is distributed in a predictive way from plants at the baseline of the food chain through the subsequent levels as herbivorous and carnivorous organisms . for example , the c and n values of collagen from herbivores are approximately 5% and 35% higher , respectively , than plants [ 20 , 63 ] . in a similar way , the c and n values of collagen from carnivores are approximately 0.81.3% and 35% higher , respectively , than herbivores . according to isotopic data available for the palaeolithic terrestrial environments , c values of plants range from 35 to 20% with a distinction between open and closed environments , and n values of plants ranged from 0 to 6% . the c and n values of collagen from herbivores range from 30 to 18% and 3 to 8% , respectively . the c and n values of collagen from carnivores range from 24 to 16% and 7 and 13% , respectively . the c and n values of collagen from freshwater fish range from 23 and 19% and 9 and 15% , respectively . following the predictive fractionation of isotope ratios through the food chain , phillips and colleagues proposed different isotopic mixing models to quantify the relative contributions of the different dietary sources to an individual [ 6971 ] . isoerror ( 2001 ) and isoconc ( 2002 ) based on the isotopic mixing models estimate the proportions for two food sources using a single isotopic element or three sources using two isotopic elements . isoerror mixing model considers the isotopic signature standard deviations in the source and mixture populations and restitutes food proportions with confidence intervals for source proportion estimates . in contrast , the isoconc mixing model is a concentration - weighted linear mixing model which considers for each element the contribution of source as proportional to the weight of the elemental concentration in that source . the isosource mixing model ( 2005 ) can estimate more than 3 sources using 2 stable isotopic elements when food sources are isotopically very different . the different isotopic mixing models have previously been applied in past foodwebs to assess the relative proportions of different sources to a single or population human mixtures [ 33 , 7278 ] . based on isotopic mixing model proposed by phillips et al . , the final aim of the research was not to determine the real contributions of specific food sources to a mixture but to compare the human dietary patterns over time and space within distinct chronological groups . simulations were run with isosource mixing models ( version 1.3.1 ) [ 69 , 79 ] . the use of mixing models to study european palaeolithic populations could be limited because the mixing models were designed from datasets composed of living north american animals . however , regardless of chronological period and geographical context , the same principle of isotopic fractionation along the trophic chain is generally applicable to all isotopic studies . furthermore , the mixing models were applied to compare different cohorts according to different parameters and not to precisely reconstruct the proportions of different resources consumed by past human populations . as such , whatever limitations exist will be similar in each group and should not affect the comparisons . for example , in this study we assume that all the hominids considered consumed the same kinds of resources . following the recommendation of phillips et al . , only three food sources were considered : fish , meat , and plant resources because they are ( i ) largely distant from the mixture and ( ii ) sufficient and reliable to consider the main food items consumed in typical omnivorous diet . due to the lack of data , plants are not often considered in previous isotopic studies whereas they are necessary for human survival . this resource was included in our simulations permitting to consider one of food items consumed by omnivorous . due to the isotopic fractionation from diet to consumer ( bone collagen ) , in following simulations , isotopic values of hominid diet , mentioned as mixing diet or mixture , were considered to be lower than isotopic values of hominid collagens of 0.81.3% for carbon and 35% for nitrogen [ 19 , 20 , 65 , 68 ] . for each cluster datasets , and according to the appropriate fractionation factor requires for running models , isotopic values of plants were estimated from available herbivorous isotopic values ( 0.81.3% for carbon and 35% for nitrogen ) ( see supplementary material si3 available online at doi : 10.4061/2011/689315 ) . isotopic values of meat and fish resources of hominid diets were derived from the literature and summarized in the database ( table si1 ) . for each cluster ( global , geographical , and environmental ) , 3 sets of simulations were performed ( one for each chronological groups : mois3 neanderthals , mois3 modern humans , and mois2 modern humans ) . to get the best inputs for running isosource and to have comparable patterns for each chronological group , a similar geometric construction was realised within the estimated carbon and nitrogen variability of each food source ( mean sd ) ( figure 2 ) . the isotopic variability for herbivorous source was calculated by averaging the mean of each faunal type isotopic signature to avoid any bias due to sample size . moreover , concerning the whole dataset and the cold environments dataset , the geometric construction usable for modelling had to be based on the largest isotopic values of sources around the mixture . concerning the south - western dataset , two sets of simulations have been performed ; the first one involves a geometric construction based on the largest isotopic values of sources around the mixture ( mean 1sd ) , and the second one involves a geometric construction based on average isotopic values of sources ( mean ) . to assess the validity of the different clusters proposed in section 1 , a set of kruskall - walis statistics kruskall - wallis statistics are nonparametric tests generally used to compare the distribution of two independent sets of values . here the aim is to test ( i ) if chronological groups are reliable for investigating the homogeneity of stable isotopic signatures of each faunal group across the time and ( ii ) if all archaeological sites associated in one cluster present homogeneous stable isotopic ratios over space . although in order to compare , in each hominid group and in each simulation , the contribution of each source to the mixture , chi - squared test with a bonferroni correction was used . to use the chi - squared test , a standardisation of results has been done based on a calculation of a resource unit related to the quantity of protein intake . a descriptive statistical analysis of the complete isotopic dataset was performed to detect whether isotopic differences existed ( i ) between the archaeological sites of the considered geographic region , ( ii ) between the archaeological sites of the considered environments as well as ( iii ) between chronological subdivisions of mois3 and mois2 . kruskal - wallis analyses were implemented on the whole dataset , on the south - western dataset , and on the cold environment dataset to test if chronological groups are efficient and if all archaeological sites associated with a chronological group ( mois3 neanderthal , mois3 modern humans , or mois2 modern humans ) could be pooled together in terms of carbon and nitrogen isotopic ratios . p values lower than 0.01 indicate statistically significant differences suggesting an isotopic heterogeneity within groups . regarding the analysis of the complete dataset ( table si4 ) , we often observed a particular dietary / isotopic behaviour for the reindeer which seems attributed to local environment . this is why we decided to perform two sets of simulations , a set including the reindeer and a set excluding from the reindeer . we identified and excluded the divergent archaeological sites for bovids ( gough 's cave and kendrick 's cave for bovids ) . since divergent archaeological sites were not identifiable for horse , in order to avoid any biases , several sets of simulations were conducted with different n values for herbivorous sources ( n 1sd ; figure 2 ) . for the south - western area ( table si5 ) , reindeer isotopic variability seems attributed to local environment whereas bovids ' seems to be heterogeneous during mois2 . to avoid any biases , divergent sites have been excluded from the simulations ( pont d'ambon , ) . as mentioned above , additional simulations have also been performed with and without reindeer . in cold environments ( table si6 ) , similarly , simulations with and without consideration of the reindeer isotopic variability within the herbivorous group were conducted . to summarise , based on the results of kruskal - wallis analyses , simulations were performed according to each cluster and each chronological group under several conditions : ( 1 ) presence of the three faunal types in the herbivorous source ( bovid , reindeer , horse ) , ( 2 ) presence of bovids and horse , in the herbivorous source , to take into account the isotopic variability of reindeer across archaeological sites ) , ( 3 ) presence of bovids and reindeer in the herbivorous source , to take into account that horses are nonruminant species . for the environmental cluster only , ( 4 ) a fourth simulation was performed considering only the reindeer in the herbivorous source since it was probably the main species consumed under these cold climates . in addition , two sets of simulations were performed to guaranty the most reliable application of isotopic biochemistry in the reconstruction of past diet : ( 1 ) one considering the totality of the hominids for each chronological group and ( 2 ) a second considering only the hominids associated with fauna . in total , 19 simulations have been performed on the whole dataset , 28 on the south - western dataset , and 16 on the cold environment dataset ( table 4 ) . simulations were implemented using three food sources selected according to isotopic dataset relative to each cluster and each chronological group . in all simulations , source 2 and source 3 isotopic signatures were determined according to isotopic dataset relative to each cluster and each chronological group . in all simulations , source 2 shows intermediate n values and high c values whereas source 3 exhibits low c and n values . the result of the simulations is driven by the relative position of the mixture compared to the three sources . regarding the mois3 neanderthals ' dataset ( table 5 ) , in all clusters and under all conditions , the contribution of source 1 is the highest ( between 48% and 67% of the mixing food ) , suggesting a consumption of food with high n values and low c values . the contribution of source 3 ( between 28% and 44% ) is lower than source 1 but higher than the contribution of source 2 ( between 1% and 19% ) , which confirms the greater consumption of food with low c signatures . more precisely , simulations without considering the reindeer isotopic values tend to increase the contribution of source 2 , whereas simulations without considering the horse isotopic values tend to decrease the contribution of source 2 . in other words , the more the isotopic value of the herbivorous source is enriched in c , the fewer source 2 contributes to the mixture . in the south - western area , even if the contribution of source 2 slightly decreases when simulations are based on average isotopic values of different sources , the differences between the two sets of simulations ( regarding a large variability versus an average variability ) are not significant . concerning the mois3 modern humans ' dataset ( table 6 ) , the results of simulations within the south - western area exhibit different dietary patterns relatively to the other two clusters ( global and environmental ) . for the three clusters , the contribution of source 1 is the highest ( between 58% and 72% ) . in the south - western area , the contribution of source 2 is higher than the contribution of source 3 ( resp . it is the opposite for the global and environmental clusters , the contribution of source 3 is higher than the contribution of source 2 ( resp . regardless of the faunal types considered in the herbivorous source , the results globally remain the same for the cold environments and the complete datasets . nevertheless , when the reindeers are absent , the contribution of source 3 is lower than the contribution of source 2 . it is the opposite when horses are not considered in the simulations . in the south - western area , similar dietary patterns are observed when the reindeers are not considered ; the contribution of source 3 clearly decreases whereas the contribution of source 2 clearly increases . as suggested for mois3 neanderthals , we can suppose that the more the isotopic values of herbivorous source are enriched in c , the fewer source 2 contributes to the mixture . the divergent results observed for the south - western area regarding the relative contribution of source 2 and 3 could be explained by a bias induced by the isotopic value of the mixture representing only by one available data in the south - western area for this period . differences between the two sets of simulations for the south - western area are low . the contribution of source 3 slightly decreases when simulations are based on average isotopic values of the three sources whereas the contribution of source 2 increases . concerning the mois2 modern humans dataset ( table 7 ) , independently of clusters and contrary to the mois3 neanderthals and modern humans , dietary patterns show a slight predominance of source 2 ( between 20% and 82% ) compared with the other two sources ( between 0 and 40% for source 3 and between 30% and 51% for source 1 ) . overall , the contribution of source 3 slightly decreases when the reindeer are excluded and increases when the horse are excluded . these different dietary patterns according to the inclusion or exclusion of reindeer and horse in herbivorous source are consistent with the observations made for mois3 neanderthals and mois3 modern humans . regarding the two sets of simulations performed for the south - western area , the contribution of source 3 decreases in conjunction with an increase of source 2 when average isotopic values are considered for sources . the contribution of source 2 also increases when the herbivorous source exhibits high c values . the results of this statistical analysis emphasize previous observations ( table 8) . with the bonferroni correction and two degrees of freedom , dietary patterns of mois2 modern humans appear statistically different than the other two chronological groups . dietary patterns of mois3 modern humans and mois3 neanderthals do not exhibit significant differences for global and environmental clusters . for geographical cluster , as observed on raw results , simulations show significant differences in dietary patterns between mois3 modern humans and mois3 neanderthals . as mentioned before , this could be explained by the unique nitrogen isotopic value available for mois3 south - western modern humans . our research on past human diet during the transition from mois3 to mois2 is based on the modelling of isotopic signatures of a mixture over time and under several clusters ( global , geographic , or environmental clusters ) . our aim has been to test the hypothesis that resource competition , analysed through isotopic modelling , may have existed between neanderthals and contemporaneous modern humans . isotopic analyses , which are generally used for studying local environments , would also seem to be applicable to a population approach . indeed , the results of our modelling illustrate that , whatever cluster is considered , the dietary behaviour of each chronological group shows similar dietary patterns . in order to compensate for the lack of reliability of certain sets of data , modelling seems to be a relevant approach . in agreement with the hypotheses underlying our models , we were able to compare mois3 modern humans with the two other hominid groups ( mois3 neanderthals & mois2 modern humans ) , even if hominid isotopic values for mois3 modern humans considered in simulations were not associated with the faunal isotopic values . indeed , results of our modelling show that whatever the conditions considered ( hominids with and without associated fauna ) , the dietary behaviour of each chronological group shows similar patterns . concerning the diachronic analysis of past dietary patterns , our study demonstrates the absence of significant differences between neanderthal diet and that of contemporaneous modern human and highlights the dietary difference among mois2 modern human . these conclusions confirm that resource competition may have occurred during mois3 between the two hominid populations living in europe . some authors have already suggested competition between the two populations by observing a correspondence between the contraction of the neanderthal ecological niche and the expansion of the ecological niche of modern humans . however , radiocarbon dates do not provide any cases of geological interstratification ( shown by mousterian , aurignacian , and transitional assemblages ) , which would support the contemporaneity of neanderthals and modern humans . it has been argued that much of europe was almost empty when the modern human expansion occurred [ 7 , 83 ] . thus , given the radiocarbon dates , little contact would occur between the two populations in europe , except in the southwest of france and in the north of spain , where encounters may have been more frequent . the divergence of mois2 modern human behaviour in relation to mois3 populations ( neanderthals and modern humans ) may be explained through use by the former population of alternative food sources , such as , for example , small fauna . our results are consistent with the observation of certain prehistorians , who suggest a relative continuity in behavior between neanderthals and contemporaneous modern humans and a behavioural modification between aurignacian and gravettian modern humans [ 84 , 85 ] . most publications on modern human dietary spectra have underlined that this population had a more diverse diet than neanderthals , consuming , beside ungulates , small game prey like fish or small mammals [ 19 , 20 , 24 , 8688 ] . due to the lack of data , stable isotopic signatures of the small prey were not considered in our simulations , and it is therefore difficult to reach any conclusions concerning potential modifications in the consumption of this type of prey . nevertheless , if small prey have similar isotopic signatures to those of reindeer , bovids , and horse used here , our results would explain the difference in patterns observed for mois2 modern humans ( with a higher contribution of source 2 ) in comparison to mois3 neanderthals and mois3 modern humans . on the contrary , the predominance of source 1 in mois3 neanderthals and mois3 modern humans could indicate a consumption of food with high n values and low c values . source 1 considered in our simulations was derived from fish isotopic values , although other species with similar isotopic value , such as mammoths , may have been taken into account and used instead of fish . furthermore , zooarchaeological and genetic studies indicate a decline in the quantity of big game and notably ungulates , beginning at about 50,000 years bp [ 8991 ] . on the basis of our results , neanderthals did not change their diet during mois3 , as richards and trinkaus have already suggested in 2009 . prior to the early modern human expansion , it is possible that neanderthals , which had a small population size [ 9398 ] , were not affected by a reduction of the population size of large mammals . by contrast , during the period of coexistence of neanderthals and modern humans , the mois3 , we can suppose that faunal contraction , associated with resource competition , might have had more serious consequences . indeed , neanderthals were probably close to their carrying capacity due to the decrease of the ungulate populations , as is suggested by certain stress markers ( dental hypoplasia , etc . ) [ 100 , 101 ] . in such conditions , the arrival of species evolving in a same ecological niche might have led to strong competition and perhaps contributed to the demise of neanderthals , although competition alone can not account for neanderthal extinction . some authors suggest that early modern humans would have possessed more varied technical abilities [ 10 , 102104 ] , were able to adjust their hunting toward a more varied prey , and would have required less food and fewer foraging returns [ 105 , 106 ] . , our study has adopted the hypothesis that all the hominids we have considered consumed the same kind of resources . provided that this hypothesis does not involve a major bias , our methodology , based upon modelling , has permitted us to address the question concerning resource competition between neanderthals and modern humans , to which zooarchaeological approaches , in view of the complexity of faunal assemblages , could not supply a clear answer . thanks to a substantial isotopic database , this study confirms the occurrence of resource competition between neanderthals and contemporaneous modern humans living in the same area . it is however not possible to reach a conclusion concerning the role of competition in neanderthals ' demise . furthermore , this study underlines the dietary changes that occurred during mois2 , as proposed by the study of lithic industry and archaeological data of faunal assemblage . it also demonstrates that modelling approaches and dietary assessment are useful for investigating ecological interaction among both present and past populations . in order to answer this last question , we are engaged in a study in progress , which uses complex mathematical models to represent , as plausibly as possible , the trophic web of neanderthals and the interaction between prey , predators , and hominid groups ( neanderthals and early modern humans ) .

during later mois3 , in europe two populations were present , autochthonous neanderthals and modern humans . ecological competition between these two populations has often been evoked but never demonstrated . our aim is to establish whether resource competition occurred . in this paper , in order to examine the possibility of ecological competition between these two populations , 599 isotopic data were subjected to rigorous statistical treatment and analysis through mixing models . the aim of this paper was to compare dietary strategies of neanderthals and modern humans over time . our conclusions suggest that neanderthals and modern humans shared dietary habits in the particular environmental context of mois3 characterised in europe by climatic deterioration . in this environmental context , the resource competition between neanderthals and modern humans may have accelerated the disappearance of the neanderthal population .

1. Introduction 2. Material and Methods 3. Results 4. Discussion and Conclusions

the neanderthals are a well - known middle pleistocene population , which was autochthonous in europe during mois 6 , 5 , and 4 . during the later part of mois3 , in the late pleistocene , europe was also populated by modern humans . the presence in europe of modern humans is inferred , according to some authors , in the oldest eastern european sites by association with protoaurignacian or aurignacian assemblages and , for later periods of mois3 , by association also with fossil remains . the dates available for the protoaurignacian , aurignacian , and late mousterian sites show that , after the arrival of modern humans , there was a period of coexistence between these two populations in europe for at least 15,000 years . this period is marked by an increase of aurignacian sites throughout europe , the appearance of so - called transitional assemblage sites ( chatelperronian , uluzzian , szeletian , lincombian , ranisian , jerzmanowician ) , and the decline of mousterian sites . after 25,000 years bp , the mousterian sites and the neanderthal population completely disappeared in europe , and only modern humans survived on this continent . despite numerous investigations , the debate concerning whether neanderthals became extinct because of climate change or competition with modern humans is still unresolved . by contrast , other authors support the existence of competitive exclusion for the same niche and argue that competition played a major role in the demise of the neanderthal population . therefore , the model used in this paper does not attempt to determine the kind of food that neanderthals and modern humans consumed but to highlight the potential differences in dietary habits characteristic of these two populations . the aim of this paper is to test the hypothesis that resource competition , analysed through isotopic modelling , was strong between neanderthals and modern humans . it assumes that if a model shows similar dietary patterns for neanderthals and modern humans , then these two populations would be in competition for resources . by contrast , if the models show differences in dietary patterns , this would signify that resource competition would be less intense . since 1990 's these methods have been increasingly used to study paleontological populations such as neanderthals or early modern humans , in order to understand their relationship with the local environment ( e.g. therefore , the literature contains a substantial number of isotopic data , mainly on carbon and nitrogen isotopic values measured on bones and dental collagen . these isotopic studies are consistent with zooarchaeological investigations which showed that neanderthals and modern humans were big game hunters ( hunting mainly big ungulates ) [ 2530 ] . the isotopic modelling used in this paper presents a new method of investigation that intends to contribute to the debate on resource competition between neanderthals and modern humans which has often been assumed but never really demonstrated . isotopic data from 51 major archaeological sites in europe ( figure 1 ) were compiled from 42 publications . of these archaeological sites , , isotopic data from 945 specimens ( faunal and human ) was assembled from the literature . furthermore , the models employed in this paper rely exclusively on three faunal types ( reindeer , horse , and bovid ) because these were the only remains present at all sites . as a result , only isotope data from 599 specimens were included in this analysis ( table 1 and tables si1 , si2 , si3 ) . as drucker has shown , local environmental context can influence isotopic signatures of plants and consequently those of consumers . as a result , the first step of our analysis was to verify isotopic modifications for each faunal type through time and space . thus , in addition to chronology ( e.g. in order to study the transition between late mois3 and mois2 , we created models by grouping faunal types and humans species into three chronological groups : ( 1 ) mois3 neanderthals , ( 2 ) mois3 modern humans , ( 3 ) mois2 modern humans . in this paper mois3 in order to nominate the coevolution period of neanderthals and modern humans in europe ; thus these groups were analysed in three different ways : ( 1 ) an absence of cluster ( global ) , ( 2 ) a geographical cluster , ( 3 ) an environmental cluster . patterns in human and animal food consumption since collagen is protein , the stable isotope ratio of this tissue provides information on the protein component of the diet over approximately the last 10 years of an individual 's life [ 63 , 64 ] . stable isotope ratios reflect the type of primary protein sources and are successively enriched in the heavy isotope ( c , n ) with each step up the food web [ 65 , 68 ] . thus , the relative isotopic variability between different organisms of a terrestrial and aquatic trophic web is distributed in a predictive way from plants at the baseline of the food chain through the subsequent levels as herbivorous and carnivorous organisms . for example , the c and n values of collagen from herbivores are approximately 5% and 35% higher , respectively , than plants [ 20 , 63 ] . in a similar way , the c and n values of collagen from carnivores are approximately 0.81.3% and 35% higher , respectively , than herbivores . according to isotopic data available for the palaeolithic terrestrial environments , c values of plants range from 35 to 20% with a distinction between open and closed environments , and n values of plants ranged from 0 to 6% . following the predictive fractionation of isotope ratios through the food chain , phillips and colleagues proposed different isotopic mixing models to quantify the relative contributions of the different dietary sources to an individual [ 6971 ] . isoerror ( 2001 ) and isoconc ( 2002 ) based on the isotopic mixing models estimate the proportions for two food sources using a single isotopic element or three sources using two isotopic elements . isoerror mixing model considers the isotopic signature standard deviations in the source and mixture populations and restitutes food proportions with confidence intervals for source proportion estimates . in contrast , the isoconc mixing model is a concentration - weighted linear mixing model which considers for each element the contribution of source as proportional to the weight of the elemental concentration in that source . , the final aim of the research was not to determine the real contributions of specific food sources to a mixture but to compare the human dietary patterns over time and space within distinct chronological groups . simulations were run with isosource mixing models ( version 1.3.1 ) [ 69 , 79 ] . the use of mixing models to study european palaeolithic populations could be limited because the mixing models were designed from datasets composed of living north american animals . however , regardless of chronological period and geographical context , the same principle of isotopic fractionation along the trophic chain is generally applicable to all isotopic studies . furthermore , the mixing models were applied to compare different cohorts according to different parameters and not to precisely reconstruct the proportions of different resources consumed by past human populations . for example , in this study we assume that all the hominids considered consumed the same kinds of resources . isotopic values of meat and fish resources of hominid diets were derived from the literature and summarized in the database ( table si1 ) . moreover , concerning the whole dataset and the cold environments dataset , the geometric construction usable for modelling had to be based on the largest isotopic values of sources around the mixture . to assess the validity of the different clusters proposed in section 1 , a set of kruskall - walis statistics kruskall - wallis statistics are nonparametric tests generally used to compare the distribution of two independent sets of values . here the aim is to test ( i ) if chronological groups are reliable for investigating the homogeneity of stable isotopic signatures of each faunal group across the time and ( ii ) if all archaeological sites associated in one cluster present homogeneous stable isotopic ratios over space . although in order to compare , in each hominid group and in each simulation , the contribution of each source to the mixture , chi - squared test with a bonferroni correction was used . a descriptive statistical analysis of the complete isotopic dataset was performed to detect whether isotopic differences existed ( i ) between the archaeological sites of the considered geographic region , ( ii ) between the archaeological sites of the considered environments as well as ( iii ) between chronological subdivisions of mois3 and mois2 . kruskal - wallis analyses were implemented on the whole dataset , on the south - western dataset , and on the cold environment dataset to test if chronological groups are efficient and if all archaeological sites associated with a chronological group ( mois3 neanderthal , mois3 modern humans , or mois2 modern humans ) could be pooled together in terms of carbon and nitrogen isotopic ratios . since divergent archaeological sites were not identifiable for horse , in order to avoid any biases , several sets of simulations were conducted with different n values for herbivorous sources ( n 1sd ; figure 2 ) . in cold environments ( table si6 ) , similarly , simulations with and without consideration of the reindeer isotopic variability within the herbivorous group were conducted . to summarise , based on the results of kruskal - wallis analyses , simulations were performed according to each cluster and each chronological group under several conditions : ( 1 ) presence of the three faunal types in the herbivorous source ( bovid , reindeer , horse ) , ( 2 ) presence of bovids and horse , in the herbivorous source , to take into account the isotopic variability of reindeer across archaeological sites ) , ( 3 ) presence of bovids and reindeer in the herbivorous source , to take into account that horses are nonruminant species . for the environmental cluster only , ( 4 ) a fourth simulation was performed considering only the reindeer in the herbivorous source since it was probably the main species consumed under these cold climates . in addition , two sets of simulations were performed to guaranty the most reliable application of isotopic biochemistry in the reconstruction of past diet : ( 1 ) one considering the totality of the hominids for each chronological group and ( 2 ) a second considering only the hominids associated with fauna . the result of the simulations is driven by the relative position of the mixture compared to the three sources . regarding the mois3 neanderthals ' dataset ( table 5 ) , in all clusters and under all conditions , the contribution of source 1 is the highest ( between 48% and 67% of the mixing food ) , suggesting a consumption of food with high n values and low c values . in other words , the more the isotopic value of the herbivorous source is enriched in c , the fewer source 2 contributes to the mixture . in the south - western area , even if the contribution of source 2 slightly decreases when simulations are based on average isotopic values of different sources , the differences between the two sets of simulations ( regarding a large variability versus an average variability ) are not significant . concerning the mois3 modern humans ' dataset ( table 6 ) , the results of simulations within the south - western area exhibit different dietary patterns relatively to the other two clusters ( global and environmental ) . for the three clusters , the contribution of source 1 is the highest ( between 58% and 72% ) . in the south - western area , the contribution of source 2 is higher than the contribution of source 3 ( resp . it is the opposite for the global and environmental clusters , the contribution of source 3 is higher than the contribution of source 2 ( resp . regardless of the faunal types considered in the herbivorous source , the results globally remain the same for the cold environments and the complete datasets . nevertheless , when the reindeers are absent , the contribution of source 3 is lower than the contribution of source 2 . it is the opposite when horses are not considered in the simulations . in the south - western area , similar dietary patterns are observed when the reindeers are not considered ; the contribution of source 3 clearly decreases whereas the contribution of source 2 clearly increases . the divergent results observed for the south - western area regarding the relative contribution of source 2 and 3 could be explained by a bias induced by the isotopic value of the mixture representing only by one available data in the south - western area for this period . the contribution of source 3 slightly decreases when simulations are based on average isotopic values of the three sources whereas the contribution of source 2 increases . concerning the mois2 modern humans dataset ( table 7 ) , independently of clusters and contrary to the mois3 neanderthals and modern humans , dietary patterns show a slight predominance of source 2 ( between 20% and 82% ) compared with the other two sources ( between 0 and 40% for source 3 and between 30% and 51% for source 1 ) . overall , the contribution of source 3 slightly decreases when the reindeer are excluded and increases when the horse are excluded . these different dietary patterns according to the inclusion or exclusion of reindeer and horse in herbivorous source are consistent with the observations made for mois3 neanderthals and mois3 modern humans . regarding the two sets of simulations performed for the south - western area , the contribution of source 3 decreases in conjunction with an increase of source 2 when average isotopic values are considered for sources . the results of this statistical analysis emphasize previous observations ( table 8) . with the bonferroni correction and two degrees of freedom , dietary patterns of mois2 modern humans appear statistically different than the other two chronological groups . dietary patterns of mois3 modern humans and mois3 neanderthals do not exhibit significant differences for global and environmental clusters . for geographical cluster , as observed on raw results , simulations show significant differences in dietary patterns between mois3 modern humans and mois3 neanderthals . as mentioned before , this could be explained by the unique nitrogen isotopic value available for mois3 south - western modern humans . our research on past human diet during the transition from mois3 to mois2 is based on the modelling of isotopic signatures of a mixture over time and under several clusters ( global , geographic , or environmental clusters ) . our aim has been to test the hypothesis that resource competition , analysed through isotopic modelling , may have existed between neanderthals and contemporaneous modern humans . indeed , the results of our modelling illustrate that , whatever cluster is considered , the dietary behaviour of each chronological group shows similar dietary patterns . in order to compensate for the lack of reliability of certain sets of data , modelling seems to be a relevant approach . in agreement with the hypotheses underlying our models , we were able to compare mois3 modern humans with the two other hominid groups ( mois3 neanderthals & mois2 modern humans ) , even if hominid isotopic values for mois3 modern humans considered in simulations were not associated with the faunal isotopic values . indeed , results of our modelling show that whatever the conditions considered ( hominids with and without associated fauna ) , the dietary behaviour of each chronological group shows similar patterns . these conclusions confirm that resource competition may have occurred during mois3 between the two hominid populations living in europe . some authors have already suggested competition between the two populations by observing a correspondence between the contraction of the neanderthal ecological niche and the expansion of the ecological niche of modern humans . however , radiocarbon dates do not provide any cases of geological interstratification ( shown by mousterian , aurignacian , and transitional assemblages ) , which would support the contemporaneity of neanderthals and modern humans . thus , given the radiocarbon dates , little contact would occur between the two populations in europe , except in the southwest of france and in the north of spain , where encounters may have been more frequent . the divergence of mois2 modern human behaviour in relation to mois3 populations ( neanderthals and modern humans ) may be explained through use by the former population of alternative food sources , such as , for example , small fauna . our results are consistent with the observation of certain prehistorians , who suggest a relative continuity in behavior between neanderthals and contemporaneous modern humans and a behavioural modification between aurignacian and gravettian modern humans [ 84 , 85 ] . due to the lack of data , stable isotopic signatures of the small prey were not considered in our simulations , and it is therefore difficult to reach any conclusions concerning potential modifications in the consumption of this type of prey . nevertheless , if small prey have similar isotopic signatures to those of reindeer , bovids , and horse used here , our results would explain the difference in patterns observed for mois2 modern humans ( with a higher contribution of source 2 ) in comparison to mois3 neanderthals and mois3 modern humans . on the contrary , the predominance of source 1 in mois3 neanderthals and mois3 modern humans could indicate a consumption of food with high n values and low c values . source 1 considered in our simulations was derived from fish isotopic values , although other species with similar isotopic value , such as mammoths , may have been taken into account and used instead of fish . furthermore , zooarchaeological and genetic studies indicate a decline in the quantity of big game and notably ungulates , beginning at about 50,000 years bp [ 8991 ] . on the basis of our results , neanderthals did not change their diet during mois3 , as richards and trinkaus have already suggested in 2009 . prior to the early modern human expansion , it is possible that neanderthals , which had a small population size [ 9398 ] , were not affected by a reduction of the population size of large mammals . by contrast , during the period of coexistence of neanderthals and modern humans , the mois3 , we can suppose that faunal contraction , associated with resource competition , might have had more serious consequences . indeed , neanderthals were probably close to their carrying capacity due to the decrease of the ungulate populations , as is suggested by certain stress markers ( dental hypoplasia , etc . ) in such conditions , the arrival of species evolving in a same ecological niche might have led to strong competition and perhaps contributed to the demise of neanderthals , although competition alone can not account for neanderthal extinction . some authors suggest that early modern humans would have possessed more varied technical abilities [ 10 , 102104 ] , were able to adjust their hunting toward a more varied prey , and would have required less food and fewer foraging returns [ 105 , 106 ] . provided that this hypothesis does not involve a major bias , our methodology , based upon modelling , has permitted us to address the question concerning resource competition between neanderthals and modern humans , to which zooarchaeological approaches , in view of the complexity of faunal assemblages , could not supply a clear answer . thanks to a substantial isotopic database , this study confirms the occurrence of resource competition between neanderthals and contemporaneous modern humans living in the same area . in order to answer this last question , we are engaged in a study in progress , which uses complex mathematical models to represent , as plausibly as possible , the trophic web of neanderthals and the interaction between prey , predators , and hominid groups ( neanderthals and early modern humans ) .

oxidative stress is a hallmark in numerous chronic and degenerative diseases associated with environmental factors . this stress can result in the formation and buildup of oxysterols ( cholesterol - derived oxidation products ) and other lipid - derived oxidation products . these lipid oxidation products exhibit toxicity and have been implicated in the pathogenesis of a variety of diseases , including atherosclerosis neurodegenerative disorders , and age - related macular degeneration . one significant source of oxidative stress is exposure to ozone , a main component of urban smog . human exposure to biologically relevant levels of ozone is clearly linked to respiratory illnesses , such as asthma . because ozone can travel in the air ozone is highly reactive with unsaturated lipids , such as cholesterol , which is present in the pulmonary surfactant lining the lungs . ozone exposure studies in human pulmonary surfactant and bronchoalveolar lavage fluid and lung tissue of mice and rats show that the double bond of cholesterol is oxidized to afford various potentially reactive compounds , including epoxides and aldehydes . these resulting oxysterols exhibit diverse biological activities , including cytoxicity , inducing apoptosis , and altering membrane properties . several of these ozone - derived oxidation products are electrophilic , meaning that they could react with the nucleophilic lysine , cysteine , and/or histidine residues of proteins to form covalent linkages . in fact , a number of studies demonstrated modification of specific proteins with cholesterol ozonolysis products . covalent modification with electrophilic lipid oxidation products can affect protein function . for example , the cholesterol ozonolysis products secosterol a ( seco a ) and/or secosterol b ( seco b ) ( both shown in figure 1 ) modify -amyloid , p53 , apolipoprotein c - ii , myelin basic protein , and antibody light chains , among other proteins . these modifications have been shown or are assumed to occur between the electrophilic aldehyde moiety of the secosterol and a nucleophilic lysine residue on the protein . the resulting secosterol adduction induces and accelerates amyloidogenesis in vitro(27 ) and may play a role in the initiation and progression of various diseases , including alzheimer s disease , cancer , atherosclerosis , and multiple sclerosis . covalent lipid modification increases the overall hydrophobicity of a modified protein ; this added hydrophobicity can affect a protein s localization . for example , palmitoylation can enable the lipidated proteins to associate with membranes and localize in lipid rafts / caveolae . modification with oxysterols also confers significant hydrophobicity to the modified protein . because of the added hydrophobicity , lipidated proteins can be challenging to study ; however , exploiting hydrophobicity is central to many methods employed to isolate and study membrane proteins . we have developed a straightforward method to test a lipid s ability to modify proteins : alkynyl lipid probes are allowed to react with protein , and any lipid - modified proteins are clicked with an azido - biotin reagent via huisgen sharpless cycloaddition and subsequently detected by a fluorescent streptavidin conjugate . here , we synthesized alkynyl analogs of several cholesterol - derived ozonolysis products and used these probes to determine the most protein - reactive oxysterol(s ) . several model adduction studies with an amino acid , peptides , and proteins explored the identity of the reacting oxysterol , revealing the potential involvement of secosterol dehydration products in adduction . as prelude for studying more complex biological samples , hydrophobic methods were developed to enrich model systems in secosterol - modified peptides , and lc - ms / ms analyses elucidated a characteristic fragmentation pattern of secosterol peptide adducts . h and c nmr spectra were collected on a 300 , 400 , or 600 mhz nmr . hrms ( high resolution mass spectrometry ) analyses for small molecules were carried out at the university of notre dame . purification by column chromatography was carried out on silica gel , and tlc plates were visualized with phosphomolybdic acid . the syntheses of alkynyl cholesterol ( a - chol ) and alkynyl 7-ketocholesterol ( a-7-ketochol ) have been previously reported . full synthetic procedures for the remaining alkynyl lipids are described in the supporting information . cholep ( 3:1 to ) , secosterols a and b , and their dehydration products were synthesized as previously described . solutions of hsa ( 1 mg / ml ) and alkynyl ( a)-oxysterol ( 20 m ) in 10 mm ammonium bicarbonate buffer ( ph 7.4 ) were incubated at room temperature overnight with stirring . for the experiment testing the a - seco a concentration dependence of hsa adduction , a - seco a concentrations of 0100 m were assayed . the samples were reduced with sodium borohydride ( 5 mm , 1 h at room temperature ) to stabilize any adducts that may have formed and neutralized with 10% hcl . the following click reagents were added to each of the samples : azido - biotin reagent ( 0.2 mm ) , tris(3-hydroxypropyltriazolylmethyl)amine ( thpta ) ligand ( 0.2 mm ) , copper sulfate ( 1 mm ) , and sodium ascorbate ( 1 mm ) , and the samples were vortexed and allowed to stir for 2 h at room temperature in the dark . biotinylated proteins were resolved using 10% nupage novex bistris gel ( life technologies , carlsbad , ca ) . precision plus protein kaleidoscope standards ( 10250 kda , bio - rad ) were electrophoresed on the same gel for reference . the proteins were transferred electrophoretically to a polyvinylidene fluoride membrane ( life technologies , grand island , ny ) and probed with streptavidin conjugated with the alexa fluor 680 fluorophore ( life technologies ) . biotinylated proteins were visualized using the odyssey infrared imaging system and odyssey software according to the manufacturer ( licor , lincoln , ne ) . a solution of actpepx ( 1 mm ) or n - aclys ( 5 mm ) and oxysterol ( 5 mm ) in 50 mm phosphate buffer ( ph 7.4):ch3cn ( 100 l , 1:1 by volume ) was incubated at 37 c for 13 h. samples containing secosterols were reduced with 2 m nabh4 ( 10 l ) and then neutralized with 10% hcl for lc - ms analysis . reverse - phase hplc was performed on a supelco discovery c18 column ( 150 mm 2.1 mm , 5 m ) using a mobile phase consisting of a : 0.05% tfa in h2o and b : 0.05% tfa in ch3cn . although tfa sometimes suppresses ionization , resolution of the peaks was optimal with tfa as the additive . the unreacted peptide and adducts were eluted with a gradient of 5% to 35% b over 20 min , then to 100% b over 5 min and held for 20 min , and back to 5% b over 5 min . the ms was operated in the positive - ion mode using electrospray ionization with conditions optimized for actpepk . for cyt c ( 40 m ) was incubated in the presence of seco a , seco a h2o , seco b h2o ( i ) , seco b h2o ( ii ) , or seco b2h2o ( 400 m ) in 10 mm nh4hco3 buffer ( ph 7.4 ) at room temperature overnight with stirring . modified cyt c sample ( 1 l ) was spotted onto a maldi target plate . a saturated solution of -cyano-4-hydroxycinnamic acid ( chca ) matrix in h2o / ch3cn / tfa ( 50/50/0.1 ) ( 1 l ) was then spotted onto the same position of the maldi plate . the modified cyt c and the matrix were immediately mixed by pipetting up and down twice and allowed to dry before sample analysis . maldi - tof - ms analyses were performed on a perseptive biosystems voyager - de str maldi - tof equipped with a pulsed n2 laser . protein spectra were collected with an accelerating voltage of 20 kv in positive ion linear mode . each spectrum was the accumulation of 1000 laser shots , with a laser intensity of 22002300 that was optimized for each spectrum to provide the best signal - to - noise ratio . for the seco a - treated cyt c sample , to enable identification of sites of secosterol modification , the cyt c mixture was trypsinized using the following protocol : trypsin ( 1:50 = w : w , trypsin : cyt c ) was added , and the protein was allowed to digest for 4 h at 37 c . prior to lc - ms / ms analysis , either folch extraction or solid phase extraction ( spe ) ( vide infra ) was employed to enrich this solution in secosterol - modified peptides . the organic layer resulting from folch extraction or the resulting spe fractions a mixture of unmodified and seco - modified peptides in 10 mm nh4hco3 buffer ( ph 7.4,100 l ) was diluted with chloroform and methanol in a ratio of 8:4:3 chloroform / methanol / buffer . the folch solution was vortexed and centrifuged briefly to allow distinct organic and aqueous layers to separate . each layer was subsequently analyzed by lc - ms using conditions described above for peptides . a c18 spe cartridge ( sep - pak vac , 1 cm , 50 mg sorbent , 55105 m particle size ; waters , milford , ma ) was conditioned with ch3cn/0.1% tfa ( 1 ml ) . the spe cartridge was then flushed with 1 ml of water/0.1% tfa . the sample ( e.g. , seco - modified angiotensin ii in 100 l of buffer ) was loaded onto the cartridge and separated using gradient elution ( 2 0.5 ml of each of the following ) : 10 , 30 , 50 , and 90% ch3cn in h2o ( 0.1%tfa ) . samples were either concentrated or analyzed directly using lc - ms / ms and/or maldi - tof - ms . for spe fractions containing < 50% ch3cn , the sample ( 0.7 l ) was spotted on a maldi target , followed by a saturated solution of chca matrix in h2o / ch3cn / tfa ( 50/50/0.1 ) ( 1 l ) . the sample and matrix were immediately mixed by pipetting up and down twice and allowed to dry before sample analysis . because of the volatility of organic solvents , sample preparation , specifically on - plate sample - matrix mixing , for maldi analysis of samples containing significant amounts of organic solvent , can be difficult . for maldi - tof - ms analysis of spe fractions containing 50% mecn , the sample ( 0.7 l ) was spotted on a maldi target , followed by a saturated solution of chca matrix in h2o / mecn / tfa ( 50/50/0.1 ) ( 1 l ) , followed by an additional 0.7 l of the sample . the sample and matrix were immediately mixed by pipetting up and down twice and allowed to dry before sample analysis . this three - layer spotting technique ensured that the organic layer and matrix were sufficiently mixed to allow for analysis . each maldi - tof - ms spectrum was the accumulation of 1000 laser shots , with a laser intensity of 19002100 that was optimized for each spectrum to provide the best signal - to - noise ratio . refer to scheme s1 for an outline of the general workflow for the experiments described here : hsa ( 1 ml , 4 mg / ml , 1x pbs ) was incubated with seco a ( 1 mm ) for 4 h at 37 c . the reaction mixture was reduced with nabh4 ( 20 mm ) at room temperature for 1 h. excess nabh4 was removed using a nanosep10k omega ( pall ) filter , and the volume of the reaction mixture was adjusted to 1 ml with 100 mm ammonium bicarbonate . dtt ( 10 mm ) was added and incubated for 10 min at 50 c , followed by iodoacetamide treatment ( 20 mm ) in the dark for 10 min at rt . trypsin ( 1:50 = w : w , trypsin : hsa ) was added to the reaction mixture and incubated at 37 c overnight . the digested peptides were loaded onto a preconditioned oasis hlb ( 60 mg , 3 cm ) following the manufacturer s protocol ( waters , milford , ma ) . after a 1 ml h2o washing step , peptides were eluted and collected as described above in spe fractionation . collected fractions were evaporated to dryness in a speedvac concentrator and resuspended in 100 l of h2o with 0.1% formic acid for lc - ms / ms analysis . the same procedure was followed for modification with seco b , except hsa was incubated with 0.2 mm seco b at 37 c overnight . the peptide solution ( secosterol - treated , trypsinized protein ) was separated using an eksigent nanolc ultra hplc and autosampler . the analytical column was packed with c18 resin ( jupiter , 3 m , 300 , phenomenex ) in a capillary column ( 20 cm length , 360 m o.d . peptides were eluted using a gradient as follows : 014.5 min , 2% b ; 14.515 min , 2 to 5% b ; 1535 min , 5 to 35% b ; 3555 min , 35 to 99% b ; 5575 min , 99% b ; and 7590 min , 99 to 2% b at a flow rate of 500 nl / min ( solvent a : 0.1% formic acid in h2o , solvent b : 0.1% formic acid in ch3cn ) . eluting peptides were mass analyzed on an ltq orbitrap velos mass spectrometer ( thermo scientific ) followed by a data dependent neutral loss ( ddnl ) ms method with dynamic exclusion enabled . full - scan ( m / z 3502000 ) spectra were acquired with the orbitrap mass analyzer ( resolution 30,000 ) , and the six most abundant ions in each ms scan were selected for fragmentation in the ltq . an isolation width of 2 m / z units , activation time of 10 ms , and 35% normalized collision energy were used to generate ms spectra followed by ms neutral loss from ms with an isolation width of 2.2 m / z units . neutral loss mass scans are enabled with the five most abundant in each ms with loss of m / z 390.349 ( singly charged species ) , 195.175 ( doubly charged species ) , and 130.120 ( triply charged species ) . dynamic exclusion settings allowed for a repeat count of 1 within a repeat duration of 15 s , and the exclusion duration time was set to 30 s. for identification of hsa peptides , tandem mass spectra were processed in bumberdash tools , version 1.4.110 , and searched by myrimatch algorithm with a custom - built subset database originated from homo_sapience_grch37 - 59_cntm_r ( 2010_11 - 18 ) . the sequence database was reversed so that each protein sequence appeared in both normal and reversed orientations , enabling false discovery rate estimation . myrimatch was configured to have dynamic modifications of + 57.021 for carbamidomethyl to cys and 400.3342 , 402.3498 , 404.3654 , 384.3342 , and 386.3498 da for [ cys , lys , his ] . ms spectra generated from ddnl were separately processed using proteowizard msconvert tool to convert to mzxml format , followed by myrimatch search . in this case the configuration was consistent with dynamic modifications of + 57.021 for carbamidomethyl to cys and + 12.00 for lys residue . a series of cholesterol - derived oxysterols , several of which are established cholesterol ozonolysis products , were prepared according to known procedures along with their corresponding alkynyl analogs ( figure 1 ) . incorporation of the triple bond in place of the c24 isopropyl group of cholesterol introduces a minimal perturbation from the endogenous lipid structure . in previous studies , we demonstrated that alkynyl cholesterol ( a - chol ) could be incorporated into neuro2a cells and metabolized to the a - chol fatty acid esters in a transformation identical to that of endogenous cholesterol . the immediate biosynthetic precursor to a - chol , a-7-dehydrocholesterol , was also converted to a - chol and its esters in cells , suggesting that alkynyl sterols are appropriate surrogates for the endogenous lipids in cell studies . the terminal alkyne moiety in our lipid probes allows for attachment of a biotin tag via click reaction with an azido - biotin reagent . the biotin tag can be used for visualization of proteins adducted with the oxysterols and/or enrichment techniques . library of cholesterol - derived oxysterols and several alkynyl sterol analogs . with a set of alkynyl oxysterols in hand , we set up assays to determine the relative reactivity of their covalent attachment with protein nucleophiles . to survey our cholesterol - derived oxysterols for protein reactivity , buffer solutions of hsa were treated with each of the alkynyl oxysterol electrophiles shown in figure 1 . the adducts were stabilized by reduction with nabh4 and then reacted with the azido - biotin reagent shown in figure 2 . any alkynyl sterol - modified proteins can then be visualized by streptavidin - alexa fluor 680 . general experimental workflow , including the azido - biotin reagent used for click biotinylation of alkynyl lipid - modified proteins . the alkynyl secosterols ( a - seco a and a - seco b ) yielded the most protein adduction of the alkynyl oxysterols tested , as determined by streptavidin fluorophore intensity ( figure 3a ) . a-7-ketochol gave no evidence of hsa adduction while a - cholep afforded relatively low levels of protein modification . the relatively low level of a - cholep protein adduction found here agrees with a previous report that the 5,6-epoxides of endogenous cholesterol are remarkably unreactive toward nucleophiles . the reactivity of the cholesterol epoxides is in contrast to the epoxide derived from 7-dehydrocholesterol , an allylic epoxide that readily adducts to cellular proteins . hsa adduction indicate the extent of hsa modification by each probe ( figure 3b ) . figure 3c and d shows the dependence of protein modification on the concentration of the a - seco a probe . thus , buffer solutions of hsa were treated with a - seco a at varying concentrations ( 0100 m ) , and biotinylated proteins were visualized using the streptavidin fluorophore ( figure 3c ) , with the integrated intensities ( figure 3d ) showing that the amount of protein lipidation increases with increasing concentration of alkynyl lipid electrophile . the results shown in figure 3b and d are extracted directly from a single gel . having confirmed that the secosterols were protein - reactive , the chemistry of adduct formation was explored in greater detail . a peptide containing one nucleophilic site ( lys ) , ac - ala - val - ala - gly - lys - ala - gly - ala - arg ( actpepk ) , was chosen as a model peptide . the peptide ( 1 mm ) was incubated in the presence of the electrophile ( 5 mm ) at 37 c for 1 h. the reaction mixture was then reduced with nabh4 to stabilize any adducts that form reversibly , and the solution was then neutralized and analyzed by lc - ms . while actpepk gave no evidence for formation of an adduct with cholep and 7-ketochol , both seco a and seco b gave rise to products with a mass corresponding to a peptide oxysterol adduct ( see figure s1a ms / ms analysis confirms that the product is a peptide with secosterol modification at the lysine residue ( vide infra ) . this is likely the product that arises from reaction of the lysine amine side chain with the seco aldehyde . sterol adducts have chromatographic behavior on reverse - phase columns more like the free oxysterol than the unmodified peptide ; both the peptide sterol adducts and free oxysterols elute after the mobile phase reaches 100% organic solvent . ( a ) streptavidin fluorophore visualization of hsa modification with alkynyl oxysterols ( 20 m ) . ( b ) integrated intensities from streptavidin fluorophore analysis of hsa modification with alkynyl oxysterols . ( c ) streptavidin fluorophore visualization of hsa modification with different concentrations of a - seco a. [ the higher weight protein ( 130 kda ) is hsa dimer . ] ( d ) integrated intensities from western blot analysis of hsa modification with different concentrations of a - seco a. alkynyl sterol - adducted protein was tagged with biotin via click reaction and labeled with the streptavidin - alexafluor 680 conjugate . the fluorophore was detected and integrated intensities were obtained using the odyssey infrared imaging system and application software . for each streptavidin fluorophore analysis , the lane with the lowest integrated intensity was set to 0 . n - acetyl lysine was chosen as a simple model nucleophile to investigate the fundamental stability and reactivity of the secosterols . in model studies , seco a and seco b were each incubated in the presence of n - acetyl lysine with subsequent nabh4 reduction , and the adduction products before and after reduction were analyzed by lc - ms . analyzing the product masses before and after reduction should shed light on the reactive moiety in the secosterol by considering the number of mass units added through reduction . reaction of seco b with n - acetyl lysine afforded a product with m / z = 589 before reduction and m / z = 591 ( [ m + h ] ) after reduction ( figure 4a and b , respectively ) , which likely results from the expected imine formation at the aldehyde with the amine side chain and subsequent reduction . product analysis of the incubation of seco a with n - acetyl lysine was less straightforward . before reduction , only one product with m / z = 589 was observed , suggesting lysine imine formation at the secosterol aldehyde ( figure s2a ) . however , there are four products observed after nabh4 reduction ( figure s2b ) . one product corresponds to the reduced seco b - lys adduct , resulting from seco a isomerization to seco b either prior to or following n - acetyl lysine modification . two diastereomeric products have m / z = 593 , a net addition of four hydrogen atoms resulting from complete reduction of the seco a - lys imine adduct . the final product with m / z = 591 is a seco a - lys adduct in which the ketone remains unreduced after treatment with nabh4 , suggesting that the ketone is resistant to reduction ( confirmed in figure s3a c ) . lc - ms analysis of seco b - lys products before and after reduction . ( a ) products of seco b incubation with n - acetyl lysine , before reduction . ( b ) products of seco b incubation with n - acetyl lysine , after reduction . using n - acetyl lysine as the limiting reagent allowed for analysis of the remaining unadducted secosterol , in addition to the lys - secosterol adducts . after incubation in the presence of n - acetyl lysine , a significant amount of seco a was converted to seco b ( figure s4 ) . isomerization of seco a to seco b proceeded with continued incubation , as shown in the lc - ms analysis after 5 h ( figure s4d ) , eventually leading to seco b as the major isomer . this facile isomerization is well established ; transient imine formation between the aldehyde of seco a and the amine groups of proteins has been shown to catalyze the conversion of seco a to seco b via an aldol reaction . there is also evidence for dehydration of seco a at longer incubation times ( data not shown ) . these studies highlight an important consideration : the initial secosterol electrophile introduced into a system may not be the primary reactive electrophile ; although we initially treat a system with seco a , the adduction results may better reflect the reactivity of seco b or some other electrophile to which seco a has converted over the course of the reaction . cyt c is an attractive model protein for adduction experiments due to its small size ( 12 kda ) , which allows for straightforward monitoring of increases in molecular weight resulting from lipid electrophile covalent attachment . incubation of cyt c and seco a in ph 7.4 buffer yielded significant protein modification that can be observed by maldi - tof - ms analysis ( figure 5 , inset ) . high resolution ftms measurement confirmed a nonreduced secosterol adduct to the cyt c tryptic peptide tgpnlhglfgr by a mass difference of 6 ppm . the b and y ions in the lc - ms / ms analysis of the trypsinized protein suggested adduction of the secosterol at his-33 , the first confirmed secosterol modification at a nonlysine residue ( figure 5 and figure s5 ) . this histidine residue has been shown to be a site of modification by other lipid electrophiles , namely 4-hydroxynonenal ( hne ) . however , unlike hne , neither seco a nor seco b contains a michael acceptor , a typical site of nucleophilic attack by histidine . high resolution ftms analysis of cyt c tryptic peptidetgpnlh(secoa - h2o)glfgr ( precursor m / z 784.9827 [ m+2h ] , mass error 1 ppm ) . doubly charged b and y ions are highlighted with purple and blue , respectively . significant water loss ions are observed at m / z 775 and 767 ( 1h2o and 2h2o , respectively ) . total loss of seco a is observed , displaying m / z 1168.7 [ m + h ] and 585.0 [ m+2h ] . b and y ions containing seco a , including b6 and y6 , indicate that the seco a is adducted at the h-33 position . inset : maldi - tof - ms analysis of secosterol - modified cyt c ( intact modified protein ) . ( ftms / ms is provided in figure s5a c . ) while secosterols a and b have electrophilic aldehyde groups , histidine attack on these moieties is not expected ; rather , lysine residues are common nucleophiles in reaction with aldehydes . indeed , previous work had established lys-22 of cyt c as the main site of seco b modification under sds micellar conditions with no evidence of histidine adduction . in an sds environment , the tertiary structure of cyt c is transformed to mimic membrane - bound cyt c ; this alternative conformation may allow the secosterol to experience previously less accessible hydrophobic interactions around lys-22 and modify cyt c at this site , whereas in the buffer solution used in this work , cyt c adopts a tertiary structure that may reduce access to lys-22 and encourage the secosterol to react with the hydrophilic , solvent - exposed his-33 . to explore the reactivity of seco a with his residues , a model peptide containing one nucleophilic site ( his ) , ac - ala - val - ala - gly - his - ala - gly - ala - arg ( actpeph ) , was incubated with either seco a or seco a lc - ms analysis of the product mixture showed evidence of a product with a mass corresponding to a peptide seco a lc - ms / ms analysis confirmed histidine as the site of modification , the most nucleophilic residue of n - terminal - protected actpeph ( figures s6c and s6d ) . after nabh4 treatment , a mixture of dehydrated seco a and seco b adducts was observed with an addition of 2 and 4 mass units , respectively , indicating that the ketone is resistant to reduction , as we observed previously ( figure s6e and s6f ) . this observation suggests that seco a must undergo dehydration prior to adduction followed by aldol reaction . maldi - tof - ms analysis of cyt c modification with secosterol dehydration products . ( e ) streptavidin fluorophore visualization of hsa modification with a - seco a compared to a - seco a h2o . ( f ) integrated intensities from streptavidin fluorophore analysis of hsa ( 1 mg / ml ) modification with a - seco a ( 20 m ) compared to a - seco a h2o ( 20 m ) . alkynyl sterol - adducted protein was tagged with biotin via click reaction and labeled with the streptavidin the fluorophore was detected and integrated intensities were obtained using the odyssey infrared imaging system and application software . for each streptavidin fluorophore analysis , the lane with the lowest integrated intensity was set to 0 . the identification of a histidine residue ( his-33 ) on cyt c as a site of secosterol modification forced a reconsideration of possible electrophiles that may be formed from seco a. seco a can undergo cyclization via an aldol reaction to form seco b , and both seco a and seco b can lose one or two water molecules , affording the dehydration products shown in figure 6 . h2o ( i ) do contain an ,-unsaturated carbonyl , a prototypical michael acceptor that might explain the observation of secosterol covalent attachment at cyt c his-33 . h2o , seco b h2o ( i ) , seco b h2o ( ii ) , and seco b2h2o were prepared according to literature procedures . maldi - tof - ms analysis indicated protein adduction of cyt c with all of the compounds except for the double dehydration product seco2h2o ( figure 6a d ) . these experiments all suggest that electrophiles generated by dehydration play a major role in the protein adduction , and this was confirmed by studies of the alkynyl analog , a - seco a both a - seco a and a - seco a h2o were prepared , and each was incubated with hsa , as previously described following the protocol outlined in figure 2 . the resulting solutions were reduced with nabh4 and treated with the azido - biotin reagent , and alkynyl sterol - modified proteins were visualized with streptavidin alexa fluor 680 . the relative intensities for the fluorescent signal indicated the extent of hsa modification by each probe ( figure 6e and f ) . under these reaction conditions , the dehydration product of a - seco a ( a - seco a h2o ) produced a comparable amount of protein adduction to the parent secosterol ( a - seco a ) itself . this result supports the notion that secosterol dehydration products contribute to the reactivity of secosterols with proteins . because dehydrated a - seco a contains an ,-unsaturated ketone , nucleophilic protein residues may react with the electrophilic secosterol through either schiff base formation at the aldehyde or michael addition . considering dehydration products containing a michael acceptor in the overall scheme of secosterol reactivity introduces the previously unrecognized possibility of protein modification at histidine side chains . sterols and the seco - sterols are hydrophobic , and methods were investigated to separate sterol - modified peptides from unmodified peptides based on hydrophobicity . thus , a mixture of secosterol - adducted actpepk and unmodified actpepk was submitted to folch extraction , and the resulting organic and aqueous layers were analyzed by lc - ms ( figure 7 ) . this analysis showed that the majority of the secosterol - modified peptide was extracted into the organic phase and suggests that hydrophobic separations may find use in the enrichment of sterol - adducted peptides and proteins . solid phase extraction with a reverse - phase ( c18 ) spe cartridge on a mixture of unmodified angiotensin ii ( asp - arg - val - tyr - ile - his - pro - phe ) and angiotensin ii modified with seco a also showed a complete separation of the modified peptide from the unmodified peptide ( figure 8) . this model study demonstrates the potential use of spe as a preparative enrichment method for peptides modified with oxysterol electrophiles . maldi - tof - ms analysis of reverse - phase spe of a secosterol - modified peptide . ( a ) mixture of unmodified and secosterol - modified angiotensin ii ( m = asp - arg - val - tyr - ile - his - pro - phe ) . ( b ) spe fractions eluted with 10% ch3cn in water with 0.1% tfa contain unmodified angiotensin ii . ( c ) spe fractions eluted with 50100% ch3cn in water with 0.1% tfa contain secosterol - modified angiotensin ii . the ms / ms fragmentation of a model peptide modified with a secosterol was investigated to establish characteristic fragmentation associated with secosterol - modified peptides ( e.g. , neutral loss of the secosterol ) to provide a basis for identifying secosterol - modified peptides in a complex proteomic analysis . in a preliminary study of the actpepk seco b lysine imine adduct , the dominant peaks observed were due to loss of water ( [ m + 2h h2o ] and [ m + 2h seco b adduct gave a dominant ion at m / z = 427.9 in the ms spectrum ( figure 9b ) . apparently , this fragment ion is due to cleavage at the c c bond between the secosterol core and the reduced imine group . cleavage in this position should result in a neutral loss of 390 da ( most of the secosterol ) , and the ability to identify seco b - modified actpepk via an lc - ms / ms / ms neutral loss experiment was confirmed , exhibiting fragment ions with + 12 m / z relative to the ions observed from the parent peptide ( figure 9a and c ) . this unique fragmentation may be useful in identifying secosterol - modified peptides during proteomic analysis . ( a ) ms of unmodified actpepk ( m / z 421.8 [ m + 2h ] ) using 30% normalized collision energy . ( b ) ms of actpepk + seco b ( = sb ) adduct ( m / z 622.9 [ m + sb + 2h ] ) . characteristic neutral loss ( m / z 390 = @ ) fragmentation ion of seco b - modified actpepk after reduction is observed at m / z 427.9 . 321.7 = [ y7 + sb @ + 2h ] , 371.4 = [ y8 + sb @ + 2h ] , 374.2 = y4 , 427.9 = [ m + sb @ + 2h ] , 514.4 = y5 + sb @ , 571.4 = y6 + sb @ , 642.5 = y7 + sb @ , 741.6 = y8 + sb @ , 842.6 = m. ( c ) ddnl ms of m / z 427.9 [ m + sb @ + 2h ] using 35% normalized collision energy aided identification of covalent modification to peptides . 212.9 = b2 , 284.0 = b3 , 303.1 = y3 , 321.8 = [ y7 + sb @ + 2h ] , 371.4 = [ y8 + sb @ + 2h ] , 374.2 = y4 , 481.4 = b5 + sb @ , 514.4 = y5 + sb @ , 571.4 = y6 + sb @ , 642.5 = y7 + sb @ , 741.5 = y8 + sb @. hsa was exposed to seco b as previously described , and the protein was trypsin - digested , with the resulting peptides fractionated by spe and subjected to lc - ms / ms ( scheme s1 ) . spectra were analyzed using the automated algorithm search described in the materials and methods section . a total of 3600 peptide spectra were filtered under stringent criteria , covering 91% of hsa . a majority ( 75% ) of the filtered spectra were eluted in the 1030% ch3cn spe fractions ( table s1 ) , which contained mostly unadducted peptides . most of the seco b peptide adducts identified were contained in the 60% ch3cn spe fraction and are listed in table 1 . all the precursor masses of seco b - modified peptides have an accuracy of < 10 ppm mass error . k-223 seems to be the most frequently modified position by oxysterols , based on the number of spectral counts . hsa was treated with either seco a or seco b followed by nabh4 reduction . the majority of unmodified peptides were contained in the 30% mecn fraction ( supporting information table 1 ) . all the identified adducted peptides were found in the 60% mecn fraction . based on ms / ms fragmentation patterns , the final form of secosterol was identified as seco b or its dehydrated products . the mass accuracy and peptide fragmentation assignments are in good agreement with their covalent modification and sites . as observed in our model studies , the seco b - lys adducts of tryptic serum albumin peptides yielded a diagnostic fragmentation pattern in the ms spectra that corresponds to the loss of 390 da ( the seco b core ) ( figure 10 and figure s8 ) . this neutral loss can be used as a signature fragment for seco b lys adducts . furthermore , this characteristic neutral loss ion can be incorporated into ddnl lc - ms analysis to identify seco b lys adducts in more complex mixtures of proteins / peptides . the residual peptide modification of m / z = + 12 da ( due to the partial fragmentation of seco b ) was observed in the resulting ms spectra . all ms spectra were extracted separately using an ms converter to generate mzxml for myrimatch searching ; the peptides are indeed identified as the same peptide sequences identified by the ms spectra ( table s2 ) . the ( a ) and ( b ) spectra are summarized in the head table . ( a ) ms spectrum of lk(402)c(57)aslqk , which is modified with seco b on lys-199 . as illustrated in figure 9 , the characteristic 390 neutral loss fragment ion is observed at m / z = 480.42 ( [ peptide + 2h + seco b 390 ] ) . ( b ) the subsequent ddnl ms spectrum of m / z 480.42 provides further evidence of seco b adduction at lys-199 in the same sequence with a shift of + 12 m / z units after loss of 390 [ lk(12)c(57)aslqk ] . the picture of seco a adduction has the potential to be more complicated than that of seco b , as described previously and outlined in figure 6 . nevertheless , a study of hsa seco a adduction was performed , evaluating spe as a method of enrichment to aid in probing the sites of seco a modification . protein adduction was carried out overnight at room temperature at 5-fold lower concentration than the aforementioned seco b whether seco a adduction occurs prior to aldol isomerization or vice versa , the identified modified peptides correspond to seco b lys adducts exhibiting ions in the ms spectra resulting from the signature 390 neutral loss , as shown in figure 10 . ms data generated from ddnl match the peptide sequences identified with the ms spectra . there are no other significant adducts observed except the dehydrated form of previously identified modified peptides ( table s2 ) . this experiment verifies spe as an effective method to enrich complex mixtures in sterol - modified peptides and also confirms the characteristic fragmentation pattern of seco b lys adducts . because of the strong biological activity and electrophilic nature of several cholesterol ozonolysis products , we synthesized alkynyl analogs of several of these cholesterol - derived oxysterols and investigated their reactivity with proteins . the trend in protein reactivity was the following : a - seco a a - seco b a - cholep > a-7-ketochol , with alkynyl secosterols a and b yielding significantly more protein adduction than the other oxysterols tested . these results agree with previous work demonstrating secosterol modification of various specific proteins . also supporting our results is a prior study that reported low reactivity of 5,6-cholesterol epoxides with amine and thiol nucleophiles . the dehydration product seco a h2o has been identified in atherosclerotic arteries , along with seco a and seco b , but the reactivity of seco a five pieces of experimental evidence suggest the potential involvement of secosterol dehydration products in the overall scheme of secosterol reactivity : ( 1 ) seco a loses a water molecule in the presence of catalytic n - acyl lysine , forming a dehydration product upon incubation at 37 c . ( 2 ) incubation of seco a in the presence of cyt c results in secosterol adduction at a histidine residue of cyt c ( his-33 ) ; however , reaction of histidine with seco a was unexpected , as seco a does not contain a typical histidine - reactive electrophile ( e.g. , a michael acceptor ) . modify cyt c after incubation at 37 c for 24 h. ( 4 ) both seco a and seco a modify model peptide actpeph at the histidine residue , and the ms spectra of the resulting adducts show the same fragment ions and isotope distributions . h2o afforded a comparable amount of protein modification to the precursor secosterol a - seco a when using hsa as a model protein . taken together , these results demonstrate that secosterols can dehydrate under mild conditions , and the resulting dehydration products are protein reactive , presumably via the ,-unsaturated carbonyl . the presence of this michael acceptor expands the reactivity of cholesterol - derived secosterols , enabling reaction with histidine nucleophiles . we note that a study of cyt c modification with trans , trans-2,4-decadienal identified his-33 adducts , affording mass increases indicative of schiff base formation rather than michael addition . because there are multiple mechanisms that could account for the histidine secosterol adduction we observed , we are performing additional experiments to unambiguously identify both the structure of the reactive secosterol(s ) and the histidine - reactive site(s ) of the identified secosterol(s ) . as demonstrated by our lc - ms analyses , modification with an oxysterol conveys considerable hydrophobicity to the adducted peptide , causing the peptide secosterol adduct to behave chromatographically more like the free oxysterol than the unmodified peptide . methods exploiting this hydrophobicity , folch extraction and spe , were employed to successfully enrich a simple mixture in ( nonalkynyl ) secosterol - modified peptide(s ) . spe enrichment was also applied to identify sites of secosterol modification in hsa , effectively removing the majority of the unmodified peptides with 30% ch3cn in water . because spe is typically one of the final sample cleanup steps before proteomic analysis , hydrophobic enrichment by spe utilizing hydrophobic enrichment in conjunction with proteomic analysis incorporating the characteristic fragmentation of secosterol adducted peptides ( neutral loss of 390 da ) may improve the likelihood of the identification of secosterol - modified peptides and proteins in complex mixtures . additionally , developing enrichment methods that use hydrophobicity , rather than tagged analogs of the oxysterols of interest , will permit the analysis of samples of endogenous sterol oxidation products . these tools may help shed light on the link between ozone exposure and diseases associated with environmental factors . in conclusion , a - seco a and a - seco b were the most protein - reactive cholesterol ozonolysis products in our study . these oxysterols can dehydrate ( under mild conditions ) to afford products containing michael acceptors , which expands the recognized reactivity of secosterols . furthermore , secosterol modification of peptides and proteins conveys sufficient lipophilicity to allow for hydrophobic enrichment of secosterol peptide adducts in complex mixtures . employing a hydrophobic enrichment method coupled with proteomic analysis incorporating the unique fragmentation of secosterol peptide adducts may ultimately provide a more comprehensive map of proteins that are modified with secosterols . it should be noted , however , that the samples tested here are not representative of protein mixtures found in tissues and fluid samples . experiments that extend the methods described here for single peptides and proteins to endogenous sources will undoubtedly pose additional challenges . experiments that apply these approaches to cells in culture are ongoing and will be reported in due course .

cholesterol undergoes ozonolysis to afford a variety of oxysterol products , including cholesterol-5,6-epoxide ( cholep ) and the isomeric aldehydes secosterol a ( seco a ) and secosterol b ( seco b ) . these oxysterols display numerous important biological activities , including protein adduction ; however , much remains to be learned about the identity of the reactive species and the range of proteins modified by these oxysterols . here , we synthesized alkynyl derivatives of cholesterol - derived oxysterols and employed a straightforward detection method to establish secosterols a and b as the most protein - reactive of the oxysterols tested . model adduction studies with an amino acid , peptides , and proteins provide evidence for the potential role of secosterol dehydration products in protein adduction . hydrophobic separation methods folch extraction and solid phase extraction ( spe)were successfully applied to enrich oxysterol - adducted peptide species , and lc - ms / ms analysis of a model peptide seco adduct revealed a unique fragmentation pattern ( neutral loss of 390 da ) for that species . coupling a hydrophobic enrichment method with proteomic analysis utilizing characteristic fragmentation patterns facilitates the identification of secosterol - modified peptides and proteins in an adducted protein . more broadly , these improved enrichment methods may give insight into the role of oxysterols and ozone exposure in the pathogenesis of a variety of diseases , including atherosclerosis , alzheimer s disease , parkinson s disease , and asthma .

Introduction Materials and Methods Results Discussion

this stress can result in the formation and buildup of oxysterols ( cholesterol - derived oxidation products ) and other lipid - derived oxidation products . these lipid oxidation products exhibit toxicity and have been implicated in the pathogenesis of a variety of diseases , including atherosclerosis neurodegenerative disorders , and age - related macular degeneration . these resulting oxysterols exhibit diverse biological activities , including cytoxicity , inducing apoptosis , and altering membrane properties . for example , the cholesterol ozonolysis products secosterol a ( seco a ) and/or secosterol b ( seco b ) ( both shown in figure 1 ) modify -amyloid , p53 , apolipoprotein c - ii , myelin basic protein , and antibody light chains , among other proteins . the resulting secosterol adduction induces and accelerates amyloidogenesis in vitro(27 ) and may play a role in the initiation and progression of various diseases , including alzheimer s disease , cancer , atherosclerosis , and multiple sclerosis . we have developed a straightforward method to test a lipid s ability to modify proteins : alkynyl lipid probes are allowed to react with protein , and any lipid - modified proteins are clicked with an azido - biotin reagent via huisgen sharpless cycloaddition and subsequently detected by a fluorescent streptavidin conjugate . here , we synthesized alkynyl analogs of several cholesterol - derived ozonolysis products and used these probes to determine the most protein - reactive oxysterol(s ) . several model adduction studies with an amino acid , peptides , and proteins explored the identity of the reacting oxysterol , revealing the potential involvement of secosterol dehydration products in adduction . as prelude for studying more complex biological samples , hydrophobic methods were developed to enrich model systems in secosterol - modified peptides , and lc - ms / ms analyses elucidated a characteristic fragmentation pattern of secosterol peptide adducts . cholep ( 3:1 to ) , secosterols a and b , and their dehydration products were synthesized as previously described . the following click reagents were added to each of the samples : azido - biotin reagent ( 0.2 mm ) , tris(3-hydroxypropyltriazolylmethyl)amine ( thpta ) ligand ( 0.2 mm ) , copper sulfate ( 1 mm ) , and sodium ascorbate ( 1 mm ) , and the samples were vortexed and allowed to stir for 2 h at room temperature in the dark . a solution of actpepx ( 1 mm ) or n - aclys ( 5 mm ) and oxysterol ( 5 mm ) in 50 mm phosphate buffer ( ph 7.4):ch3cn ( 100 l , 1:1 by volume ) was incubated at 37 c for 13 h. samples containing secosterols were reduced with 2 m nabh4 ( 10 l ) and then neutralized with 10% hcl for lc - ms analysis . for the seco a - treated cyt c sample , to enable identification of sites of secosterol modification , the cyt c mixture was trypsinized using the following protocol : trypsin ( 1:50 = w : w , trypsin : cyt c ) was added , and the protein was allowed to digest for 4 h at 37 c . prior to lc - ms / ms analysis , either folch extraction or solid phase extraction ( spe ) ( vide infra ) was employed to enrich this solution in secosterol - modified peptides . for maldi - tof - ms analysis of spe fractions containing 50% mecn , the sample ( 0.7 l ) was spotted on a maldi target , followed by a saturated solution of chca matrix in h2o / mecn / tfa ( 50/50/0.1 ) ( 1 l ) , followed by an additional 0.7 l of the sample . refer to scheme s1 for an outline of the general workflow for the experiments described here : hsa ( 1 ml , 4 mg / ml , 1x pbs ) was incubated with seco a ( 1 mm ) for 4 h at 37 c . collected fractions were evaporated to dryness in a speedvac concentrator and resuspended in 100 l of h2o with 0.1% formic acid for lc - ms / ms analysis . dynamic exclusion settings allowed for a repeat count of 1 within a repeat duration of 15 s , and the exclusion duration time was set to 30 s. for identification of hsa peptides , tandem mass spectra were processed in bumberdash tools , version 1.4.110 , and searched by myrimatch algorithm with a custom - built subset database originated from homo_sapience_grch37 - 59_cntm_r ( 2010_11 - 18 ) . a series of cholesterol - derived oxysterols , several of which are established cholesterol ozonolysis products , were prepared according to known procedures along with their corresponding alkynyl analogs ( figure 1 ) . library of cholesterol - derived oxysterols and several alkynyl sterol analogs . to survey our cholesterol - derived oxysterols for protein reactivity , buffer solutions of hsa were treated with each of the alkynyl oxysterol electrophiles shown in figure 1 . the alkynyl secosterols ( a - seco a and a - seco b ) yielded the most protein adduction of the alkynyl oxysterols tested , as determined by streptavidin fluorophore intensity ( figure 3a ) . the peptide ( 1 mm ) was incubated in the presence of the electrophile ( 5 mm ) at 37 c for 1 h. the reaction mixture was then reduced with nabh4 to stabilize any adducts that form reversibly , and the solution was then neutralized and analyzed by lc - ms . while actpepk gave no evidence for formation of an adduct with cholep and 7-ketochol , both seco a and seco b gave rise to products with a mass corresponding to a peptide oxysterol adduct ( see figure s1a ms / ms analysis confirms that the product is a peptide with secosterol modification at the lysine residue ( vide infra ) . ( d ) integrated intensities from western blot analysis of hsa modification with different concentrations of a - seco a. alkynyl sterol - adducted protein was tagged with biotin via click reaction and labeled with the streptavidin - alexafluor 680 conjugate . in model studies , seco a and seco b were each incubated in the presence of n - acetyl lysine with subsequent nabh4 reduction , and the adduction products before and after reduction were analyzed by lc - ms . lc - ms analysis of seco b - lys products before and after reduction . isomerization of seco a to seco b proceeded with continued incubation , as shown in the lc - ms analysis after 5 h ( figure s4d ) , eventually leading to seco b as the major isomer . this facile isomerization is well established ; transient imine formation between the aldehyde of seco a and the amine groups of proteins has been shown to catalyze the conversion of seco a to seco b via an aldol reaction . the b and y ions in the lc - ms / ms analysis of the trypsinized protein suggested adduction of the secosterol at his-33 , the first confirmed secosterol modification at a nonlysine residue ( figure 5 and figure s5 ) . inset : maldi - tof - ms analysis of secosterol - modified cyt c ( intact modified protein ) . to explore the reactivity of seco a with his residues , a model peptide containing one nucleophilic site ( his ) , ac - ala - val - ala - gly - his - ala - gly - ala - arg ( actpeph ) , was incubated with either seco a or seco a lc - ms analysis of the product mixture showed evidence of a product with a mass corresponding to a peptide seco a lc - ms / ms analysis confirmed histidine as the site of modification , the most nucleophilic residue of n - terminal - protected actpeph ( figures s6c and s6d ) . after nabh4 treatment , a mixture of dehydrated seco a and seco b adducts was observed with an addition of 2 and 4 mass units , respectively , indicating that the ketone is resistant to reduction , as we observed previously ( figure s6e and s6f ) . maldi - tof - ms analysis of cyt c modification with secosterol dehydration products . the identification of a histidine residue ( his-33 ) on cyt c as a site of secosterol modification forced a reconsideration of possible electrophiles that may be formed from seco a. seco a can undergo cyclization via an aldol reaction to form seco b , and both seco a and seco b can lose one or two water molecules , affording the dehydration products shown in figure 6 . maldi - tof - ms analysis indicated protein adduction of cyt c with all of the compounds except for the double dehydration product seco2h2o ( figure 6a d ) . these experiments all suggest that electrophiles generated by dehydration play a major role in the protein adduction , and this was confirmed by studies of the alkynyl analog , a - seco a both a - seco a and a - seco a h2o were prepared , and each was incubated with hsa , as previously described following the protocol outlined in figure 2 . under these reaction conditions , the dehydration product of a - seco a ( a - seco a h2o ) produced a comparable amount of protein adduction to the parent secosterol ( a - seco a ) itself . thus , a mixture of secosterol - adducted actpepk and unmodified actpepk was submitted to folch extraction , and the resulting organic and aqueous layers were analyzed by lc - ms ( figure 7 ) . this analysis showed that the majority of the secosterol - modified peptide was extracted into the organic phase and suggests that hydrophobic separations may find use in the enrichment of sterol - adducted peptides and proteins . solid phase extraction with a reverse - phase ( c18 ) spe cartridge on a mixture of unmodified angiotensin ii ( asp - arg - val - tyr - ile - his - pro - phe ) and angiotensin ii modified with seco a also showed a complete separation of the modified peptide from the unmodified peptide ( figure 8) . maldi - tof - ms analysis of reverse - phase spe of a secosterol - modified peptide . ( a ) mixture of unmodified and secosterol - modified angiotensin ii ( m = asp - arg - val - tyr - ile - his - pro - phe ) . the ms / ms fragmentation of a model peptide modified with a secosterol was investigated to establish characteristic fragmentation associated with secosterol - modified peptides ( e.g. , neutral loss of the secosterol ) to provide a basis for identifying secosterol - modified peptides in a complex proteomic analysis . in a preliminary study of the actpepk seco b lysine imine adduct , the dominant peaks observed were due to loss of water ( [ m + 2h h2o ] and [ m + 2h seco b adduct gave a dominant ion at m / z = 427.9 in the ms spectrum ( figure 9b ) . cleavage in this position should result in a neutral loss of 390 da ( most of the secosterol ) , and the ability to identify seco b - modified actpepk via an lc - ms / ms / ms neutral loss experiment was confirmed , exhibiting fragment ions with + 12 m / z relative to the ions observed from the parent peptide ( figure 9a and c ) . this unique fragmentation may be useful in identifying secosterol - modified peptides during proteomic analysis . hsa was exposed to seco b as previously described , and the protein was trypsin - digested , with the resulting peptides fractionated by spe and subjected to lc - ms / ms ( scheme s1 ) . based on ms / ms fragmentation patterns , the final form of secosterol was identified as seco b or its dehydrated products . as observed in our model studies , the seco b - lys adducts of tryptic serum albumin peptides yielded a diagnostic fragmentation pattern in the ms spectra that corresponds to the loss of 390 da ( the seco b core ) ( figure 10 and figure s8 ) . furthermore , this characteristic neutral loss ion can be incorporated into ddnl lc - ms analysis to identify seco b lys adducts in more complex mixtures of proteins / peptides . the ( a ) and ( b ) spectra are summarized in the head table . ( b ) the subsequent ddnl ms spectrum of m / z 480.42 provides further evidence of seco b adduction at lys-199 in the same sequence with a shift of + 12 m / z units after loss of 390 [ lk(12)c(57)aslqk ] . the picture of seco a adduction has the potential to be more complicated than that of seco b , as described previously and outlined in figure 6 . protein adduction was carried out overnight at room temperature at 5-fold lower concentration than the aforementioned seco b whether seco a adduction occurs prior to aldol isomerization or vice versa , the identified modified peptides correspond to seco b lys adducts exhibiting ions in the ms spectra resulting from the signature 390 neutral loss , as shown in figure 10 . this experiment verifies spe as an effective method to enrich complex mixtures in sterol - modified peptides and also confirms the characteristic fragmentation pattern of seco b lys adducts . because of the strong biological activity and electrophilic nature of several cholesterol ozonolysis products , we synthesized alkynyl analogs of several of these cholesterol - derived oxysterols and investigated their reactivity with proteins . the trend in protein reactivity was the following : a - seco a a - seco b a - cholep > a-7-ketochol , with alkynyl secosterols a and b yielding significantly more protein adduction than the other oxysterols tested . the dehydration product seco a h2o has been identified in atherosclerotic arteries , along with seco a and seco b , but the reactivity of seco a five pieces of experimental evidence suggest the potential involvement of secosterol dehydration products in the overall scheme of secosterol reactivity : ( 1 ) seco a loses a water molecule in the presence of catalytic n - acyl lysine , forming a dehydration product upon incubation at 37 c . ( 2 ) incubation of seco a in the presence of cyt c results in secosterol adduction at a histidine residue of cyt c ( his-33 ) ; however , reaction of histidine with seco a was unexpected , as seco a does not contain a typical histidine - reactive electrophile ( e.g. modify cyt c after incubation at 37 c for 24 h. ( 4 ) both seco a and seco a modify model peptide actpeph at the histidine residue , and the ms spectra of the resulting adducts show the same fragment ions and isotope distributions . taken together , these results demonstrate that secosterols can dehydrate under mild conditions , and the resulting dehydration products are protein reactive , presumably via the ,-unsaturated carbonyl . because there are multiple mechanisms that could account for the histidine secosterol adduction we observed , we are performing additional experiments to unambiguously identify both the structure of the reactive secosterol(s ) and the histidine - reactive site(s ) of the identified secosterol(s ) . as demonstrated by our lc - ms analyses , modification with an oxysterol conveys considerable hydrophobicity to the adducted peptide , causing the peptide secosterol adduct to behave chromatographically more like the free oxysterol than the unmodified peptide . because spe is typically one of the final sample cleanup steps before proteomic analysis , hydrophobic enrichment by spe utilizing hydrophobic enrichment in conjunction with proteomic analysis incorporating the characteristic fragmentation of secosterol adducted peptides ( neutral loss of 390 da ) may improve the likelihood of the identification of secosterol - modified peptides and proteins in complex mixtures . in conclusion , a - seco a and a - seco b were the most protein - reactive cholesterol ozonolysis products in our study . furthermore , secosterol modification of peptides and proteins conveys sufficient lipophilicity to allow for hydrophobic enrichment of secosterol peptide adducts in complex mixtures . employing a hydrophobic enrichment method coupled with proteomic analysis incorporating the unique fragmentation of secosterol peptide adducts may ultimately provide a more comprehensive map of proteins that are modified with secosterols .

rheumatoid arthritis ( ra ) is an inflammatory polyarthritis with a variable course and prognosis . the prevalence is approximately 1% and affects females more commonly than males ( 3:1 ) . upwards of 80% of patients report foot problems during the course of the disease . acquired pes planovalgus ( ppv ) with associated involvement of the tibialis posterior ( tp ) tendon is common . pes planovalgus is a complex multi - planar deformity characterised by valgus alignment of the rearfoot with corresponding midfoot collapse and forefoot abduction . the cause is unknown but inflammatory damage in the peri - talar complex , tibialis posterior ( tp ) tendinopathy and altered foot mechanics frequently co - exist in published cohorts [ 36 ] . currently only one study has investigated electromyographic ( emg ) activity of tp in ra . here the magnitude of activity of tp in stance was increased in patients with valgus rearfoot alignment compared to those without . these findings have been replicated in non - ra cohorts with tp tendon dysfunction and flat foot . despite preliminary evidence regarding differences in emg activity of tp with varying foot posture ; there is minimal evidence in terms of how these features respond to intervention or how they behave over time in a chronic inflammatory joint disease such as ra . the consequence of tp dysfunction can also be indirectly assessed through structural and functional changes , for example by 3d kinematics of the peri - talar complex via multi - segmented foot models . we aim to develop and understand the reliability characteristics of an extended multi - segmented foot model which includes a midfoot segment combined with fine - wire emg to tp and surface emg to selected lower limb muscles applied in patients with ra and ppv . in particular , the motivation to investigate between - day reliability is a prerequisite for accurately quantifying progressive change in muscle and joint function associated with chronic inflammatory disease , and as a technique to assess , monitor and evaluate function and outcome for non - pharmacological , pharmacological and surgical interventions . patients were recruited from outpatient clinics at glasgow royal infirmary and gartnavel general hospital , glasgow uk . patients were eligible for inclusion if they had a confirmed diagnosis of ra based on the 1987acr criteria and passively correctable ppv . patients were excluded if they had a history of a significant neurological or musculoskeletal condition affecting the lower limb in terms of gait or muscle function or if they were taking anti - coagulant medication . the study was conducted in accordance with the declaration of helsinki and ethical approval was obtained from the west of scotland local research ethics committee ( 09/s0704/44 ) and nhs greater glasgow and clyde research and development ( gn09rh373 ) . participant age , gender and disease duration were recorded . to account for changes in foot function , symptoms or treatment status as a result of the disease process , these included : a tender and swollen foot joint count undertaken by a single clinician ( rb ) ; foot posture using the structural index ; foot related impairment and disability using the foot impact scale ( fis ) for ra and global disability using the health assessment questionnaire ( haq ) . visual analogue scales ( 100 mm vas ) were used to record foot pain , general health and arthritis pain and change in therapy between time points was recorded . twenty spherical reflective markers ( 5 and 10 mm diameter ) were located on anatomical landmarks during static calibration . this was reduced to 13 and 11 tracking markers for the barefoot and shod conditions respectively ( detailed description of the model is contained within supplementary material ) . a 12 camera 120 hz motion analysis system ( qualisys oqus , gothenburg , sweden ) was used to track the motion of the markers during gait trials . visual 3d software ( c - motion , inc . , rockville , md , usa ) was used to build the multi segmented foot model . the model comprised five segments for the barefoot condition comprising the whole foot , shank , rearfoot , midfoot and forefoot . the midfoot segment could not be recorded in shoe so the model was reduced to four segments in the shod condition . five trials were recorded and the ensemble average created for each condition on two occasions 1 week apart . the limb selected for analysis was based on clinical severity of foot posture . previous work in inflammatory joint disease has informed the selection of a core set of discrete kinematic variables which best represent foot dysfunction . these include initial foot contact angle and terminal stance plantarflexion angle , peak rearfoot eversion angle , peak midfoot inversion angle , lowest navicular height ( mm ) , peak forefoot dorsiflexion angle and peak forefoot abduction angle . a shoe ( flextop diabetic shoe , reed medical ltd . , uk ) was modified for the shod trials . windows were cut to allow the tracking of markers for the rear and forefoot segments and to record vertical height of the navicular . the shoe was selected for the soft leather upper and flexible vamp in order to accommodate forefoot deformity . velcro straps were stitched to the superior aspect of the heel counter in order to maintain stability during walking ( supplementary file ) . in order to avoid undertaking an invasive procedure on subjects potentially at risk of infection , intramuscular emg tibialis anterior , soleus , peroneus longus and medial gastrocnemius emg signals were recorded using trigno wireless surface electrodes ( delsys inc . , surface electrodes had a single differential configuration , inter - electrode distance of 10 mm , 4-bar formation , bandwidth of 20450 hz and 99.9% silver contact material . intramuscular emg of tp was undertaken using bi - polar stainless steel nylon coated fine wire electrodes ( 0.051 mm diameter ) inserted via 50 mm length 15 gauge needles ( motion lab systems inc . the raw signal was passed through a differential amplifier with a gain of 1000 and sampled at a frequency of 2400 hz . electrodes were inserted under ultrasound guidance ( esaote mylab 70 ) using a 134 mhz linear array transducer via the posterior - medial approach at 50% of the distance between the medial malleolus and the medial joint line of the knee . placement of the electrode was verified by checking the signal while applying manual resistance in the direction of dorsiflexion and eversion while participants plantarflexed and inverted ; the signal was also checked when participants flexed their toes to ensure the electrode was not placed inadvertently in the flexor digitorum longus muscle . walking time over 5.5 m was recorded using timing gates ( brower timing systems , utah , usa ) and trials exceeding 5% of the self - selected speed were excluded . walking speed on the second visit all emg signals were high pass filtered with a cut off frequency of 20 hz . all emg data ( including mvics ) were subject to a root mean squared ( rms ) moving average of 25 ms in order to create a wave envelope . emg data was normalised to maximum voluntary isometric contractions ( mvic ) ; three mvics were recorded for each muscle following completion of walking trials . the mvic data was recorded against manual resistance for 5 s with a gradual build up of 2 s prior to maximal effort for the final 3 s. the peak value from a 500 ms window obtained from the 3-s maximal effort of the mvic was used as the reference value similar to the methods used by bogey et al . and murley et al . . all participants were verbally encouraged in a standard manner during the mvics and a 1-min recovery period was set between repetitions . kinematic data were subject to a fourth order butterworth low pass filter with a cut off of 6 hz . demographic and group characteristics were summarised with the mean , and either standard deviation ( sd ) or range . biomechanical and emg data were normalised to 100% of stance and analysed using the absolute coefficient of multiple correlation ( cmc ) . within subject variation was assessed using two way mixed model intra class correlation coefficients ( icc ) with 95% confidence intervals ( ci ) . the descriptors poor , fair - to - good , and excellent reliability were assigned to icc cut off values of < 0.40 , between 0.40 and 0.75 , and > 0.75 respectively , similar cut - offs were used for the cmc values . the standard error of measurement ( sem ) with 95% ci was calculated to express the level of error in original and clinically meaningful units . patients were recruited from outpatient clinics at glasgow royal infirmary and gartnavel general hospital , glasgow uk . patients were eligible for inclusion if they had a confirmed diagnosis of ra based on the 1987acr criteria and passively correctable ppv . patients were excluded if they had a history of a significant neurological or musculoskeletal condition affecting the lower limb in terms of gait or muscle function or if they were taking anti - coagulant medication . the study was conducted in accordance with the declaration of helsinki and ethical approval was obtained from the west of scotland local research ethics committee ( 09/s0704/44 ) and nhs greater glasgow and clyde research and development ( gn09rh373 ) . participant age , gender and disease duration were recorded . to account for changes in foot function , symptoms or treatment status as a result of the disease process , these included : a tender and swollen foot joint count undertaken by a single clinician ( rb ) ; foot posture using the structural index ; foot related impairment and disability using the foot impact scale ( fis ) for ra and global disability using the health assessment questionnaire ( haq ) . visual analogue scales ( 100 mm vas ) were used to record foot pain , general health and arthritis pain and change in therapy between time points was recorded . twenty spherical reflective markers ( 5 and 10 mm diameter ) were located on anatomical landmarks during static calibration . this was reduced to 13 and 11 tracking markers for the barefoot and shod conditions respectively ( detailed description of the model is contained within supplementary material ) . a 12 camera 120 hz motion analysis system ( qualisys oqus , gothenburg , sweden ) was used to track the motion of the markers during gait trials . visual 3d software ( c - motion , rockville , md , usa ) was used to build the multi segmented foot model . the model comprised five segments for the barefoot condition comprising the whole foot , shank , rearfoot , midfoot and forefoot . the midfoot segment could not be recorded in shoe so the model was reduced to four segments in the shod condition . five trials were recorded and the ensemble average created for each condition on two occasions 1 week apart . the limb selected for analysis was based on clinical severity of foot posture . previous work in inflammatory joint disease has informed the selection of a core set of discrete kinematic variables which best represent foot dysfunction . these include initial foot contact angle and terminal stance plantarflexion angle , peak rearfoot eversion angle , peak midfoot inversion angle , lowest navicular height ( mm ) , peak forefoot dorsiflexion angle and peak forefoot abduction angle . a shoe ( flextop diabetic shoe , reed medical ltd . , uk ) was modified for the shod trials . windows were cut to allow the tracking of markers for the rear and forefoot segments and to record vertical height of the navicular . the shoe was selected for the soft leather upper and flexible vamp in order to accommodate forefoot deformity . velcro straps were stitched to the superior aspect of the heel counter in order to maintain stability during walking ( supplementary file ) . in order to avoid undertaking an invasive procedure on subjects potentially at risk of infection , intramuscular emg was restricted to the tp muscle because of its inaccessibility via surface electrodes . tibialis anterior , soleus , peroneus longus and medial gastrocnemius emg signals were recorded using trigno wireless surface electrodes ( delsys inc . , surface electrodes had a single differential configuration , inter - electrode distance of 10 mm , 4-bar formation , bandwidth of 20450 hz and 99.9% silver contact material . intramuscular emg of tp was undertaken using bi - polar stainless steel nylon coated fine wire electrodes ( 0.051 mm diameter ) inserted via 50 mm length 15 gauge needles ( motion lab systems inc . the raw signal was passed through a differential amplifier with a gain of 1000 and sampled at a frequency of 2400 hz . electrodes were inserted under ultrasound guidance ( esaote mylab 70 ) using a 134 mhz linear array transducer via the posterior - medial approach at 50% of the distance between the medial malleolus and the medial joint line of the knee . placement of the electrode was verified by checking the signal while applying manual resistance in the direction of dorsiflexion and eversion while participants plantarflexed and inverted ; the signal was also checked when participants flexed their toes to ensure the electrode was not placed inadvertently in the flexor digitorum longus muscle . walking time over 5.5 m was recorded using timing gates ( brower timing systems , utah , usa ) and trials exceeding 5% of the self - selected speed were excluded . walking speed on the second visit all emg signals were high pass filtered with a cut off frequency of 20 hz . all emg data ( including mvics ) were subject to a root mean squared ( rms ) moving average of 25 ms in order to create a wave envelope . emg data was normalised to maximum voluntary isometric contractions ( mvic ) ; three mvics were recorded for each muscle following completion of walking trials . the mvic data was recorded against manual resistance for 5 s with a gradual build up of 2 s prior to maximal effort for the final 3 s. the peak value from a 500 ms window obtained from the 3-s maximal effort of the mvic was used as the reference value similar to the methods used by bogey et al . and murley et al . . all participants were verbally encouraged in a standard manner during the mvics and a 1-min recovery period was set between repetitions . kinematic data were subject to a fourth order butterworth low pass filter with a cut off of 6 hz . statistical analyses were performed using spss 17.0 ( spss inc . , chicago , il , usa ) . demographic and group characteristics were summarised with the mean , and either standard deviation ( sd ) or range . biomechanical and emg data were normalised to 100% of stance and analysed using the absolute coefficient of multiple correlation ( cmc ) . within subject variation was assessed using two way mixed model intra class correlation coefficients ( icc ) with 95% confidence intervals ( ci ) . the descriptors poor , fair - to - good , and excellent reliability were assigned to icc cut off values of < 0.40 , between 0.40 and 0.75 , and > 0.75 respectively , similar cut - offs were used for the cmc values . the standard error of measurement ( sem ) with 95% ci was calculated to express the level of error in original and clinically meaningful units . five patients with ra ( 3f:2 m ) and ppv with a mean ( sd ) age of 53 ( 9 ) years and mean disease duration of 7 ( 5 ) years were recruited . numerical differences were recorded between time points in global and foot related measures of disability ( table 1 ) . analyses were restricted to the stance phase of gait as preliminary analyses indicated that cmcs can be falsely inflated due to muscle inactivity during the swing phase . within- and between - day reliability of muscle activation patterns are presented in table 2 . within - day reliability was mostly excellent with only two muscles falling marginally below mean cmc values of 0.75 ; tp and peroneus longus . the lowest mean value was 0.68 for peroneus longus . in all cases , both within- and between - days , the cmc values were greater in the shod condition than the barefoot condition . between - day reliability ranged from moderate - to - good to excellent ; values for peroneus longus and tp again fell below 0.75 . discrete emg variables in terms of timing and magnitude of contraction were investigated between - days ( table 3 ) . a number of icc values fell into the poor category ( < 0.40 ) and negative values were found in tp and peroneus longus . a negative icc value is suggestive of greater levels of intra - session variation than inter session variation ; where negative values occurred these are represented by 0 . for the majority of studied variables the icc results were poor , only four variables were in the excellent category and these were all in the temporal domain . the sem varied across both the temporal and the amplitude characteristics in all muscle groups ; however there was a trend towards reduced levels of error in the temporal domain . the sem for magnitude of tp contraction ranged from 8% to 27% of mvic across both phases of stance . for timing of peak tp activity the sem ranged from 1% to 4% of the stance phase . despite the smaller levels of error in the temporal domain , in almost all cases the 95% ci crossed zero across both domains . within - day reliability was excellent with all kinematic variables reaching mean cmc values > 0.75 in both conditions ( table 4 ) . there was a general trend towards improved reliability with higher cmc values in the shod condition compared to barefoot for the majority of variables . between - day reliability ranged from fair - to - good to excellent for all kinematic variables with the lowest value of 0.548 recorded for the rearfoot in the transverse plane there was a trend towards higher cmc values in the sagittal plane compared to frontal and transverse planes within- and between - days . values were excellent ranging from 0.89 to 0.99 for all variables with low sem values however , in the majority of cases the 95% ci of the sem crossed zero . five patients with ra ( 3f:2 m ) and ppv with a mean ( sd ) age of 53 ( 9 ) years and mean disease duration of 7 ( 5 ) years were recruited . numerical differences were recorded between time points in global and foot related measures of disability ( table 1 ) . analyses were restricted to the stance phase of gait as preliminary analyses indicated that cmcs can be falsely inflated due to muscle inactivity during the swing phase . within- and between - day reliability of muscle activation patterns are presented in table 2 . within - day reliability was mostly excellent with only two muscles falling marginally below mean cmc values of 0.75 ; tp and peroneus longus . the lowest mean value was 0.68 for peroneus longus . in all cases , both within- and between - days , the cmc values were greater in the shod condition than the barefoot condition . between - day reliability ranged from moderate - to - good to excellent discrete emg variables in terms of timing and magnitude of contraction were investigated between - days ( table 3 ) . a number of icc values fell into the poor category ( < 0.40 ) and negative values were found in tp and peroneus longus . a negative icc value is suggestive of greater levels of intra - session variation than inter session variation ; where negative values occurred these are represented by 0 . for the majority of studied variables the icc results were poor , only four variables were in the excellent category and these were all in the temporal domain . the sem varied across both the temporal and the amplitude characteristics in all muscle groups ; however there was a trend towards reduced levels of error in the temporal domain . the sem for magnitude of tp contraction ranged from 8% to 27% of mvic across both phases of stance . for timing of peak tp activity the sem ranged from 1% to 4% of the stance phase . despite the smaller levels of error in the temporal domain , in almost all cases the 95% ci crossed zero across both domains . within - day reliability was excellent with all kinematic variables reaching mean cmc values > 0.75 in both conditions ( table 4 ) . there was a general trend towards improved reliability with higher cmc values in the shod condition compared to barefoot for the majority of variables . between - day reliability ranged from fair - to - good to excellent for all kinematic variables with the lowest value of 0.548 recorded for the rearfoot in the transverse plane . there was a trend towards higher cmc values in the sagittal plane compared to frontal and transverse planes within- and between - days . values were excellent ranging from 0.89 to 0.99 for all variables with low sem values however , in the majority of cases the 95% ci of the sem crossed zero . this study set out to assess the within- and between - day reliability of emg activity patterns for tp and other selected lower limb muscles as well as 3d multi - segmented foot kinematics in an ra cohort with ppv . the emg and kinematic results demonstrated superior within - day reliability to between - day reliability in this small cohort . however , in this patient cohort discrete variables for muscle timing and amplitude demonstrated unacceptably poor levels of reliability between - days . tibialis posterior is the most powerful supinator of the rearfoot , it also adducts the forefoot and contributes to ankle plantarflexion ; in addition it acts as a dynamic stabiliser of the medial longitudinal arch . increased emg signal amplitude and activity during the stance phase of gait has been consistently demonstrated in ra and non - ra ppv cohorts [ 79 ] this suggests the muscle is working to counteract the motion tendencies of eversion and dorsiflexion in the ankle / subtalar complex and forefoot abduction which characterise ppv . we recorded tp emg patterns in ra patients with ppv and demonstrated that this could be reliably measured using a fine - wire , indwelling electrode approach in a single session both barefoot and in - shoe . however , while the overall emg muscle pattern was similar 1 week later , discrete timing and magnitude variables were not reliable . altered muscle activity has also been described for other lower leg muscles including gastrocnemius , peroneus longus , peroneus brevis and flexors digitorum and hallucis longus [ 79 ] . this evidence suggests these muscles must be studied if the complexity of the primary impairment and compensatory mechanisms are to be fully understood . however , we observed the same trend for poor reliability of other lower limb muscles as for tp : this confirms and extends the observations of murley et al . to include an inflammatory joint disease . factors related to emg technique , the effects of ra on emg measurement , and the effects of ra directly on muscle structure and function should be considered when interpreting these results . potential sources of error with emg are well described and include subcutaneous soft - tissue volume , cross talk , motion artefact , intramuscular bleeding , accuracy of electrode placement , skin preparation and retraction of electrodes during dynamic tasks . in an attempt to minimise their effect however , there are specific issues related to ra which may compound these potential sources of error . moreover , muscle activation capacity can be reduced depending on disease activity state and joint pain and effusion . structural muscle changes such as decreased volume may have influenced the precision of surface and fine - wire electrode placement and signal detection . effusions or pain in the ankle , subtalar or midtarsal joints where the lower leg muscle tendons cross to insert into the foot may have influenced muscle activation patterns . changes in ra disease activity state with up or down regulation of inflammatory cytokines may have altered muscle physiology and emg signal detection . we did not measure muscle cross - sectional areas or volume so can not account for the influence of structural changes . however , we attempted to limit error by guiding the electrode to the tp muscle belly using ultrasound ; a technique we have shown to be highly accurate . ra is a disease with a variable course , characterised by flares and remissions with associated fluctuations in patient symptoms . we found evidence that self - reported disease state and pain changed between testing periods so this may have changed muscle physiology , muscle activity , and subsequently emg signal detection . muscle - specific force and muscle activation patterns have been found to be normal in ra patients with stable disease , even with significant muscle loss . in our cohort disease activity may have fluctuated but a firmer conclusion can only be reached on this if objective measures were employed . finally we detected difference in joint tenderness between time points and this may have adversely influenced reproducibility of muscle activity capacity during mvic tests , as well as muscle function during gait . ra patients seldom walk barefoot and this may explain the superior reliability in the shod condition . combining emg muscle activation patterns with multi - segmented foot kinematics presents an opportunity to understand the relationship between muscle function and joint motion . the reliability of the kinematic data in this study was consistent with that reported for other inflammatory joint conditions . we confirmed previous observations where larger foot segments have greater reliability both within- and between - days in the sagittal plane in comparison with smaller segments with smaller ranges of motion , particularly in the frontal and transverse planes . inflammatory joint diseases present challenges for identification of surface landmarks , in particular when joints are swollen , tender and deformed . nevertheless , the model presented here showed excellent reliability for discrete variables both barefoot and shod . firstly the complexity and time burden of the protocol restricted the sample size , particularly in a heterogeneous disease such as ra . future work should attempt to recruit a larger sample in order to draw more meaningful conclusions . secondly , normalising the emg signals to mvics has inherent limitations in a patient population with fluctuating symptoms , particularly joint pain and effusions . thirdly , while the use of the intensity and duration of muscle activation are appealing variables to study for clinical and research applications , other forms of analysis may show greater reliability . in conclusion , patterns of muscle activation and kinematic motion appeared more consistent than discrete variables and absolute measures of error . the findings demonstrate that , in this cohort of ra patients using these emg variables , the use of discrete emg variables between time points is not supported either barefoot or shod . within session reliability is greater than between session and this should be considered when planning intervention or longitudinal studies ; a single session evaluation or alternative analysis may be more appropriate due to the lack of measurement precision . kinematic reliability has been established in the presence of pathology in this cohort but should be interpreted within the error limits .

highlights we use ultrasound to guide fine wires into tibialis posterior and record emg signal . we simultaneously recorded emg and multi - segment foot kinematics . we examine reliability over time in flat footed rheumatoid arthritis patients . between - day reliability of discrete emg variables is questionable . within- and between - day reliability of kinematic variables were acceptable .

Background Methods Patients Demographic, disease and clinical assessment Biomechanical analysis EMG analysis Data processing Statistical analyses Results Group characteristics EMG reliability Kinematic reliability Discussion Conflict of interest Supplementary data

rheumatoid arthritis ( ra ) is an inflammatory polyarthritis with a variable course and prognosis . the prevalence is approximately 1% and affects females more commonly than males ( 3:1 ) . acquired pes planovalgus ( ppv ) with associated involvement of the tibialis posterior ( tp ) tendon is common . pes planovalgus is a complex multi - planar deformity characterised by valgus alignment of the rearfoot with corresponding midfoot collapse and forefoot abduction . the cause is unknown but inflammatory damage in the peri - talar complex , tibialis posterior ( tp ) tendinopathy and altered foot mechanics frequently co - exist in published cohorts [ 36 ] . currently only one study has investigated electromyographic ( emg ) activity of tp in ra . here the magnitude of activity of tp in stance was increased in patients with valgus rearfoot alignment compared to those without . these findings have been replicated in non - ra cohorts with tp tendon dysfunction and flat foot . despite preliminary evidence regarding differences in emg activity of tp with varying foot posture ; there is minimal evidence in terms of how these features respond to intervention or how they behave over time in a chronic inflammatory joint disease such as ra . the consequence of tp dysfunction can also be indirectly assessed through structural and functional changes , for example by 3d kinematics of the peri - talar complex via multi - segmented foot models . we aim to develop and understand the reliability characteristics of an extended multi - segmented foot model which includes a midfoot segment combined with fine - wire emg to tp and surface emg to selected lower limb muscles applied in patients with ra and ppv . in particular , the motivation to investigate between - day reliability is a prerequisite for accurately quantifying progressive change in muscle and joint function associated with chronic inflammatory disease , and as a technique to assess , monitor and evaluate function and outcome for non - pharmacological , pharmacological and surgical interventions . the study was conducted in accordance with the declaration of helsinki and ethical approval was obtained from the west of scotland local research ethics committee ( 09/s0704/44 ) and nhs greater glasgow and clyde research and development ( gn09rh373 ) . to account for changes in foot function , symptoms or treatment status as a result of the disease process , these included : a tender and swollen foot joint count undertaken by a single clinician ( rb ) ; foot posture using the structural index ; foot related impairment and disability using the foot impact scale ( fis ) for ra and global disability using the health assessment questionnaire ( haq ) . visual analogue scales ( 100 mm vas ) were used to record foot pain , general health and arthritis pain and change in therapy between time points was recorded . twenty spherical reflective markers ( 5 and 10 mm diameter ) were located on anatomical landmarks during static calibration . this was reduced to 13 and 11 tracking markers for the barefoot and shod conditions respectively ( detailed description of the model is contained within supplementary material ) . a 12 camera 120 hz motion analysis system ( qualisys oqus , gothenburg , sweden ) was used to track the motion of the markers during gait trials . visual 3d software ( c - motion , inc . the model comprised five segments for the barefoot condition comprising the whole foot , shank , rearfoot , midfoot and forefoot . previous work in inflammatory joint disease has informed the selection of a core set of discrete kinematic variables which best represent foot dysfunction . these include initial foot contact angle and terminal stance plantarflexion angle , peak rearfoot eversion angle , peak midfoot inversion angle , lowest navicular height ( mm ) , peak forefoot dorsiflexion angle and peak forefoot abduction angle . , uk ) was modified for the shod trials . the shoe was selected for the soft leather upper and flexible vamp in order to accommodate forefoot deformity . , surface electrodes had a single differential configuration , inter - electrode distance of 10 mm , 4-bar formation , bandwidth of 20450 hz and 99.9% silver contact material . the raw signal was passed through a differential amplifier with a gain of 1000 and sampled at a frequency of 2400 hz . placement of the electrode was verified by checking the signal while applying manual resistance in the direction of dorsiflexion and eversion while participants plantarflexed and inverted ; the signal was also checked when participants flexed their toes to ensure the electrode was not placed inadvertently in the flexor digitorum longus muscle . walking time over 5.5 m was recorded using timing gates ( brower timing systems , utah , usa ) and trials exceeding 5% of the self - selected speed were excluded . walking speed on the second visit all emg signals were high pass filtered with a cut off frequency of 20 hz . all emg data ( including mvics ) were subject to a root mean squared ( rms ) moving average of 25 ms in order to create a wave envelope . the mvic data was recorded against manual resistance for 5 s with a gradual build up of 2 s prior to maximal effort for the final 3 s. the peak value from a 500 ms window obtained from the 3-s maximal effort of the mvic was used as the reference value similar to the methods used by bogey et al . and murley et al . . all participants were verbally encouraged in a standard manner during the mvics and a 1-min recovery period was set between repetitions . the descriptors poor , fair - to - good , and excellent reliability were assigned to icc cut off values of < 0.40 , between 0.40 and 0.75 , and > 0.75 respectively , similar cut - offs were used for the cmc values . the standard error of measurement ( sem ) with 95% ci was calculated to express the level of error in original and clinically meaningful units . patients were eligible for inclusion if they had a confirmed diagnosis of ra based on the 1987acr criteria and passively correctable ppv . patients were excluded if they had a history of a significant neurological or musculoskeletal condition affecting the lower limb in terms of gait or muscle function or if they were taking anti - coagulant medication . the study was conducted in accordance with the declaration of helsinki and ethical approval was obtained from the west of scotland local research ethics committee ( 09/s0704/44 ) and nhs greater glasgow and clyde research and development ( gn09rh373 ) . to account for changes in foot function , symptoms or treatment status as a result of the disease process , these included : a tender and swollen foot joint count undertaken by a single clinician ( rb ) ; foot posture using the structural index ; foot related impairment and disability using the foot impact scale ( fis ) for ra and global disability using the health assessment questionnaire ( haq ) . visual analogue scales ( 100 mm vas ) were used to record foot pain , general health and arthritis pain and change in therapy between time points was recorded . twenty spherical reflective markers ( 5 and 10 mm diameter ) were located on anatomical landmarks during static calibration . this was reduced to 13 and 11 tracking markers for the barefoot and shod conditions respectively ( detailed description of the model is contained within supplementary material ) . a 12 camera 120 hz motion analysis system ( qualisys oqus , gothenburg , sweden ) was used to track the motion of the markers during gait trials . visual 3d software ( c - motion , rockville , md , usa ) was used to build the multi segmented foot model . the model comprised five segments for the barefoot condition comprising the whole foot , shank , rearfoot , midfoot and forefoot . five trials were recorded and the ensemble average created for each condition on two occasions 1 week apart . the limb selected for analysis was based on clinical severity of foot posture . previous work in inflammatory joint disease has informed the selection of a core set of discrete kinematic variables which best represent foot dysfunction . these include initial foot contact angle and terminal stance plantarflexion angle , peak rearfoot eversion angle , peak midfoot inversion angle , lowest navicular height ( mm ) , peak forefoot dorsiflexion angle and peak forefoot abduction angle . a shoe ( flextop diabetic shoe , reed medical ltd . , uk ) was modified for the shod trials . windows were cut to allow the tracking of markers for the rear and forefoot segments and to record vertical height of the navicular . velcro straps were stitched to the superior aspect of the heel counter in order to maintain stability during walking ( supplementary file ) . in order to avoid undertaking an invasive procedure on subjects potentially at risk of infection , intramuscular emg was restricted to the tp muscle because of its inaccessibility via surface electrodes . tibialis anterior , soleus , peroneus longus and medial gastrocnemius emg signals were recorded using trigno wireless surface electrodes ( delsys inc . , surface electrodes had a single differential configuration , inter - electrode distance of 10 mm , 4-bar formation , bandwidth of 20450 hz and 99.9% silver contact material . intramuscular emg of tp was undertaken using bi - polar stainless steel nylon coated fine wire electrodes ( 0.051 mm diameter ) inserted via 50 mm length 15 gauge needles ( motion lab systems inc . the raw signal was passed through a differential amplifier with a gain of 1000 and sampled at a frequency of 2400 hz . electrodes were inserted under ultrasound guidance ( esaote mylab 70 ) using a 134 mhz linear array transducer via the posterior - medial approach at 50% of the distance between the medial malleolus and the medial joint line of the knee . walking time over 5.5 m was recorded using timing gates ( brower timing systems , utah , usa ) and trials exceeding 5% of the self - selected speed were excluded . walking speed on the second visit all emg signals were high pass filtered with a cut off frequency of 20 hz . emg data was normalised to maximum voluntary isometric contractions ( mvic ) ; three mvics were recorded for each muscle following completion of walking trials . the mvic data was recorded against manual resistance for 5 s with a gradual build up of 2 s prior to maximal effort for the final 3 s. the peak value from a 500 ms window obtained from the 3-s maximal effort of the mvic was used as the reference value similar to the methods used by bogey et al . . all participants were verbally encouraged in a standard manner during the mvics and a 1-min recovery period was set between repetitions . kinematic data were subject to a fourth order butterworth low pass filter with a cut off of 6 hz . demographic and group characteristics were summarised with the mean , and either standard deviation ( sd ) or range . biomechanical and emg data were normalised to 100% of stance and analysed using the absolute coefficient of multiple correlation ( cmc ) . within subject variation was assessed using two way mixed model intra class correlation coefficients ( icc ) with 95% confidence intervals ( ci ) . the descriptors poor , fair - to - good , and excellent reliability were assigned to icc cut off values of < 0.40 , between 0.40 and 0.75 , and > 0.75 respectively , similar cut - offs were used for the cmc values . analyses were restricted to the stance phase of gait as preliminary analyses indicated that cmcs can be falsely inflated due to muscle inactivity during the swing phase . within- and between - day reliability of muscle activation patterns are presented in table 2 . within - day reliability was mostly excellent with only two muscles falling marginally below mean cmc values of 0.75 ; tp and peroneus longus . in all cases , both within- and between - days , the cmc values were greater in the shod condition than the barefoot condition . between - day reliability ranged from moderate - to - good to excellent ; values for peroneus longus and tp again fell below 0.75 . discrete emg variables in terms of timing and magnitude of contraction were investigated between - days ( table 3 ) . for the majority of studied variables the icc results were poor , only four variables were in the excellent category and these were all in the temporal domain . for timing of peak tp activity the sem ranged from 1% to 4% of the stance phase . within - day reliability was excellent with all kinematic variables reaching mean cmc values > 0.75 in both conditions ( table 4 ) . there was a general trend towards improved reliability with higher cmc values in the shod condition compared to barefoot for the majority of variables . between - day reliability ranged from fair - to - good to excellent for all kinematic variables with the lowest value of 0.548 recorded for the rearfoot in the transverse plane there was a trend towards higher cmc values in the sagittal plane compared to frontal and transverse planes within- and between - days . values were excellent ranging from 0.89 to 0.99 for all variables with low sem values however , in the majority of cases the 95% ci of the sem crossed zero . numerical differences were recorded between time points in global and foot related measures of disability ( table 1 ) . within- and between - day reliability of muscle activation patterns are presented in table 2 . within - day reliability was mostly excellent with only two muscles falling marginally below mean cmc values of 0.75 ; tp and peroneus longus . in all cases , both within- and between - days , the cmc values were greater in the shod condition than the barefoot condition . between - day reliability ranged from moderate - to - good to excellent discrete emg variables in terms of timing and magnitude of contraction were investigated between - days ( table 3 ) . for the majority of studied variables the icc results were poor , only four variables were in the excellent category and these were all in the temporal domain . the sem varied across both the temporal and the amplitude characteristics in all muscle groups ; however there was a trend towards reduced levels of error in the temporal domain . the sem for magnitude of tp contraction ranged from 8% to 27% of mvic across both phases of stance . despite the smaller levels of error in the temporal domain , in almost all cases the 95% ci crossed zero across both domains . within - day reliability was excellent with all kinematic variables reaching mean cmc values > 0.75 in both conditions ( table 4 ) . there was a general trend towards improved reliability with higher cmc values in the shod condition compared to barefoot for the majority of variables . between - day reliability ranged from fair - to - good to excellent for all kinematic variables with the lowest value of 0.548 recorded for the rearfoot in the transverse plane . there was a trend towards higher cmc values in the sagittal plane compared to frontal and transverse planes within- and between - days . values were excellent ranging from 0.89 to 0.99 for all variables with low sem values however , in the majority of cases the 95% ci of the sem crossed zero . this study set out to assess the within- and between - day reliability of emg activity patterns for tp and other selected lower limb muscles as well as 3d multi - segmented foot kinematics in an ra cohort with ppv . the emg and kinematic results demonstrated superior within - day reliability to between - day reliability in this small cohort . however , in this patient cohort discrete variables for muscle timing and amplitude demonstrated unacceptably poor levels of reliability between - days . tibialis posterior is the most powerful supinator of the rearfoot , it also adducts the forefoot and contributes to ankle plantarflexion ; in addition it acts as a dynamic stabiliser of the medial longitudinal arch . increased emg signal amplitude and activity during the stance phase of gait has been consistently demonstrated in ra and non - ra ppv cohorts [ 79 ] this suggests the muscle is working to counteract the motion tendencies of eversion and dorsiflexion in the ankle / subtalar complex and forefoot abduction which characterise ppv . however , while the overall emg muscle pattern was similar 1 week later , discrete timing and magnitude variables were not reliable . this evidence suggests these muscles must be studied if the complexity of the primary impairment and compensatory mechanisms are to be fully understood . however , we observed the same trend for poor reliability of other lower limb muscles as for tp : this confirms and extends the observations of murley et al . to include an inflammatory joint disease . factors related to emg technique , the effects of ra on emg measurement , and the effects of ra directly on muscle structure and function should be considered when interpreting these results . in an attempt to minimise their effect however , there are specific issues related to ra which may compound these potential sources of error . moreover , muscle activation capacity can be reduced depending on disease activity state and joint pain and effusion . changes in ra disease activity state with up or down regulation of inflammatory cytokines may have altered muscle physiology and emg signal detection . we did not measure muscle cross - sectional areas or volume so can not account for the influence of structural changes . however , we attempted to limit error by guiding the electrode to the tp muscle belly using ultrasound ; a technique we have shown to be highly accurate . ra is a disease with a variable course , characterised by flares and remissions with associated fluctuations in patient symptoms . we found evidence that self - reported disease state and pain changed between testing periods so this may have changed muscle physiology , muscle activity , and subsequently emg signal detection . in our cohort disease activity may have fluctuated but a firmer conclusion can only be reached on this if objective measures were employed . finally we detected difference in joint tenderness between time points and this may have adversely influenced reproducibility of muscle activity capacity during mvic tests , as well as muscle function during gait . ra patients seldom walk barefoot and this may explain the superior reliability in the shod condition . combining emg muscle activation patterns with multi - segmented foot kinematics presents an opportunity to understand the relationship between muscle function and joint motion . the reliability of the kinematic data in this study was consistent with that reported for other inflammatory joint conditions . we confirmed previous observations where larger foot segments have greater reliability both within- and between - days in the sagittal plane in comparison with smaller segments with smaller ranges of motion , particularly in the frontal and transverse planes . inflammatory joint diseases present challenges for identification of surface landmarks , in particular when joints are swollen , tender and deformed . nevertheless , the model presented here showed excellent reliability for discrete variables both barefoot and shod . firstly the complexity and time burden of the protocol restricted the sample size , particularly in a heterogeneous disease such as ra . future work should attempt to recruit a larger sample in order to draw more meaningful conclusions . secondly , normalising the emg signals to mvics has inherent limitations in a patient population with fluctuating symptoms , particularly joint pain and effusions . in conclusion , patterns of muscle activation and kinematic motion appeared more consistent than discrete variables and absolute measures of error . the findings demonstrate that , in this cohort of ra patients using these emg variables , the use of discrete emg variables between time points is not supported either barefoot or shod . kinematic reliability has been established in the presence of pathology in this cohort but should be interpreted within the error limits .

the eutherian comparative genomic analysis protocol included gene annotations , phylogenetic analysis and protein molecular evolution analysis . the bioedit 7.0.5.3 program was used in nucleotide and protein sequence analyses ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) . the ncbi 's blast programs were used in identification of genes in eutherian genomic sequence assemblies downloaded from ncbi ( ftp://ftp.ncbi.nlm.nih.gov/blast/ and ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/eukaryotes/vertebrates_mammals/ ) , . alternatively , the ensembl genome browser 's blast or blat web tools were used in gene identifications ( http://www.ensembl.org ) . the gene feature analyses included direct evidence of eutherian gene annotations deposited in ncbi 's nr , est_human , est_mouse and est_others databases ( http://www.ncbi.nlm.nih.gov ) . the protocol tested potential growth hormone ( gh ) coding sequences using tests of reliability of eutherian public genomic sequences . using ncbi 's blast programs and primary sequence reads deposited in ncbi 's trace archive ( http://www.ncbi.nlm.nih.gov/traces/trace.cgi ) , the first test step analysed nucleotide sequence coverage of potential coding sequences . the potential coding sequences were described as complete coding sequences in second test step only if consensus trace sequence coverage was available for every nucleotide . only the complete gh coding sequences were deposited in european nucleotide archive ( http://www.ebi.ac.uk/ena/about/tpa-policy ) and used in phylogenetic and protein molecular evolution analyses . in gene descriptions , the guidelines of human and mouse gene nomenclature were followed ( http://www.genenames.org/about/guidelines and http://www.informatics.jax.org/mgihome/nomen/gene.shtml ) . there were 100 complete eutherian gh coding sequences , among 146 potential coding sequences ( fig . 1 ) ( supplementary data file 1 ) . the most comprehensive third party data gene data set of eutherian gh genes annotated 15 gha genes , 36 ghb genes , 5 ghc genes , 39 ghd genes and 5 ghe genes . the eutherian gha genes were described as prolactin prl orthologues , eutherian ghb genes were described as growth hormone gh orthologues and paralogues , domesticated guinea pig ghc genes were first described in present work , ghd genes were described as prolactin paralogues in mouse and brown rat and ghe genes were described as prolactin paralogues in domestic cattle , , . the masking of transposable elements using repeatmasker version open-4.0.3 was included as preparatory step in multiple pairwise genomic sequence alignments , using default settings except simple repeats and low complexity elements were not masked ( sensitive mode , cross_match version 1.080812 , repbase update 20130422 , rm database version 20130422 ) ( http://www.repeatmasker.org/ ) . in genomic sequence alignments , the mvista web tool was used , using avid alignment program and default settings ( http://genome.lbl.gov/vista/index.shtml ) . using clustalw implemented in bioedit 7.0.5.3 , the common predicted promoter genomic sequence regions were aligned at nucleotide sequence level and then manually corrected . the pairwise nucleotide sequence identities of common predicted promoter genomic sequence regions were calculated using bioedit 7.0.5.3 , and used in statistical analysis ( microsoft office excel ) . the common predicted promoter genomic sequence regions of eutherian gha and ghb genes were described ( supplementary data file 2 , supplementary data file 3 ) . for example , among primates , the calculated patterns of average pairwise nucleotide sequence identities of common predicted promoter genomic sequence regions exceeded empirically determined cut - offs of detection of common genomic sequence regions . whereas the average pairwise nucleotide sequence identity of primate gha common predicted promoter genomic sequence regions was = 0,872 ( amax = 0,986 , amin = 0,767 , ad = 0,074 ) ( supplementary data file 2 , supplementary data file 3 ) , average pairwise nucleotide sequence identity of primate ghb common predicted promoter genomic sequence regions was = 0,844 ( amax = 0,989 , amin = 0,252 , ad = 0,111 ) ( supplementary data file 2 , supplementary data file 3 ) . the translated complete eutherian gh coding sequences were aligned at amino acid level using clustalw implemented in bioedit 7.0.5.3 . then the protein sequence alignments were manually corrected , as well as nucleotide sequence alignments ( supplementary data file 4 ) . in phylogenetic tree calculations , the mega 6.06 program was used ( http://www.megasoftware.net ) , using neighbour - joining method ( default settings , except gaps / missing data treatment = pairwise deletion ) ( data not shown ) , minimum evolution method ( default settings , except gaps / missing data treatment = pairwise deletion ) and maximum parsimony method ( default settings , except gaps / missing data treatment = use all sites ) ( data not shown ) . however , the maximum likelihood methods were not used in present analysis because their homogeneity and stationarity assumptions were not satisfied ( data not shown ) . the pairwise nucleotide sequence identities of complete eutherian gh coding sequences were calculated using bioedit 7.0.5.3 , and used in statistical analysis ( microsoft office excel ) . the present work first described 5 eutherian gha - ghe major gene clusters ( fig . 1 ) . there were evidence of differential gene expansions in all eutherian gh major gene clusters , except gha major gene cluster included orthologues only . for example , the present study confirmed that there were differential gene expansions of primate ghb paralogues , , mouse and brown rat ghd paralogues , and domestic cattle ghe paralogues . of note , the present phylogenetic analysis first included completed eutherian gh gene data set . for example , the phylogenies of eutherian gha and ghb major gene clusters , as well as phylogenies of domesticated guinea pig ghc and domestic cattle ghe major gene clusters were first described . the present phylogenetic analysis of primate ghb paralogues was in agreement with previous analyses , . in addition , the overall grouping within ghd major gene cluster agreed with analysis of soares et al . . the calculated average pairwise nucleotide sequence identity of entire data set of eutherian gh homologues was = 0,448 ( amax = 0,995 , amin = 0,224 , ad = 0,141 ) . indeed , the updated and revised eutherian gh gene classification was confirmed by calculated patterns of pairwise nucleotide sequence identities of eutherian gh genes ( supplementary data file 5 ) . first , whereas the eutherian gha major gene cluster showed nucleotide sequence identities typical in comparisons between eutherian orthologues , eutherian ghb major gene cluster showed nucleotide sequence identities typical in comparisons between eutherian orthologues and paralogues . next , the nucleotide sequence identities of eutherian ghc and ghe major gene clusters respectively were typical in comparisons between eutherian paralogues . however , there were calculated nucleotide sequence identity patterns of ghd major gene cluster distant eutherian paralogues . finally , there were nucleotide sequence identities of close eutherian homologues in comparisons between eutherian gha , ghc , ghd and ghe major gene clusters . yet , in comparisons between eutherian ghb major gene cluster and other major gene clusters , there were nucleotide sequence identities of typical eutherian homologues . the tests of protein molecular evolution integrated patterns of nucleotide sequence similarities with protein tertiary structures . in codon the ratios between observed and expected amino acid codon counts determined relative synonymous codon usage statistics ( r ) . the not preferable amino acid codons with r 0.7 were tta ( 0,28 ) , ttg ( 0,56 ) , cta ( 0,54 ) , ata ( 0,62 ) , gta ( 0,38 ) , tcg ( 0,39 ) , ccg ( 0,34 ) , acg ( 0,41 ) , gcg ( 0,15 ) , tgt ( 0,55 ) , cgt ( 0,54 ) , cga ( 0,54 ) , agt ( 0,63 ) and ggt ( 0,56 ) . accordingly , the reference protein sequence amino acid sites were indicated as invariant amino acid sites ( invariant alignment positions ) , forward amino acid sites ( variant alignment positions that did not include amino acid codons with r 0.7 ) or compensatory amino acid sites ( variant alignment positions that included amino acid codons with r 0.7 ) . the presence of preferable amino acid codons , as well as absence of not preferable amino acid codons indicated that forward amino acid sites could have major influence on protein tertiary structure and function . the deepview / swiss - pdbviever 4.1.0 ( http://spdbv.vital-it.ch/ ) was used in analysis of human gha1 tertiary structure 1n9d , . in prediction of n - terminal signal peptides , the present study first described 5 eutherian gh major protein clusters ( fig . 1 ) . there were 6 common cysteine amino acid residues 16 present in eutherian gh proteins ( fig . 1b ) ( supplementary data file 4 ) . whereas the eutherian ghb major protein cluster included 4 common cys amino acid residues 36 , there were 6 common cys amino acid residues 16 present in other eutherian gh major protein clusters . yet , in present eutherian gh protein data set , there were substitutions at common cys residues 1 , 2 and 5 ( c33 , c40 and c220 in human gha1 ) but not at invariant common cys amino acid residues 3 , 4 and 6 ( c87 , c203 and c228 in human gha1 ) . whereas the n - terminal signal peptides were predicted in all eutherian gh major protein clusters ( data not shown ) , no invariant common potential n - glycosylation sites were found in eutherian gh major protein clusters . the present tests of protein molecular evolution included entire eutherian gh homologue data set ( fig . the human gha1 protein primary structure was used as reference protein amino acid sequence in analysis of human gha1 tertiary structure 1n9d ( supplementary data file 6 ) . first , there were 4 invariant amino acid sites among 228 reference protein amino acid residues . for example , the invariant common cys amino acid residues 3 and 4 ( c87 and c203 in human gha1 ) were implicated in disulfide linkage . second , there were 13 forward amino acid sites described in reference protein amino acid sequence . for example , the human gha1 amino acid sites h75 and h88 were designated as major tertiary structure determinant amino acid residues . the bioedit 7.0.5.3 program was used in nucleotide and protein sequence analyses ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) . the ncbi 's blast programs were used in identification of genes in eutherian genomic sequence assemblies downloaded from ncbi ( ftp://ftp.ncbi.nlm.nih.gov/blast/ and ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/eukaryotes/vertebrates_mammals/ ) , . alternatively , the ensembl genome browser 's blast or blat web tools were used in gene identifications ( http://www.ensembl.org ) . the gene feature analyses included direct evidence of eutherian gene annotations deposited in ncbi 's nr , est_human , est_mouse and est_others databases ( http://www.ncbi.nlm.nih.gov ) . the protocol tested potential growth hormone ( gh ) coding sequences using tests of reliability of eutherian public genomic sequences . using ncbi 's blast programs and primary sequence reads deposited in ncbi 's trace archive ( http://www.ncbi.nlm.nih.gov/traces/trace.cgi ) , the first test step analysed nucleotide sequence coverage of potential coding sequences . the potential coding sequences were described as complete coding sequences in second test step only if consensus trace sequence coverage was available for every nucleotide . only the complete gh coding sequences were deposited in european nucleotide archive ( http://www.ebi.ac.uk/ena/about/tpa-policy ) and used in phylogenetic and protein molecular evolution analyses . in gene descriptions , the guidelines of human and mouse gene nomenclature were followed ( http://www.genenames.org/about/guidelines and http://www.informatics.jax.org/mgihome/nomen/gene.shtml ) . there were 100 complete eutherian gh coding sequences , among 146 potential coding sequences ( fig . 1 ) ( supplementary data file 1 ) . the most comprehensive third party data gene data set of eutherian gh genes annotated 15 gha genes , 36 ghb genes , 5 ghc genes , 39 ghd genes and 5 ghe genes . the eutherian gha genes were described as prolactin prl orthologues , eutherian ghb genes were described as growth hormone gh orthologues and paralogues , domesticated guinea pig ghc genes were first described in present work , ghd genes were described as prolactin paralogues in mouse and brown rat and ghe genes were described as prolactin paralogues in domestic cattle , , . the masking of transposable elements using repeatmasker version open-4.0.3 was included as preparatory step in multiple pairwise genomic sequence alignments , using default settings except simple repeats and low complexity elements were not masked ( sensitive mode , cross_match version 1.080812 , repbase update 20130422 , rm database version 20130422 ) ( http://www.repeatmasker.org/ ) . in genomic sequence alignments , the mvista web tool was used , using avid alignment program and default settings ( http://genome.lbl.gov/vista/index.shtml ) . using clustalw implemented in bioedit 7.0.5.3 , the common predicted promoter genomic sequence regions were aligned at nucleotide sequence level and then manually corrected . the pairwise nucleotide sequence identities of common predicted promoter genomic sequence regions were calculated using bioedit 7.0.5.3 , and used in statistical analysis ( microsoft office excel ) . the common predicted promoter genomic sequence regions of eutherian gha and ghb genes were described ( supplementary data file 2 , supplementary data file 3 ) . for example , among primates , the calculated patterns of average pairwise nucleotide sequence identities of common predicted promoter genomic sequence regions exceeded empirically determined cut - offs of detection of common genomic sequence regions . whereas the average pairwise nucleotide sequence identity of primate gha common predicted promoter genomic sequence regions was = 0,872 ( amax = 0,986 , amin = 0,767 , ad = 0,074 ) ( supplementary data file 2 , supplementary data file 3 ) , average pairwise nucleotide sequence identity of primate ghb common predicted promoter genomic sequence regions was = 0,844 ( amax = 0,989 , amin = 0,252 , ad = 0,111 ) ( supplementary data file 2 , supplementary data file 3 ) . the translated complete eutherian gh coding sequences were aligned at amino acid level using clustalw implemented in bioedit 7.0.5.3 . then the protein sequence alignments were manually corrected , as well as nucleotide sequence alignments ( supplementary data file 4 ) . in phylogenetic tree calculations , the mega 6.06 program was used ( http://www.megasoftware.net ) , using neighbour - joining method ( default settings , except gaps / missing data treatment = pairwise deletion ) ( data not shown ) , minimum evolution method ( default settings , except gaps / missing data treatment = pairwise deletion ) and maximum parsimony method ( default settings , except gaps / missing data treatment = use all sites ) ( data not shown ) . however , the maximum likelihood methods were not used in present analysis because their homogeneity and stationarity assumptions were not satisfied ( data not shown ) . the pairwise nucleotide sequence identities of complete eutherian gh coding sequences were calculated using bioedit 7.0.5.3 , and used in statistical analysis ( microsoft office excel ) . the present work first described 5 eutherian gha - ghe major gene clusters ( fig . 1 ) . there were evidence of differential gene expansions in all eutherian gh major gene clusters , except gha major gene cluster included orthologues only . for example , the present study confirmed that there were differential gene expansions of primate ghb paralogues , , mouse and brown rat ghd paralogues , and domestic cattle ghe paralogues . of note , for example , the phylogenies of eutherian gha and ghb major gene clusters , as well as phylogenies of domesticated guinea pig ghc and domestic cattle ghe major gene clusters were first described . the present phylogenetic analysis of primate ghb paralogues was in agreement with previous analyses , . in addition , the overall grouping within ghd major gene cluster agreed with analysis of soares et al . . the calculated average pairwise nucleotide sequence identity of entire data set of eutherian gh homologues was = 0,448 ( amax = 0,995 , amin = 0,224 , ad = 0,141 ) . indeed , the updated and revised eutherian gh gene classification was confirmed by calculated patterns of pairwise nucleotide sequence identities of eutherian gh genes ( supplementary data file 5 ) . first , whereas the eutherian gha major gene cluster showed nucleotide sequence identities typical in comparisons between eutherian orthologues , eutherian ghb major gene cluster showed nucleotide sequence identities typical in comparisons between eutherian orthologues and paralogues . next , the nucleotide sequence identities of eutherian ghc and ghe major gene clusters respectively were typical in comparisons between eutherian paralogues . however , there were calculated nucleotide sequence identity patterns of ghd major gene cluster distant eutherian paralogues . finally , there were nucleotide sequence identities of close eutherian homologues in comparisons between eutherian gha , ghc , ghd and ghe major gene clusters . yet , in comparisons between eutherian ghb major gene cluster and other major gene clusters , there were nucleotide sequence identities of typical eutherian homologues . the tests of protein molecular evolution integrated patterns of nucleotide sequence similarities with protein tertiary structures . in codon the ratios between observed and expected amino acid codon counts determined relative synonymous codon usage statistics ( r ) . the not preferable amino acid codons with r 0.7 were tta ( 0,28 ) , ttg ( 0,56 ) , cta ( 0,54 ) , ata ( 0,62 ) , gta ( 0,38 ) , tcg ( 0,39 ) , ccg ( 0,34 ) , acg ( 0,41 ) , gcg ( 0,15 ) , tgt ( 0,55 ) , cgt ( 0,54 ) , cga ( 0,54 ) , agt ( 0,63 ) and ggt ( 0,56 ) . accordingly , the reference protein sequence amino acid sites were indicated as invariant amino acid sites ( invariant alignment positions ) , forward amino acid sites ( variant alignment positions that did not include amino acid codons with r 0.7 ) or compensatory amino acid sites ( variant alignment positions that included amino acid codons with r 0.7 ) . the presence of preferable amino acid codons , as well as absence of not preferable amino acid codons indicated that forward amino acid sites could have major influence on protein tertiary structure and function . the deepview / swiss - pdbviever 4.1.0 ( http://spdbv.vital-it.ch/ ) was used in analysis of human gha1 tertiary structure 1n9d , . in prediction of n - terminal signal peptides , the present study first described 5 eutherian gh major protein clusters ( fig . 1 ) . there were 6 common cysteine amino acid residues 16 present in eutherian gh proteins ( fig . whereas the eutherian ghb major protein cluster included 4 common cys amino acid residues 36 , there were 6 common cys amino acid residues 16 present in other eutherian gh major protein clusters . yet , in present eutherian gh protein data set , there were substitutions at common cys residues 1 , 2 and 5 ( c33 , c40 and c220 in human gha1 ) but not at invariant common cys amino acid residues 3 , 4 and 6 ( c87 , c203 and c228 in human gha1 ) . whereas the n - terminal signal peptides were predicted in all eutherian gh major protein clusters ( data not shown ) , no invariant common potential n - glycosylation sites were found in eutherian gh major protein clusters . the present tests of protein molecular evolution included entire eutherian gh homologue data set ( fig . the human gha1 protein primary structure was used as reference protein amino acid sequence in analysis of human gha1 tertiary structure 1n9d ( supplementary data file 6 ) . first , there were 4 invariant amino acid sites among 228 reference protein amino acid residues . for example , the invariant common cys amino acid residues 3 and 4 ( c87 and c203 in human gha1 ) were implicated in disulfide linkage . second , there were 13 forward amino acid sites described in reference protein amino acid sequence . for example , the human gha1 amino acid sites h75 and h88 were designated as major tertiary structure determinant amino acid residues . the eutherian gh third party data gene data set included genes implicated in major physiological processes , , , , , , . for example , the human gh homologues were recorded in world anti - doping code 's prohibited list ( http://list.wada-ama.org/ ) . the present updated gene classification and nomenclature of eutherian gh genes integrated gene annotations , phylogenetic analysis and protein molecular evolution analysis into new framework of future experiments . the following are the supplementary data related to this article.supplementary data file 1third party data gene data set of eutherian growth hormone genes.supplementary data file 2pairwise genomic sequence alignments of eutherian growth hormone genes . the translated sequence regions were displayed as indigo rectangles and untranslated sequence regions were displayed as cyan rectangles in base sequences ( top ) . the genomic sequence regions that showed conservation levels exceeding empirical cut - offs of detection of common genomic sequence regions were shown accordingly in pairwise alignments . using rectangles , the common predicted promoter genomic sequence regions ( p ) were labelled . ( a ) the cut - offs of detection of common genomic sequence regions in pairwise alignments with homo sapiens gha1 were 90% per 100 bp ( pongo abelii , nomascus leucogenys ) , 85% per 100 bp ( macaca mulatta ) , 80% per 100 bp ( callithrix jacchus ) , 75% per 100 bp ( microcebus murinus , otolemur garnettii ) , 65% per 100 bp ( rodentia ) or 70% per 100 bp in other pairwise alignments . ( b ) the cut - offs of detection of common genomic sequence regions in pairwise alignments with homo sapiens ghb1 were 80% per 100 bp ( haplorrhini ) , 75% per 100 bp ( otolemur garnettii ) , 65% per 100 bp ( rodentia ) or 70% per 100 bp in other pairwise alignments . the exons in base sequences ( top ) were annotated using transcripts bc088370.1 ( a ) and bc035965.1 ( b).supplementary data file 3nucleotide sequence alignments of common predicted promoter genomic sequence regions . the 5-terminal exons were annotated as in supplementary data file 2 , and labelled by xs below alignments . the translation start sites were marked by black triangles above alignments . according to conservation levels , the nucleotide positions were labelled by white letters on black background ( 100% conservation level ) , white letters on dark grey background ( 85% conservation level ) or black letters on grey background ( 70% conservation level).supplementary data file 4protein sequence alignments of eutherian growth hormone proteins . in reference human gha1 protein amino acid sequence ( top ) , the 4 invariant amino acid sites were shown using white letters on violet backgrounds and 13 forward amino acid sites were shown using white letters on red backgrounds . according to conservation levels , the amino acid positions were labelled by white letters on black background ( 100% conservation level ) , white letters on dark grey background ( 75% conservation level ) or black letters on grey background ( 50% conservation level ) . the stop codons were indicated by & s.supplementary data file 5pairwise nucleotide sequence identities of eutherian growth hormone genes.supplementary data file 6protein molecular evolution analysis of eutherian growth hormone proteins . the 4 invariant amino acid sites were shown using white letters on violet backgrounds and 13 forward amino acid sites were shown using white letters on red backgrounds . the common cys amino acid residues 16 were labelled below reference protein amino acid sequence , as well as 14 predicted functional amino acid residues ( # ) . the ribbon representation of human gha1 tertiary structure 1n9d . whereas the invariant amino acid sites were labelled violet , forward amino acid sites were labelled red . the translated sequence regions were displayed as indigo rectangles and untranslated sequence regions were displayed as cyan rectangles in base sequences ( top ) . the genomic sequence regions that showed conservation levels exceeding empirical cut - offs of detection of common genomic sequence regions were shown accordingly in pairwise alignments . using rectangles , the common predicted promoter genomic sequence regions ( p ) were labelled . ( a ) the cut - offs of detection of common genomic sequence regions in pairwise alignments with homo sapiens gha1 were 90% per 100 bp ( pongo abelii , nomascus leucogenys ) , 85% per 100 bp ( macaca mulatta ) , 80% per 100 bp ( callithrix jacchus ) , 75% per 100 bp ( microcebus murinus , otolemur garnettii ) , 65% per 100 bp ( rodentia ) or 70% per 100 bp in other pairwise alignments . ( b ) the cut - offs of detection of common genomic sequence regions in pairwise alignments with homo sapiens ghb1 were 80% per 100 bp ( haplorrhini ) , 75% per 100 bp ( otolemur garnettii ) , 65% per 100 bp ( rodentia ) or 70% per 100 bp in other pairwise alignments . the exons in base sequences ( top ) were annotated using transcripts bc088370.1 ( a ) and bc035965.1 ( b ) . the 5-terminal exons were annotated as in supplementary data file 2 , and labelled by xs below alignments . the translation start sites were marked by black triangles above alignments . according to conservation levels , the nucleotide positions were labelled by white letters on black background ( 100% conservation level ) , white letters on dark grey background ( 85% conservation level ) or black letters on grey background ( 70% conservation level ) . protein sequence alignments of eutherian growth hormone proteins . in reference human gha1 protein amino acid sequence ( top ) , the 4 invariant amino acid sites were shown using white letters on violet backgrounds and 13 forward amino acid sites were shown using white letters on red backgrounds . according to conservation levels , the amino acid positions were labelled by white letters on black background ( 100% conservation level ) , white letters on dark grey background ( 75% conservation level ) or black letters on grey background ( 50% conservation level ) . the stop codons were indicated by & s. pairwise nucleotide sequence identities of eutherian growth hormone genes . the 4 invariant amino acid sites were shown using white letters on violet backgrounds and 13 forward amino acid sites were shown using white letters on red backgrounds . the common cys amino acid residues 16 were labelled below reference protein amino acid sequence , as well as 14 predicted functional amino acid residues ( # ) . the ribbon representation of human gha1 tertiary structure 1n9d . whereas the invariant amino acid sites were labelled violet , forward amino acid sites were labelled red .

among 146 potential coding sequences , the most comprehensive eutherian growth hormone gene data set annotated 100 complete coding sequences . the eutherian comparative genomic analysis protocol first described 5 major gene clusters of eutherian growth hormone genes . the present updated gene classification and nomenclature of eutherian growth hormone genes integrated gene annotations , phylogenetic analysis and protein molecular evolution analysis into new framework of future experiments . the curated third party data gene data set of eutherian growth hormone genes was deposited in european nucleotide archive under accession numbers lm644135lm644234 .

Direct link to deposited data Experimental design, materials and methods Gene annotations Phylogenetic analysis Protein molecular evolution analysis Discussion

the eutherian comparative genomic analysis protocol included gene annotations , phylogenetic analysis and protein molecular evolution analysis . the bioedit 7.0.5.3 program was used in nucleotide and protein sequence analyses ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) . alternatively , the ensembl genome browser 's blast or blat web tools were used in gene identifications ( http://www.ensembl.org ) . the gene feature analyses included direct evidence of eutherian gene annotations deposited in ncbi 's nr , est_human , est_mouse and est_others databases ( http://www.ncbi.nlm.nih.gov ) . the protocol tested potential growth hormone ( gh ) coding sequences using tests of reliability of eutherian public genomic sequences . using ncbi 's blast programs and primary sequence reads deposited in ncbi 's trace archive ( http://www.ncbi.nlm.nih.gov/traces/trace.cgi ) , the first test step analysed nucleotide sequence coverage of potential coding sequences . the potential coding sequences were described as complete coding sequences in second test step only if consensus trace sequence coverage was available for every nucleotide . only the complete gh coding sequences were deposited in european nucleotide archive ( http://www.ebi.ac.uk/ena/about/tpa-policy ) and used in phylogenetic and protein molecular evolution analyses . in gene descriptions , the guidelines of human and mouse gene nomenclature were followed ( http://www.genenames.org/about/guidelines and http://www.informatics.jax.org/mgihome/nomen/gene.shtml ) . there were 100 complete eutherian gh coding sequences , among 146 potential coding sequences ( fig . the most comprehensive third party data gene data set of eutherian gh genes annotated 15 gha genes , 36 ghb genes , 5 ghc genes , 39 ghd genes and 5 ghe genes . the eutherian gha genes were described as prolactin prl orthologues , eutherian ghb genes were described as growth hormone gh orthologues and paralogues , domesticated guinea pig ghc genes were first described in present work , ghd genes were described as prolactin paralogues in mouse and brown rat and ghe genes were described as prolactin paralogues in domestic cattle , , . in genomic sequence alignments , the mvista web tool was used , using avid alignment program and default settings ( http://genome.lbl.gov/vista/index.shtml ) . using clustalw implemented in bioedit 7.0.5.3 , the common predicted promoter genomic sequence regions were aligned at nucleotide sequence level and then manually corrected . the common predicted promoter genomic sequence regions of eutherian gha and ghb genes were described ( supplementary data file 2 , supplementary data file 3 ) . for example , among primates , the calculated patterns of average pairwise nucleotide sequence identities of common predicted promoter genomic sequence regions exceeded empirically determined cut - offs of detection of common genomic sequence regions . the translated complete eutherian gh coding sequences were aligned at amino acid level using clustalw implemented in bioedit 7.0.5.3 . in phylogenetic tree calculations , the mega 6.06 program was used ( http://www.megasoftware.net ) , using neighbour - joining method ( default settings , except gaps / missing data treatment = pairwise deletion ) ( data not shown ) , minimum evolution method ( default settings , except gaps / missing data treatment = pairwise deletion ) and maximum parsimony method ( default settings , except gaps / missing data treatment = use all sites ) ( data not shown ) . however , the maximum likelihood methods were not used in present analysis because their homogeneity and stationarity assumptions were not satisfied ( data not shown ) . the pairwise nucleotide sequence identities of complete eutherian gh coding sequences were calculated using bioedit 7.0.5.3 , and used in statistical analysis ( microsoft office excel ) . the present work first described 5 eutherian gha - ghe major gene clusters ( fig . there were evidence of differential gene expansions in all eutherian gh major gene clusters , except gha major gene cluster included orthologues only . for example , the present study confirmed that there were differential gene expansions of primate ghb paralogues , , mouse and brown rat ghd paralogues , and domestic cattle ghe paralogues . of note , the present phylogenetic analysis first included completed eutherian gh gene data set . for example , the phylogenies of eutherian gha and ghb major gene clusters , as well as phylogenies of domesticated guinea pig ghc and domestic cattle ghe major gene clusters were first described . the present phylogenetic analysis of primate ghb paralogues was in agreement with previous analyses , . in addition , the overall grouping within ghd major gene cluster agreed with analysis of soares et al . the calculated average pairwise nucleotide sequence identity of entire data set of eutherian gh homologues was = 0,448 ( amax = 0,995 , amin = 0,224 , ad = 0,141 ) . indeed , the updated and revised eutherian gh gene classification was confirmed by calculated patterns of pairwise nucleotide sequence identities of eutherian gh genes ( supplementary data file 5 ) . first , whereas the eutherian gha major gene cluster showed nucleotide sequence identities typical in comparisons between eutherian orthologues , eutherian ghb major gene cluster showed nucleotide sequence identities typical in comparisons between eutherian orthologues and paralogues . next , the nucleotide sequence identities of eutherian ghc and ghe major gene clusters respectively were typical in comparisons between eutherian paralogues . however , there were calculated nucleotide sequence identity patterns of ghd major gene cluster distant eutherian paralogues . finally , there were nucleotide sequence identities of close eutherian homologues in comparisons between eutherian gha , ghc , ghd and ghe major gene clusters . yet , in comparisons between eutherian ghb major gene cluster and other major gene clusters , there were nucleotide sequence identities of typical eutherian homologues . the tests of protein molecular evolution integrated patterns of nucleotide sequence similarities with protein tertiary structures . accordingly , the reference protein sequence amino acid sites were indicated as invariant amino acid sites ( invariant alignment positions ) , forward amino acid sites ( variant alignment positions that did not include amino acid codons with r 0.7 ) or compensatory amino acid sites ( variant alignment positions that included amino acid codons with r 0.7 ) . in prediction of n - terminal signal peptides , the present study first described 5 eutherian gh major protein clusters ( fig . whereas the eutherian ghb major protein cluster included 4 common cys amino acid residues 36 , there were 6 common cys amino acid residues 16 present in other eutherian gh major protein clusters . yet , in present eutherian gh protein data set , there were substitutions at common cys residues 1 , 2 and 5 ( c33 , c40 and c220 in human gha1 ) but not at invariant common cys amino acid residues 3 , 4 and 6 ( c87 , c203 and c228 in human gha1 ) . the present tests of protein molecular evolution included entire eutherian gh homologue data set ( fig . the human gha1 protein primary structure was used as reference protein amino acid sequence in analysis of human gha1 tertiary structure 1n9d ( supplementary data file 6 ) . for example , the invariant common cys amino acid residues 3 and 4 ( c87 and c203 in human gha1 ) were implicated in disulfide linkage . for example , the human gha1 amino acid sites h75 and h88 were designated as major tertiary structure determinant amino acid residues . the bioedit 7.0.5.3 program was used in nucleotide and protein sequence analyses ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) . alternatively , the ensembl genome browser 's blast or blat web tools were used in gene identifications ( http://www.ensembl.org ) . the gene feature analyses included direct evidence of eutherian gene annotations deposited in ncbi 's nr , est_human , est_mouse and est_others databases ( http://www.ncbi.nlm.nih.gov ) . the protocol tested potential growth hormone ( gh ) coding sequences using tests of reliability of eutherian public genomic sequences . using ncbi 's blast programs and primary sequence reads deposited in ncbi 's trace archive ( http://www.ncbi.nlm.nih.gov/traces/trace.cgi ) , the first test step analysed nucleotide sequence coverage of potential coding sequences . the potential coding sequences were described as complete coding sequences in second test step only if consensus trace sequence coverage was available for every nucleotide . only the complete gh coding sequences were deposited in european nucleotide archive ( http://www.ebi.ac.uk/ena/about/tpa-policy ) and used in phylogenetic and protein molecular evolution analyses . in gene descriptions , the guidelines of human and mouse gene nomenclature were followed ( http://www.genenames.org/about/guidelines and http://www.informatics.jax.org/mgihome/nomen/gene.shtml ) . there were 100 complete eutherian gh coding sequences , among 146 potential coding sequences ( fig . the most comprehensive third party data gene data set of eutherian gh genes annotated 15 gha genes , 36 ghb genes , 5 ghc genes , 39 ghd genes and 5 ghe genes . the eutherian gha genes were described as prolactin prl orthologues , eutherian ghb genes were described as growth hormone gh orthologues and paralogues , domesticated guinea pig ghc genes were first described in present work , ghd genes were described as prolactin paralogues in mouse and brown rat and ghe genes were described as prolactin paralogues in domestic cattle , , . in genomic sequence alignments , the mvista web tool was used , using avid alignment program and default settings ( http://genome.lbl.gov/vista/index.shtml ) . using clustalw implemented in bioedit 7.0.5.3 , the common predicted promoter genomic sequence regions were aligned at nucleotide sequence level and then manually corrected . the common predicted promoter genomic sequence regions of eutherian gha and ghb genes were described ( supplementary data file 2 , supplementary data file 3 ) . for example , among primates , the calculated patterns of average pairwise nucleotide sequence identities of common predicted promoter genomic sequence regions exceeded empirically determined cut - offs of detection of common genomic sequence regions . the translated complete eutherian gh coding sequences were aligned at amino acid level using clustalw implemented in bioedit 7.0.5.3 . then the protein sequence alignments were manually corrected , as well as nucleotide sequence alignments ( supplementary data file 4 ) . in phylogenetic tree calculations , the mega 6.06 program was used ( http://www.megasoftware.net ) , using neighbour - joining method ( default settings , except gaps / missing data treatment = pairwise deletion ) ( data not shown ) , minimum evolution method ( default settings , except gaps / missing data treatment = pairwise deletion ) and maximum parsimony method ( default settings , except gaps / missing data treatment = use all sites ) ( data not shown ) . however , the maximum likelihood methods were not used in present analysis because their homogeneity and stationarity assumptions were not satisfied ( data not shown ) . the pairwise nucleotide sequence identities of complete eutherian gh coding sequences were calculated using bioedit 7.0.5.3 , and used in statistical analysis ( microsoft office excel ) . the present work first described 5 eutherian gha - ghe major gene clusters ( fig . there were evidence of differential gene expansions in all eutherian gh major gene clusters , except gha major gene cluster included orthologues only . for example , the present study confirmed that there were differential gene expansions of primate ghb paralogues , , mouse and brown rat ghd paralogues , and domestic cattle ghe paralogues . of note , for example , the phylogenies of eutherian gha and ghb major gene clusters , as well as phylogenies of domesticated guinea pig ghc and domestic cattle ghe major gene clusters were first described . the present phylogenetic analysis of primate ghb paralogues was in agreement with previous analyses , . in addition , the overall grouping within ghd major gene cluster agreed with analysis of soares et al . the calculated average pairwise nucleotide sequence identity of entire data set of eutherian gh homologues was = 0,448 ( amax = 0,995 , amin = 0,224 , ad = 0,141 ) . indeed , the updated and revised eutherian gh gene classification was confirmed by calculated patterns of pairwise nucleotide sequence identities of eutherian gh genes ( supplementary data file 5 ) . first , whereas the eutherian gha major gene cluster showed nucleotide sequence identities typical in comparisons between eutherian orthologues , eutherian ghb major gene cluster showed nucleotide sequence identities typical in comparisons between eutherian orthologues and paralogues . next , the nucleotide sequence identities of eutherian ghc and ghe major gene clusters respectively were typical in comparisons between eutherian paralogues . however , there were calculated nucleotide sequence identity patterns of ghd major gene cluster distant eutherian paralogues . finally , there were nucleotide sequence identities of close eutherian homologues in comparisons between eutherian gha , ghc , ghd and ghe major gene clusters . yet , in comparisons between eutherian ghb major gene cluster and other major gene clusters , there were nucleotide sequence identities of typical eutherian homologues . the tests of protein molecular evolution integrated patterns of nucleotide sequence similarities with protein tertiary structures . accordingly , the reference protein sequence amino acid sites were indicated as invariant amino acid sites ( invariant alignment positions ) , forward amino acid sites ( variant alignment positions that did not include amino acid codons with r 0.7 ) or compensatory amino acid sites ( variant alignment positions that included amino acid codons with r 0.7 ) . in prediction of n - terminal signal peptides , the present study first described 5 eutherian gh major protein clusters ( fig . whereas the eutherian ghb major protein cluster included 4 common cys amino acid residues 36 , there were 6 common cys amino acid residues 16 present in other eutherian gh major protein clusters . yet , in present eutherian gh protein data set , there were substitutions at common cys residues 1 , 2 and 5 ( c33 , c40 and c220 in human gha1 ) but not at invariant common cys amino acid residues 3 , 4 and 6 ( c87 , c203 and c228 in human gha1 ) . the present tests of protein molecular evolution included entire eutherian gh homologue data set ( fig . the human gha1 protein primary structure was used as reference protein amino acid sequence in analysis of human gha1 tertiary structure 1n9d ( supplementary data file 6 ) . for example , the invariant common cys amino acid residues 3 and 4 ( c87 and c203 in human gha1 ) were implicated in disulfide linkage . second , there were 13 forward amino acid sites described in reference protein amino acid sequence . for example , the human gha1 amino acid sites h75 and h88 were designated as major tertiary structure determinant amino acid residues . the eutherian gh third party data gene data set included genes implicated in major physiological processes , , , , , , . for example , the human gh homologues were recorded in world anti - doping code 's prohibited list ( http://list.wada-ama.org/ ) . the present updated gene classification and nomenclature of eutherian gh genes integrated gene annotations , phylogenetic analysis and protein molecular evolution analysis into new framework of future experiments . the following are the supplementary data related to this article.supplementary data file 1third party data gene data set of eutherian growth hormone genes.supplementary data file 2pairwise genomic sequence alignments of eutherian growth hormone genes . using rectangles , the common predicted promoter genomic sequence regions ( p ) were labelled . according to conservation levels , the nucleotide positions were labelled by white letters on black background ( 100% conservation level ) , white letters on dark grey background ( 85% conservation level ) or black letters on grey background ( 70% conservation level).supplementary data file 4protein sequence alignments of eutherian growth hormone proteins . in reference human gha1 protein amino acid sequence ( top ) , the 4 invariant amino acid sites were shown using white letters on violet backgrounds and 13 forward amino acid sites were shown using white letters on red backgrounds . according to conservation levels , the amino acid positions were labelled by white letters on black background ( 100% conservation level ) , white letters on dark grey background ( 75% conservation level ) or black letters on grey background ( 50% conservation level ) . the stop codons were indicated by & s.supplementary data file 5pairwise nucleotide sequence identities of eutherian growth hormone genes.supplementary data file 6protein molecular evolution analysis of eutherian growth hormone proteins . using rectangles , the common predicted promoter genomic sequence regions ( p ) were labelled . ( a ) the cut - offs of detection of common genomic sequence regions in pairwise alignments with homo sapiens gha1 were 90% per 100 bp ( pongo abelii , nomascus leucogenys ) , 85% per 100 bp ( macaca mulatta ) , 80% per 100 bp ( callithrix jacchus ) , 75% per 100 bp ( microcebus murinus , otolemur garnettii ) , 65% per 100 bp ( rodentia ) or 70% per 100 bp in other pairwise alignments . according to conservation levels , the nucleotide positions were labelled by white letters on black background ( 100% conservation level ) , white letters on dark grey background ( 85% conservation level ) or black letters on grey background ( 70% conservation level ) . protein sequence alignments of eutherian growth hormone proteins . in reference human gha1 protein amino acid sequence ( top ) , the 4 invariant amino acid sites were shown using white letters on violet backgrounds and 13 forward amino acid sites were shown using white letters on red backgrounds . according to conservation levels , the amino acid positions were labelled by white letters on black background ( 100% conservation level ) , white letters on dark grey background ( 75% conservation level ) or black letters on grey background ( 50% conservation level ) . the stop codons were indicated by & s. pairwise nucleotide sequence identities of eutherian growth hormone genes .

chronic obstructive pulmonary disease ( copd ) is the fourth leading cause of death in the world . tobacco smoking using water pipe ( wp ) ( narghile , hubble bubble or hookah ) is a traditional way of smoking that is prevalent in middle east and other parts of asia . usually smokers use 10 - 20 g of tobacco during smoking of wp and the time of smoking is 40 - 50 minutes . most users and even physicians believe that water filters the toxic components considerably and is less harmful than cigarette smoking . previous studies on main stream smoke of wp showed that the nicotine content of wp tobacco is more than cigarettes ( 2% to 4% , in comparison with 1% to 3% for cigarettes ) and carbon mono oxide ( co ) in wp is produced three times more than cigarette . the amount of tar in smoke of wp is less than cigarette smoke , but pyronsynthesized and polycyclic aromatic hydrocarbons are present in the tar despite the low temperatures characteristic of the tobacco in the wp . several studies reported the effect of wp smoke on pulmonary function tests , small air way function and tonicity of bronchial trees . therefore , in the present study the prevalence of wp smoking in the city of mashhad and the effect of quantity and duration of smoking on pulmonary functional tests ( pfts ) and the respiratory symptoms were examined . six hundred seventy - three ( 673 ) subjects ( 372 males aged 43.29 13.23 and 301 females aged 41.84 12.35 ) were interviewed from 10 randomly selected areas in the city of mashhad using the cluster sampling method . the data of interviewed subjects regarding the prevalence of smoking of the wp were collected . the city of mashhad is a holy city located in the north east of iran with a population of two - million people , many of whom are immigrants from other parts of the country . respiratory symptoms and pft values of wp smokers ( 58 subjects including 24 males and 34 females aged 40.94 11.97 ) were compared with 50 non - smokers ( 22 males and 28 females , aged 39.68 12.73 , control group ) which were matched regarding age , job , race / ethnicity , place of living / working . a farsi questionnaire was used to assess the prevalence of wp smoking among population of the city of mashhad and the respiratory symptoms . the questionnaire had two different parts : part ( a ) questions on wp smoking , number of wp / week ) , duration of smoking and total amount of smoking ( amount x duration)part ( b ) respiratory symptoms ( wheezing , breathlessness , cough and sputum ) . in addition , a non - smoker ( control ) group from the same area of the city was also interviewed regarding the respiratory symptoms . part ( a ) questions on wp smoking , number of wp / week ) , duration of smoking and total amount of smoking ( amount x duration ) part ( b ) respiratory symptoms ( wheezing , breathlessness , cough and sputum ) . in addition , a non - smoker ( control ) group from the same area of the city was also interviewed regarding the respiratory symptoms . questionnaire on respiratory symptoms was designed in accordance with several previous questionnaires of similar studies by expert groups [ table 1 ] . the respiratory symptoms severity score pulmonary function tests of smokers and control groups were measured using a spirometer with a pneumotachograph sensor ( model st90 , fukuda , sangyo co. , ltd , japan ) . prior to pulmonary function testing , the required maneuver was demonstrated by the operator , and subjects were encouraged and supervised throughout test performance . pulmonary function testing was performed using the acceptability standards outlined by the american thoracic society ( ats ) with subjects in a standing position and wearing nose clips . the highest level for forced vital capacity ( fvc ) , forced expiratory volume in one second ( fev1 ) , peak expiratory flow ( pef ) , maximal mid expiratory flow ( mmef ) and maximal expiratory flow at 75% , 50% , and 25% of the fvc ( mef75 , mef50 , and mef25 , respectively ) were taken independently from the three curves . the data of pft values and age were expressed as mean sd and data of smoking and respiratory symptoms as percentage of each group having the corresponding symptom . differences in the prevalence of symptoms between smokers and control groups were tested by the chi - square test . pft values between smokers and control groups were compared using the unpaired t test . the correlation between pft values and respiratory symptoms with duration and quantity of smoking . a two - sided p value of 0.05 was the criterion for statistical significance . six hundred seventy - three ( 673 ) subjects ( 372 males aged 43.29 13.23 and 301 females aged 41.84 12.35 ) were interviewed from 10 randomly selected areas in the city of mashhad using the cluster sampling method . the data of interviewed subjects regarding the prevalence of smoking of the wp were collected . the city of mashhad is a holy city located in the north east of iran with a population of two - million people , many of whom are immigrants from other parts of the country . respiratory symptoms and pft values of wp smokers ( 58 subjects including 24 males and 34 females aged 40.94 11.97 ) were compared with 50 non - smokers ( 22 males and 28 females , aged 39.68 12.73 , control group ) which were matched regarding age , job , race / ethnicity , place of living / working . a farsi questionnaire was used to assess the prevalence of wp smoking among population of the city of mashhad and the respiratory symptoms . the questionnaire had two different parts : part ( a ) questions on wp smoking , number of wp / week ) , duration of smoking and total amount of smoking ( amount x duration)part ( b ) respiratory symptoms ( wheezing , breathlessness , cough and sputum ) . in addition , a non - smoker ( control ) group from the same area of the city was also interviewed regarding the respiratory symptoms . part ( a ) questions on wp smoking , number of wp / week ) , duration of smoking and total amount of smoking ( amount x duration ) part ( b ) respiratory symptoms ( wheezing , breathlessness , cough and sputum ) . in addition , a non - smoker ( control ) group from the same area of the city was also interviewed regarding the respiratory symptoms . questionnaire on respiratory symptoms was designed in accordance with several previous questionnaires of similar studies by expert groups [ table 1 ] . the respiratory symptoms severity score pulmonary function tests of smokers and control groups were measured using a spirometer with a pneumotachograph sensor ( model st90 , fukuda , sangyo co. , ltd , japan ) . prior to pulmonary function testing , the required maneuver was demonstrated by the operator , and subjects were encouraged and supervised throughout test performance . pulmonary function testing was performed using the acceptability standards outlined by the american thoracic society ( ats ) with subjects in a standing position and wearing nose clips . the highest level for forced vital capacity ( fvc ) , forced expiratory volume in one second ( fev1 ) , peak expiratory flow ( pef ) , maximal mid expiratory flow ( mmef ) and maximal expiratory flow at 75% , 50% , and 25% of the fvc ( mef75 , mef50 , and mef25 , respectively ) were taken independently from the three curves . the data of pft values and age were expressed as mean sd and data of smoking and respiratory symptoms as percentage of each group having the corresponding symptom . differences in the prevalence of symptoms between smokers and control groups were tested by the chi - square test . the correlation between pft values and respiratory symptoms with duration and quantity of smoking was assessed using least - square regression . all analyses were performed with spss software ( version 11.5 , spss in . , usa ) . the prevalence of wp smoking among studied subjects was 8.6% ( 58 out of 673 subjects ) including 6.5% of males ( 24 out of 372 subjects ) and 11.3% of females ( 34 out of 301 subjects ) . the prevalence of wp smoking was higher in the low socioeconomic areas of the city ( kalat road , south motahari street and eshrat abad ) than the high socioeconomic areas ( ahmad abad , pirozi and vakil abad blvd ) [ table 2 ] . total studied subjects and prevalence of water pipe smoking among different areas of mashhad city the prevalence of wp smoking was higher in younger individuals ( 20 - 40 years old ) and lower among 50 - 60 years old population . the wp smoking was lower among women of 50 - 60 and 20 - 30 years aged and was higher in other age groups [ table 3 ] . prevalence of water pipe smoking among different age groups in studied population the most and least prevalent respiratory symptoms among wp smokers were chest tightness ( 37% ) and sputum production ( 13% ) , respectively . the most and least prevalent respiratory symptoms among non - smokers were also chest tightness ( 6.6% ) and cough ( 13% ) , respectively . the prevalence of all respiratory symptoms among wp smokers ( except for the chronic sputum production ) , ( p < 0.05 to p < 0.001 ) , was higher as compared to the control group [ figure 1a ] . in addition , the severity of respiratory symptoms among wp smokers ( except for chronic sputum production ) , ( p < 0.05 to p < 0.001 ) , was also higher as compared to the control group [ figure 1b ] . comparison of prevalence ( a ) and severity ( b ) of respiratory symptoms between water pipe ( wp ) smokers and those of non - smokers . 0.001 all values of pft in wp smokers were significantly lower than those of non - smokers ( p < 0.05 to p < 0.001 ) [ figure 2 ] . comparison of pulmonary functional tests ( pft ) between water pipe ( wp ) smokers and non smokers ( controls ) subjects . values were presented as mean sd of percent predicted ( for smokers and non smokers n = 58 and 50 respectively ) . fvc = forced vital capacity , fev1 = forced expiratory volume in one second , mmef = maximal mid expiratory flow , pef = peak expiratory flow , mef75 , mef50 , and mef25 = maximal expiratory flow at 75% , 50% , and 25% of the fvc , respectively . * = p < 0.005 , * = p < 0.001 there was no significant correlation between respiratory symptoms and duration or total amount of smoking in wp smokers [ table 4 ] . however , there was significant negative correlation between pft values and duration as well as total amount of wp smoking except for mef25 ( p < 0.05 to p < 0.001 ) . this was also true for the amount of smoking with only pef and mef75 ( p < 0.05 for both cases ) [ table 5 ] . correlation between respiratory symptoms , smoking duration ( year ) , amount and total smoking in water pipe smokers correlation between pft values , smoking duration ( year ) , amount and total smoking of water pipe smokers the prevalence of wp smoking among studied subjects was 8.6% ( 58 out of 673 subjects ) including 6.5% of males ( 24 out of 372 subjects ) and 11.3% of females ( 34 out of 301 subjects ) . the prevalence of wp smoking was higher in the low socioeconomic areas of the city ( kalat road , south motahari street and eshrat abad ) than the high socioeconomic areas ( ahmad abad , pirozi and vakil abad blvd ) [ table 2 ] . total studied subjects and prevalence of water pipe smoking among different areas of mashhad city the prevalence of wp smoking was higher in younger individuals ( 20 - 40 years old ) and lower among 50 - 60 years old population . the wp smoking was lower among women of 50 - 60 and 20 - 30 years aged and was higher in other age groups [ table 3 ] . the most and least prevalent respiratory symptoms among wp smokers were chest tightness ( 37% ) and sputum production ( 13% ) , respectively . the most and least prevalent respiratory symptoms among non - smokers were also chest tightness ( 6.6% ) and cough ( 13% ) , respectively . the prevalence of all respiratory symptoms among wp smokers ( except for the chronic sputum production ) , ( p < 0.05 to p < 0.001 ) , was higher as compared to the control group [ figure 1a ] . in addition , the severity of respiratory symptoms among wp smokers ( except for chronic sputum production ) , ( p < 0.05 to p < 0.001 ) , was also higher as compared to the control group [ figure 1b ] . comparison of prevalence ( a ) and severity ( b ) of respiratory symptoms between water pipe ( wp ) smokers and those of non - smokers . amount of smoking was expressed in no . of wp / week and total amount of smoking is amount x duration . * = p < 0.05 , * * * = p < 0.001 all values of pft in wp smokers were significantly lower than those of non - smokers ( p < 0.05 to p < 0.001 ) [ figure 2 ] . comparison of pulmonary functional tests ( pft ) between water pipe ( wp ) smokers and non smokers ( controls ) subjects . values were presented as mean sd of percent predicted ( for smokers and non smokers n = 58 and 50 respectively ) . fvc = forced vital capacity , fev1 = forced expiratory volume in one second , mmef = maximal mid expiratory flow , pef = peak expiratory flow , mef75 , mef50 , and mef25 = maximal expiratory flow at 75% , 50% , and 25% of the fvc , respectively . * = p < 0.005 , * = p < 0.001 there was no significant correlation between respiratory symptoms and duration or total amount of smoking in wp smokers [ table 4 ] . however , there was significant negative correlation between pft values and duration as well as total amount of wp smoking except for mef25 ( p < 0.05 to p < 0.001 ) . this was also true for the amount of smoking with only pef and mef75 ( p < 0.05 for both cases ) [ table 5 ] . correlation between respiratory symptoms , smoking duration ( year ) , amount and total smoking in water pipe smokers correlation between pft values , smoking duration ( year ) , amount and total smoking of water pipe smokers in the present study , the prevalence of wp smoking in the city of mashhad ( north east iran ) was studied . the respiratory symptoms and pft values of wp smokers were also compared with non - smoker subjects . the results showed that 8.6% of the population of the mashhad city was wp smoker . the prevalence of smoking was lower in male ( 6.5% ) than female ( 11.3% ) subjects . the prevalence of wp smoking was higher in low socioeconomical regions ( kalat road , south motahari street and eshrat abad ) compared to the high socioeconomical areas ( ahmad abad , pirozi and vakil abad blvd ) . the prevalence of wp smoking was higher in younger aged groups ( 20 - 40 years old ) and was lowest in the 50 - 60 years old population . the wp smoking was lower among women in studied subjects with 50 - 60 and 20 - 30 years old but was higher in other age groups . previous studies showed that the prevalence of wp in 13 - 15 years aged population of 5 different arabic countries was 10% . it was also shown that 25.5% of male and 4.9% of female students in syria were wp smokers . in addition , the prevalence of wp smoking in arab young population was demonstrated to be 26.6% . the prevalence of this type of smoking in kuwait was 57% for male and 69% for female subjects . the results of other studies showed that the prevalence of wp smoking in middle east countries was 11 - 32% which showed a progressive increase . the present study , which is a population - based survey , showed that the prevalence of this type of smoking was lower in the iranian population as compared to arabic and other middle east countries . in fact , the results of a study evaluating the prevalence of wp smoking in patients referred to two hospitals in tehran showed 9.3% prevalence of wp smoking . the results of these two later studies support the findings of our study regarding the prevalence of wp smoking in iran . the prevalence of wp smoking among students of birmingham city was also 8% indicating the similar prevalence of this type of smoking in western countries as shown in the present study . it was shown that the prevalence of wp smoking among iranian university students was 18.7% which was increased in 2005 compared with 2003 . it was also shown that 22% of the male and 8% of the female students had a history of at least one episode of smoking and the most important substance used was cigarette followed by wp . the results of the present study also showed higher prevalence of wp smoking which was higher among male compared female subjects . the prevalence of wp smoking among iranian athletes was shown to be 10.5% which was very similar to the results of the present study . the prevalence of wp smoking among lor migrating tribes in mamasani , southern iran ( age range , 21 - 80 years ) , was 79.9% . the results of a comprehensive study among the population of all regions of iran showed the prevalence of water pipe smoking of 2.7% ( 3.5% males and 1.9% females ) . the prevalence of wp smoking among rural pregnant women in southern iran was 8% , and among general population in south iran region ( bandar abbas ) was reported to be 10.4% . although the results of the later study regarding the prevalence of wp smoking is similar to the results of the present study , the results of three former studies were different from the results of ours . the reason of these differences is most likely to be due to race / ethnicity differences among study participants . regarding the factors that promote use of wp smoking or cigarette smoking , a study on adult residents of shiraz , iran , showed that the prevalence of cigarette smoking was 9.7% and that of hookah smoking was 11.9% . a higher perceived level of stress , a non - manual occupation , and sedentary lifestyle were positively associated with cigarette smoking . manual labor occupations , housewife / jobless status , and going frequently to restaurants were positive predictors of hookah smoking . although cigarette smoking was 31 times more common in men , the prevalence of hookah smoking in women was the same as men . in a meta - analysis in an adult population of west of iran in 25,990 subjects the prevalence smoking was 22.9 ( 20.6 - 25.2 ) and 0.6 ( 0.3 - 0.9 ) in men and women , respectively . therefore , it was found that more than one - fifth of men from 15 year to 64-year - old of west of iran smoked cigarette . our previous study also showed the prevalence of smoking of 12.7% ( 17.2% in male and 2.5 in female ) in a study population of 1435 individuals in north east iran . when we look at the data from india , a cross - sectional study in east delhi showed that the prevalence of current smoking was 24.6% . a majority of current smokers smoked bidi exclusively , and on an average 13.5 bidi / cigarette were smoked per day . multivariate analysis showed that the factors associated with current smoking were male sex , advancing age , illiteracy , skilled occupation , low socio- economic status , and low bmi ( p < 0.001 ) . low educational status was associated with poor hazard awareness and quitting behavior . since 1990 , hookah use has increased markedly , spreading from middle east to the other parts of the world , particularly among younger people , such as college and university students . studies suggest age , sex and socioeconomic status , as well as close relationship with smokers in family or friend group , emotional stress and history of alcohol and illicit drug use as predictors of smoking . it was also shown that wp smoking was higher in younger age groups , female gender and in poor population . the reason of higher prevalence of wp smoking in younger age groups , females and unemployed population is perhaps because this type of smoking is a group smoking behavior . when we look at the data from different parts of world , a systematic review showed that the prevalence of current wp smoking among university students was high in the persian gulf region ( 6% ) , the united kingdom ( 8% ) , the united states ( 10% ) , syria ( 15% ) , lebanon ( 28% ) , and pakistan ( 33% ) . the prevalence of wp smoking among adults of different countries was as the follows : pakistan ( 6% ) , persian gulf region ( 4%-12% ) , australia ( 11% in arab speaking adults ) , syria ( 9%-12% ) , and lebanon ( 15% ) which was similar to the results of our study . the water pip smoking among us population was reported as 29% , among turkey university students 32.7% and among jordan university students was also 36.8% ( 61.9% in male and 10.7% in female students ) . the prevalence rate of wp smoking among indian population was reported between 2.6% and 50% . these data showed lower prevalence of wp smoking in the population of the mashhad compared to the arab countries but higher than some regions of india . the results also showed an increased frequency of respiratory symptoms and reduction of all values of pulmonary function tests in wp smokers compared to those of non - smoker subjects . the increased respiratory symptoms and reduction of pft values in wp smokers showed the effect of this type of smoking on the respiratory system . however , those pft values indicating the diameter of smaller airways ( mmef , mef50 and mef25 ) were less affected in smokers . there was no significant relationship between quantity and duration of wp smoking and respiratory symptoms which was perhaps due to subjective notion of the data of respiratory symptoms . however , there was a significant and negative correlation between all pft values and both quantity and duration of smoking . the relationship between respiratory symptoms and pft values with quantity and duration of smoking are further confirmation of the significant effect of wp smoking on the respiratory system . it was also shown that wp smoking has a similar effect on respiratory symptoms and pft values as deep inspiration cigarette smoking . kiter et al . , also evaluated the effect of wp smoking on pft values . a more rapid effect of wp smoking on pft values as compared to cigarette smoking was also shown which may be due to higher nicotine content of wp smoke . confounding factors like age , job , race / ethnicity , place of living / working , active / passive smoking and other related variables ( other than wp ) , chemical exposure however , wp smokers and control groups were recruited from the same region of the city with similar age , job , race / ethnicity , place of living / working . therefore , it seems that these factors have no or minor effect on respiratory symptoms and pulmonary function tests in two groups . the comparison of prevalence of wp smoking among different socioeconomic populations , among people of different jobs and educational level and also rural versus urban population is important and needs to be evaluated further . in conclusion , the results of the present study showed 8.6% prevalence of wp smoking among population of the city of mashhad which was much higher in female as compared to male subjects .

background : the prevalence of water pipe ( wp ) smoking was studied using a standard questionnaire . pulmonary function tests were also compared between wp smokers and non-smokers.materials and methods : the prevalence of wp smoking was studied using a standard questionnaire . pulmonary function tests including forced vital capacity ( fvc ) , forced expiratory volume in one second ( fev1 ) , maximal mid - expiratory flow ( mmef ) , peak expiratory flow ( pef ) , maximal expiratory flow at 75% , 50% , and 25% of the fvc ( mef75,50,25 ) were compared between wp smokers and non-smokers.results:a total of 673 individuals including 372 males and 301 females were interviewed . the number of wp smokers was 58 ( 8.6% ) including 24 males ( 6.5% ) and 34 females ( 11.3% ) . all pulmonary functional test ( pft ) values in wp smokers were lower as compared to the non - smokers ( p < 0.05 to p < 0.001 ) . the prevalence and severity of respiratory symptoms ( rs ) in wp smokers were higher than non - smokers ( p < 0.05 to p < 0.001 ) . there were negative correlations between pft values and positive correlation between rs and duration , rate , as well as total smoking ( duration x rate ) ( p < 0.05 to p < 0.001).conclusion : in this study the prevalence of wp smoking in mashhad city was evaluated for the first time . the results also showed a significant effect of wp smoking on pft values and respiratory symptoms .

INTRODUCTION MATERIALS AND METHODS Study area and population Protocol Statistical analysis RESULTS Prevalence of water pipe smoking Respiratory symptoms Pulmonary function tests Correlation between smoking duration (year) and amount (pack/year) with pulmonary function tests and respiratory symptoms of smokers DISCUSION CONCLUSION

tobacco smoking using water pipe ( wp ) ( narghile , hubble bubble or hookah ) is a traditional way of smoking that is prevalent in middle east and other parts of asia . previous studies on main stream smoke of wp showed that the nicotine content of wp tobacco is more than cigarettes ( 2% to 4% , in comparison with 1% to 3% for cigarettes ) and carbon mono oxide ( co ) in wp is produced three times more than cigarette . several studies reported the effect of wp smoke on pulmonary function tests , small air way function and tonicity of bronchial trees . therefore , in the present study the prevalence of wp smoking in the city of mashhad and the effect of quantity and duration of smoking on pulmonary functional tests ( pfts ) and the respiratory symptoms were examined . six hundred seventy - three ( 673 ) subjects ( 372 males aged 43.29 13.23 and 301 females aged 41.84 12.35 ) were interviewed from 10 randomly selected areas in the city of mashhad using the cluster sampling method . the data of interviewed subjects regarding the prevalence of smoking of the wp were collected . respiratory symptoms and pft values of wp smokers ( 58 subjects including 24 males and 34 females aged 40.94 11.97 ) were compared with 50 non - smokers ( 22 males and 28 females , aged 39.68 12.73 , control group ) which were matched regarding age , job , race / ethnicity , place of living / working . a farsi questionnaire was used to assess the prevalence of wp smoking among population of the city of mashhad and the respiratory symptoms . the questionnaire had two different parts : part ( a ) questions on wp smoking , number of wp / week ) , duration of smoking and total amount of smoking ( amount x duration)part ( b ) respiratory symptoms ( wheezing , breathlessness , cough and sputum ) . in addition , a non - smoker ( control ) group from the same area of the city was also interviewed regarding the respiratory symptoms . part ( a ) questions on wp smoking , number of wp / week ) , duration of smoking and total amount of smoking ( amount x duration ) part ( b ) respiratory symptoms ( wheezing , breathlessness , cough and sputum ) . in addition , a non - smoker ( control ) group from the same area of the city was also interviewed regarding the respiratory symptoms . the respiratory symptoms severity score pulmonary function tests of smokers and control groups were measured using a spirometer with a pneumotachograph sensor ( model st90 , fukuda , sangyo co. , ltd , japan ) . the highest level for forced vital capacity ( fvc ) , forced expiratory volume in one second ( fev1 ) , peak expiratory flow ( pef ) , maximal mid expiratory flow ( mmef ) and maximal expiratory flow at 75% , 50% , and 25% of the fvc ( mef75 , mef50 , and mef25 , respectively ) were taken independently from the three curves . the data of pft values and age were expressed as mean sd and data of smoking and respiratory symptoms as percentage of each group having the corresponding symptom . differences in the prevalence of symptoms between smokers and control groups were tested by the chi - square test . pft values between smokers and control groups were compared using the unpaired t test . the correlation between pft values and respiratory symptoms with duration and quantity of smoking . six hundred seventy - three ( 673 ) subjects ( 372 males aged 43.29 13.23 and 301 females aged 41.84 12.35 ) were interviewed from 10 randomly selected areas in the city of mashhad using the cluster sampling method . the data of interviewed subjects regarding the prevalence of smoking of the wp were collected . respiratory symptoms and pft values of wp smokers ( 58 subjects including 24 males and 34 females aged 40.94 11.97 ) were compared with 50 non - smokers ( 22 males and 28 females , aged 39.68 12.73 , control group ) which were matched regarding age , job , race / ethnicity , place of living / working . a farsi questionnaire was used to assess the prevalence of wp smoking among population of the city of mashhad and the respiratory symptoms . the questionnaire had two different parts : part ( a ) questions on wp smoking , number of wp / week ) , duration of smoking and total amount of smoking ( amount x duration)part ( b ) respiratory symptoms ( wheezing , breathlessness , cough and sputum ) . in addition , a non - smoker ( control ) group from the same area of the city was also interviewed regarding the respiratory symptoms . part ( a ) questions on wp smoking , number of wp / week ) , duration of smoking and total amount of smoking ( amount x duration ) part ( b ) respiratory symptoms ( wheezing , breathlessness , cough and sputum ) . in addition , a non - smoker ( control ) group from the same area of the city was also interviewed regarding the respiratory symptoms . the respiratory symptoms severity score pulmonary function tests of smokers and control groups were measured using a spirometer with a pneumotachograph sensor ( model st90 , fukuda , sangyo co. , ltd , japan ) . the highest level for forced vital capacity ( fvc ) , forced expiratory volume in one second ( fev1 ) , peak expiratory flow ( pef ) , maximal mid expiratory flow ( mmef ) and maximal expiratory flow at 75% , 50% , and 25% of the fvc ( mef75 , mef50 , and mef25 , respectively ) were taken independently from the three curves . the data of pft values and age were expressed as mean sd and data of smoking and respiratory symptoms as percentage of each group having the corresponding symptom . differences in the prevalence of symptoms between smokers and control groups were tested by the chi - square test . the correlation between pft values and respiratory symptoms with duration and quantity of smoking was assessed using least - square regression . the prevalence of wp smoking among studied subjects was 8.6% ( 58 out of 673 subjects ) including 6.5% of males ( 24 out of 372 subjects ) and 11.3% of females ( 34 out of 301 subjects ) . the prevalence of wp smoking was higher in the low socioeconomic areas of the city ( kalat road , south motahari street and eshrat abad ) than the high socioeconomic areas ( ahmad abad , pirozi and vakil abad blvd ) [ table 2 ] . total studied subjects and prevalence of water pipe smoking among different areas of mashhad city the prevalence of wp smoking was higher in younger individuals ( 20 - 40 years old ) and lower among 50 - 60 years old population . prevalence of water pipe smoking among different age groups in studied population the most and least prevalent respiratory symptoms among wp smokers were chest tightness ( 37% ) and sputum production ( 13% ) , respectively . the most and least prevalent respiratory symptoms among non - smokers were also chest tightness ( 6.6% ) and cough ( 13% ) , respectively . the prevalence of all respiratory symptoms among wp smokers ( except for the chronic sputum production ) , ( p < 0.05 to p < 0.001 ) , was higher as compared to the control group [ figure 1a ] . in addition , the severity of respiratory symptoms among wp smokers ( except for chronic sputum production ) , ( p < 0.05 to p < 0.001 ) , was also higher as compared to the control group [ figure 1b ] . comparison of prevalence ( a ) and severity ( b ) of respiratory symptoms between water pipe ( wp ) smokers and those of non - smokers . 0.001 all values of pft in wp smokers were significantly lower than those of non - smokers ( p < 0.05 to p < 0.001 ) [ figure 2 ] . comparison of pulmonary functional tests ( pft ) between water pipe ( wp ) smokers and non smokers ( controls ) subjects . fvc = forced vital capacity , fev1 = forced expiratory volume in one second , mmef = maximal mid expiratory flow , pef = peak expiratory flow , mef75 , mef50 , and mef25 = maximal expiratory flow at 75% , 50% , and 25% of the fvc , respectively . * = p < 0.005 , * = p < 0.001 there was no significant correlation between respiratory symptoms and duration or total amount of smoking in wp smokers [ table 4 ] . however , there was significant negative correlation between pft values and duration as well as total amount of wp smoking except for mef25 ( p < 0.05 to p < 0.001 ) . this was also true for the amount of smoking with only pef and mef75 ( p < 0.05 for both cases ) [ table 5 ] . correlation between respiratory symptoms , smoking duration ( year ) , amount and total smoking in water pipe smokers correlation between pft values , smoking duration ( year ) , amount and total smoking of water pipe smokers the prevalence of wp smoking among studied subjects was 8.6% ( 58 out of 673 subjects ) including 6.5% of males ( 24 out of 372 subjects ) and 11.3% of females ( 34 out of 301 subjects ) . the prevalence of wp smoking was higher in the low socioeconomic areas of the city ( kalat road , south motahari street and eshrat abad ) than the high socioeconomic areas ( ahmad abad , pirozi and vakil abad blvd ) [ table 2 ] . total studied subjects and prevalence of water pipe smoking among different areas of mashhad city the prevalence of wp smoking was higher in younger individuals ( 20 - 40 years old ) and lower among 50 - 60 years old population . the most and least prevalent respiratory symptoms among wp smokers were chest tightness ( 37% ) and sputum production ( 13% ) , respectively . the most and least prevalent respiratory symptoms among non - smokers were also chest tightness ( 6.6% ) and cough ( 13% ) , respectively . the prevalence of all respiratory symptoms among wp smokers ( except for the chronic sputum production ) , ( p < 0.05 to p < 0.001 ) , was higher as compared to the control group [ figure 1a ] . in addition , the severity of respiratory symptoms among wp smokers ( except for chronic sputum production ) , ( p < 0.05 to p < 0.001 ) , was also higher as compared to the control group [ figure 1b ] . comparison of prevalence ( a ) and severity ( b ) of respiratory symptoms between water pipe ( wp ) smokers and those of non - smokers . * = p < 0.05 , * * * = p < 0.001 all values of pft in wp smokers were significantly lower than those of non - smokers ( p < 0.05 to p < 0.001 ) [ figure 2 ] . comparison of pulmonary functional tests ( pft ) between water pipe ( wp ) smokers and non smokers ( controls ) subjects . fvc = forced vital capacity , fev1 = forced expiratory volume in one second , mmef = maximal mid expiratory flow , pef = peak expiratory flow , mef75 , mef50 , and mef25 = maximal expiratory flow at 75% , 50% , and 25% of the fvc , respectively . * = p < 0.005 , * = p < 0.001 there was no significant correlation between respiratory symptoms and duration or total amount of smoking in wp smokers [ table 4 ] . however , there was significant negative correlation between pft values and duration as well as total amount of wp smoking except for mef25 ( p < 0.05 to p < 0.001 ) . this was also true for the amount of smoking with only pef and mef75 ( p < 0.05 for both cases ) [ table 5 ] . correlation between respiratory symptoms , smoking duration ( year ) , amount and total smoking in water pipe smokers correlation between pft values , smoking duration ( year ) , amount and total smoking of water pipe smokers in the present study , the prevalence of wp smoking in the city of mashhad ( north east iran ) was studied . the respiratory symptoms and pft values of wp smokers were also compared with non - smoker subjects . the results showed that 8.6% of the population of the mashhad city was wp smoker . the prevalence of smoking was lower in male ( 6.5% ) than female ( 11.3% ) subjects . the prevalence of wp smoking was higher in low socioeconomical regions ( kalat road , south motahari street and eshrat abad ) compared to the high socioeconomical areas ( ahmad abad , pirozi and vakil abad blvd ) . the prevalence of wp smoking was higher in younger aged groups ( 20 - 40 years old ) and was lowest in the 50 - 60 years old population . previous studies showed that the prevalence of wp in 13 - 15 years aged population of 5 different arabic countries was 10% . in addition , the prevalence of wp smoking in arab young population was demonstrated to be 26.6% . the results of other studies showed that the prevalence of wp smoking in middle east countries was 11 - 32% which showed a progressive increase . the present study , which is a population - based survey , showed that the prevalence of this type of smoking was lower in the iranian population as compared to arabic and other middle east countries . in fact , the results of a study evaluating the prevalence of wp smoking in patients referred to two hospitals in tehran showed 9.3% prevalence of wp smoking . the results of these two later studies support the findings of our study regarding the prevalence of wp smoking in iran . the prevalence of wp smoking among students of birmingham city was also 8% indicating the similar prevalence of this type of smoking in western countries as shown in the present study . it was shown that the prevalence of wp smoking among iranian university students was 18.7% which was increased in 2005 compared with 2003 . the results of the present study also showed higher prevalence of wp smoking which was higher among male compared female subjects . the prevalence of wp smoking among iranian athletes was shown to be 10.5% which was very similar to the results of the present study . the prevalence of wp smoking among lor migrating tribes in mamasani , southern iran ( age range , 21 - 80 years ) , was 79.9% . the results of a comprehensive study among the population of all regions of iran showed the prevalence of water pipe smoking of 2.7% ( 3.5% males and 1.9% females ) . the prevalence of wp smoking among rural pregnant women in southern iran was 8% , and among general population in south iran region ( bandar abbas ) was reported to be 10.4% . although the results of the later study regarding the prevalence of wp smoking is similar to the results of the present study , the results of three former studies were different from the results of ours . regarding the factors that promote use of wp smoking or cigarette smoking , a study on adult residents of shiraz , iran , showed that the prevalence of cigarette smoking was 9.7% and that of hookah smoking was 11.9% . although cigarette smoking was 31 times more common in men , the prevalence of hookah smoking in women was the same as men . in a meta - analysis in an adult population of west of iran in 25,990 subjects the prevalence smoking was 22.9 ( 20.6 - 25.2 ) and 0.6 ( 0.3 - 0.9 ) in men and women , respectively . our previous study also showed the prevalence of smoking of 12.7% ( 17.2% in male and 2.5 in female ) in a study population of 1435 individuals in north east iran . multivariate analysis showed that the factors associated with current smoking were male sex , advancing age , illiteracy , skilled occupation , low socio- economic status , and low bmi ( p < 0.001 ) . studies suggest age , sex and socioeconomic status , as well as close relationship with smokers in family or friend group , emotional stress and history of alcohol and illicit drug use as predictors of smoking . the reason of higher prevalence of wp smoking in younger age groups , females and unemployed population is perhaps because this type of smoking is a group smoking behavior . when we look at the data from different parts of world , a systematic review showed that the prevalence of current wp smoking among university students was high in the persian gulf region ( 6% ) , the united kingdom ( 8% ) , the united states ( 10% ) , syria ( 15% ) , lebanon ( 28% ) , and pakistan ( 33% ) . the prevalence of wp smoking among adults of different countries was as the follows : pakistan ( 6% ) , persian gulf region ( 4%-12% ) , australia ( 11% in arab speaking adults ) , syria ( 9%-12% ) , and lebanon ( 15% ) which was similar to the results of our study . the prevalence rate of wp smoking among indian population was reported between 2.6% and 50% . these data showed lower prevalence of wp smoking in the population of the mashhad compared to the arab countries but higher than some regions of india . the results also showed an increased frequency of respiratory symptoms and reduction of all values of pulmonary function tests in wp smokers compared to those of non - smoker subjects . the increased respiratory symptoms and reduction of pft values in wp smokers showed the effect of this type of smoking on the respiratory system . however , those pft values indicating the diameter of smaller airways ( mmef , mef50 and mef25 ) were less affected in smokers . there was no significant relationship between quantity and duration of wp smoking and respiratory symptoms which was perhaps due to subjective notion of the data of respiratory symptoms . however , there was a significant and negative correlation between all pft values and both quantity and duration of smoking . the relationship between respiratory symptoms and pft values with quantity and duration of smoking are further confirmation of the significant effect of wp smoking on the respiratory system . , also evaluated the effect of wp smoking on pft values . a more rapid effect of wp smoking on pft values as compared to cigarette smoking was also shown which may be due to higher nicotine content of wp smoke . confounding factors like age , job , race / ethnicity , place of living / working , active / passive smoking and other related variables ( other than wp ) , chemical exposure however , wp smokers and control groups were recruited from the same region of the city with similar age , job , race / ethnicity , place of living / working . therefore , it seems that these factors have no or minor effect on respiratory symptoms and pulmonary function tests in two groups . the comparison of prevalence of wp smoking among different socioeconomic populations , among people of different jobs and educational level and also rural versus urban population is important and needs to be evaluated further . in conclusion , the results of the present study showed 8.6% prevalence of wp smoking among population of the city of mashhad which was much higher in female as compared to male subjects .

peptide - centric techniques are gaining a lot of interest for the search of new protein biomarkers , surrogate endpoints , or markers for classification of diseases . typically , such techniques extensively use mass spectrometry ( ms ) for protein - expression profiling , because they promote the high - throughput quantitative characterization of a proteome . ms allows to separate peptides , present in a sample , according to their masses . it also provides a measure of abundance of the peptides . by comparing the protein abundances for different samples , differentially expressed proteins can be found . by analyzing the proteins , important information about , for example , we consider the problem of the quantification of overlapping peptides in a high - resolution matrix - assisted laser desorption and ionisation / time - of - flight ( maldi - tof ) mass spectrum ( ms ) . peptides are chains of amino acids and are composed of atoms of five chemical elements : carbon ( c ) , hydrogen ( h ) , nitrogen ( n ) , oxygen ( o ) , and sulphur ( s ) . because the chemical elements have different isotopes , peptides can have different isotopic variants , which differ with respect to their weights . for a peptide of a known chemical composition it follows that , in a singly charged high - resolution mass spectrum , a peptide produces a series of peaks that are separated by one mass unit ( dalton , da ) and that correspond to different isotopic variants of the peptide . a cluster of peaks observed in a mass spectrum can be produced by more than one peptide . this happens if two peptides differ in mass by at most a few mass units . the resulting observed joint spectrum is presented in figure 1(b ) , with a our key interest is to quantify the true underlying peptides , as displayed in figure 1(a ) . the quantification means a proper assessment of ( 1 ) the number of overlapping peptides ( components ) , ( 2 ) the monoisotopic masses of the peptides , that is , the masses of the isotopic variants that contain the most abundant isotopes of chemical elements constructing the peptides , and ( 3 ) the corresponding abundances of the peptides . developed a peak - picking algorithm by means of a wavelet function , combined with a greedy search to quantify the overlapping peptides . this method has two limitations : ( 1 ) often no unique solution can be found for the wavelet functions to fit to the peptide profiles , and ( 2 ) greedy search is often problematic in that it can either include noise peaks as peptide peaks or discard peptide peaks , depending on the fit to the wavelet functions . breen et al . suggested to model the isotopic distribution by a poisson approximation , which can also be used to identify overlapping peptides . this limits the application of the method , for example , by not allowing for the estimation of the mass locations of these peptides . moreover , the use of the summary statistics could result in severe inefficiency of the parameter estimates . especially when there is discrepancy between the true isotopic distribution and the poisson - approximated one , it would incur biased quantification for the parameters of interest , for example , the relative abundance(s ) of these peptides . the quantification of the overlapping peptides is a difficult problem , because the data may contain a very limited amount of information that can be used to distinguish between different configurations of the number , location , and abundances of the peptides , which might have led to the observed joint spectrum . a possible solution is to use prior information that could increase the information content of the data . to this aim , in the paper we propose to use a bayesian model to analyze high - resolution mass spectra with overlapping peptides . the model allows to use the prior information about , for example , the possible location of the peptides and about their isotopic distribution . it is important to note that the prior information reflects a prior knowledge that can be helpful in analyzing the data but does not necessarily need to exactly represent the data . in other words , the prior and the data can come from different types of proteins . this is because , in the bayesian framework , the posterior is ( combined ) information of the prior and the data , being closer to the source that contains more information . section 2 presents the shape representation of the ms data that we will consider for our modeling . in section 3 , we discuss the prior information that can be used in analyzing ms data with overlapping peptides . the details of the bayesian modeling approach are formulated in section 4 . in section 5 , results of an analysis of real - life data sets and a simulation study are presented . finally , concluding remarks are given in section 6 . for the data representation , used for the modeling approach , one way is to base the analysis on the summarized information of the ms data by , for example , using only one data point representing one observed peak . in principle , this implies a severe information reduction and may consequently cause biased estimation . in order to avoid the problem and retain all the information from the data , we consider the original setting of the ms data by means of the ( peak-)shape representation . to work with the shape representation , all mass coordinates and their corresponding intensities are considered . assume that , for a peak cluster observed in a mass spectrum , as shown in figure 1(b ) , we have got n intensity measurements , denoted by yj ( j = 1 , , the intensity at mass coordinate xj is a sum of intensity measurements of all the isotopic peaks of the peptides that are present at that coordinate . thus , for the example shown in figure 1 , yj = h1(xj ; 1 , s ) + h2(xj ; 2 , s ) + h3(xj ; 3 , s ) , where (x ; , s ) is a suitable function capturing the shape of the peak envelope like , for example , the normal function ( either cdf or pdf ) with q and hq denoting , respectively , a mass location and an overall abundance parameter for the qth overlapping peptide , and s , a dispersion parameter . the shape representation uses the full content of the ms data and therefore , in principle , allows for a more efficient inference . as mentioned in section 1 , the use of prior information could increase the information content of the ms data and allow for a more efficient quantification of overlapping peptides . the refseq database of the ncbi , available at http://www.ncbi.nlm.nih.gov/refseq/ , provides the monoisotopic masses of human peptides . when accessed on february 27 , 2008 , for the human proteome , the database contained amino acid sequences for 132,292 proteins . performing an in silico digest by trypsine resulted in 2,616,371 peptides with monoisotopic masses between 400 and 4000 da , with 306,427 unique atomic compositions . figure 2 presents the number of peptides with monoisotopic masses appearing in small intervals of 0.01 da around the mass range of 2000 da . this prior information can be quantified by using an appropriate prior distribution in modelling ms data . the ncbi data can also be used to extract information about possible forms of the isotopic distribution of peptides . note that , to compute the isotopic distribution , information about the chemical composition of the peptide is needed . however , one can predict the distribution by considering the variability of the distribution of peptides with a similar monoisotopic mass . to this aim , the isotopic distribution can be modeled by using a polynomial model , with the monoisotopic mass as a covariate [ 4 , 5 ] . the model is applied to the isotopic ratios , which are defined as follows . let p1 , p2 , p3 , and so forth denote the probability of occurrence of , respectively , the first ( monoisotopic ) , second , third , and so forth ( with respect to the increasing mass ) , isotopic variant of a peptide , given by the isotopic distribution . the common reference ratios are defined as follows : r1 = p1/p1 = 1 , r2 = p2/p1 , and so forth . thus , they give the probability of occurrence of an isotopic variant relative to the probability of the monoisotopic variant . the consecutive isotopic ratios are defined as follows : c1 = p1/p1 , c2 = p2/p1 , c3 = p3/p2 , and so forth . thus , they give the probability of occurrence of an isotopic variant relative to the previous variant . note that the two sets of ratios are equivalent , because rl = c1c2 cl . we used the approach by fitting the following model to the logarithms of the consecutive ratios of the isotopic distributions of peptides from the ncbi data set : ( 1)log cl=k=04k(m1000)k+ , where m is the monoisotopic mass of the peptide and ~ n(0 , l ) . model ( 1 ) was applied to the logarithms of ratios cl , and not to ratios rl , because the assumptions of the model were more appropriate for the former set of ratios . the estimated coefficients of the model for isotopic ratios l = 2 to 8 are shown in table 1 . they allow to infer the form of the isotopic distribution of a peptide with monoisotopic mass m. again , this prior information can be quantified by using an appropriate prior distribution in modelling ms data . due to the fact that rl = c1c2 cl , the overestimation of consecutive ratio c1 would result in the over - estimation of all the common reference ratios r1 to rl . to circumvent the problem , the ratios can be reparameterized . recall that pl is the probability of occurrence of the lthe isotopic variant and thus i=1pl = 1 , where l is the total number of isotopic variants , observed for a peptide . hence , instead of putting a prior on r2 , we use the equality relationship in ( 2 ) and define a prior for ( 1 p1)/p1 . this is because the increase of the other ratios , as shown in ( 2 ) , would result in the shrinkage of r2 ( given p1 ) . the prior for ( 1 p1)/p1 was obtained by fitting a model with monoisotopic mass m as a covariate to the isotopic distributions of the ncbi data set . a linear relationship between the logarithm of p1 and m , which can be expressed as p1 = + m + , it can then be transformed to the log - odds scale of p1 . after the transformation , the residuals thus , the model takes the form ( 3)log ( 1p1p1)=log [ 1exp ( +m)](+m)+ , where ~ n(0 , p1 ) . the resulting prior for the common - reference ( isotopic ) ratio rlq is lognormal , that is , ( 4)rlq~log - normal(i=1li,i=1li2 ) , where l=3, ,l , and r2q = ( 1 p1)/p1 l=3rlq . the prior for ( 1 p1)/p1 can be obtained from the estimates of the model shown in ( 3 ) . more specifically , ( 5)(1p1)p1~log - normal(p1,p12),where p1=log [ 1exp ( +m)](+m ) . in this section , we consider a model for the peak - shape representation of a mass spectrum . essentially , the model formulation is based on the definition in section 2 . for the observed intensity n ) , we assume the following model : ( 6)yj~n(e(yj),2 ) , with ( 7)e(yj)=f(h , r , m,s2,s)=q=1q l=1lhqrlq(xj;mq+(l1)s,s2 ) , where xj is the mass coordinate corresponding to intensity yj , m = ( m1 , m1 , , mq ) is a vector of monoisotopic masses of the q overlapping peptides , and s is the difference in mass locations between two neighboring isotopic peaks of the same peptide , assumed to be constant over all the isotopic peaks for all the overlapping peptides . in ( 7 ) , hq is the abundance of the qth overlapping peptide ( q = 1,2 , , parameter rlq is the lth common reference isotopic ratio for the qth peptide , and r = ( r11 , r21 , , rl1 ; r12 , r22 , , rl2 ; ; r1q , r2q , , rlq ) is a vector containing the isotopic ratios for all peptides . the function (x ; , s ) is a function of a chosen distribution , defined for the shape of the peaks . in this respect , either the difference of a cdf ( cumulative distribution function ) between two neighboring mass coordinates or a pdf ( probability distribution function ) can be used . to approximate the ( underlying ) continuous mass coordinate , we chose to use the cdf , which is also believed to be a more accurate approximation of area under the curve especially when the dispersion parameter s takes very small values . for a normal distribution function , the area under the curve between two neighboring mass coordinates is ( 8)(xj;mq+(l1)s,s2 ) = { (xj mq+(l1)s,s2 ) (xj1 mq+(l1)s,s2 ) if j2,(xj mq+(l1)s,s2 ) (0 mq+(l1 ) s,s2 ) if j=1 , with (xj | mq + ( l 1)s , s ) denoting the value of the normal cdf function with mean mq + ( l 1)s and variance s , calculated at xj . thus , an alternative is to approximate the shape by a function that accounts for an asymmetric shape . asymmetric laplace function can serve for this purpose . in this case , an extra shape parameter the shape function takes the following form : ( 9)(xj;mq+(l1)s,s, ) = { f(xj mq+(l1)s,s,)f(xj1 mq+(l1)s,s, ) if j2,f(xj mq+(l1 ) s,s,)f(0 mq+(l1)s,s, ) if j=1 , with f(xj | mq + ( l 1)s , s , ) denoting the value of cdf function of an asymmetric laplace distribution with mean mq + ( l 1)s and standard deviation s , calculated at xj , that is , ( 10)f(xj mq+(l1 ) s,s, ) = { 21+2exp [ 2s|xj(mq+(l1)s)| ] if xj < mq+(l1)s,111+2exp [ 2s|xj(mq+(l1)s)| ] if xjmq+(l1)s . for hq , , s , s , and , we use the following noninformative or weak - informative priors : ( 11)hq~n(0,1)i ( hq0 ) with ~(,),2~(,),s~n(0,106)i ( 0s0.5),s~n(1,1s ) with s~(,)i ( s1600),~u(0.01,0.99 ) , where , , * , * , * * , and * * are positive constants close to zero . to avoid numerical problems , the peak - width parameter s is constrained to be positive and not larger than 0.5 , because peaks observed in a spectrum are clearly separated from each other , with the width of a peak not larger than 1 da . parameter s reflects the average difference in molecular weight of the isotopes and is usually very close to one . thus , s is constrained to be close to one by setting a lower bound for the precision parameter s . the skewness parameter ( for the asymmetric laplace function ) is constrained to be smaller than one since the peak envelops are always right skewed ( at least in the maldi - tof data ) . the informative priors for the isotopic ratios are defined by ( 4 ) and ( 5 ) . this suggests that a suitable choice for the prior distribution of m , at a specific cluster for the possible mass range of m , may be a normal distribution . thus , the prior for the monoisotopic mass of the qth peptide is defined as follows : ( 12)mq~n(g,m2 ) , where g = 1 , , g , with g being the number of clusters , at which the monoisotopic masses are likely to occur . for instance , assuming that the monoisotopic mass of a certain peptide can vary in the mass range of [ 1997.5,2002.5 ] da , as shown in figure 2 , then g = 5 , as there are five clusters shown in the figure . mean g and variance m can be estimated from the ncbi data ( as illustrated in figure 3 ) . to consider all the g possible locations clusters , which can possibly contain the true value of the monoisotopic mass of the overlapping peptide , a bayesian model averaging approach can be considered . more specifically , g candidate models are fitted , each with a normal prior n(g , m ) , and g = 1 , , g. the resulting parameter estimates are a weighted sum of the g candidate models . this means that the point estimate of a parameter is obtained as the weighted average of the model - specific estimates ^g ( 13)^=g=1gwg^g , where wg is the weight of the gth model . based on the dic ( deviance information criterion ) of each model , wg can be computed as follows : ( 14)wg = exp ( (1/2)dicg)[g=1gexp ( ( 1/2)dicg ) ] , where dicg = dicg min g(dicg ) . the standard error can be computed as [ 6 , 7 ] ( 15)^()=g=1gwg^g()2+(^g^)2 , where ^g and ^g( ) are , respectively , the point estimate and the standard error for parameter in the gth candidate model . the conditional posterior distributions of hq and can be obtained analytically . on the other hand , because of nonlinearity , there are no analytical solutions for the conditional posterior distributions for s , , s , mq , and rlq . these distributions therefore need to be evaluated by numerical ( sampling ) methods , for example , a metropolis - hasting algorithm with acception - rejection rules . to investigate the performance of the proposed modeling approach , we applied the model to real - life and simulated data . the model was fitted by using the r package r2winbugs , built in r to automatically call the ( winbugs1.4 ) software , which allows to fit bayesian models . the model was applied to a data set of replicated joint mass spectra obtained for peptides of bovine cytochrome c from lc packings . it is a chain of 105 amino acids . a peptide mixture of tryptic digested bovine cytochrome c was purchased from lc packings and mixed with five internal standards from laser biolabs used for the calibration of the mass spectrometer . according to the data sheets of the suppliers , the mixture should contain 17 protein fragments . one part was enzymatically labeled with a stable o - isotope , with trypsine as a catalyst , while the other part remained unlabeled . in the first case , three units from the unlabeled part were mixed with one unit from the labeled part , which should result in the relative abundance of 1/3 . in the second case , three units from the labeled part were mixed with one unit from the unlabeled part , what should result in the relative abundance of 3/1 . in both cases , the composed mixture was automatically spotted six times on one stainless steel plate by a robot . the plate was processed by a 4800 maldi - tof / tof analyzer ( applied biosystems ) mass spectrometer and yielded six spectra for the 1/3 mixture and six spectra for the 3/1 mixture . in the o labeling strategy , the labeled peptide ideally receives two o - atoms at its carboxyl terminus , which leads to a four - da mass shift of the corresponding peptide peaks when analyzed by a mass spectrometer . thus , each spectrum can be treated as containing pairs ( q = 2 ) of overlapping peptides with the difference in the monoisotopic masses equal to four units of mass difference between two neighboring isotopic peaks , that is , m2 = m1 + 4s = m1 + 4 1.0015 ( see the notation of section 4 ) . for the analysis purposes , we chose two peptides with monoisotopic masses of 1456.66 da and 1584.76 da . for each peptide , we considered one spectrum for each of two different relative abundances ( 1/3 or 3/1 ) of the o and o labeled peptides . this results in the following four ( sub-)data sets : data 1 : m1 = 1456.66248 , h2/h1 = 3/1 , data 2 : m1 = 1456.66248 , h2/h1 = 1/3 , data 3 : m1 = 1584.75744 , h2/h1 = 3/1 , data 4 : m1 = 1584.75744 , h2/h1 = 1/3 . a graphical representation of data sets 1 and 2 is shown in figure 4 . tables 2 and 3 show the means and the standard errors for the parameters of model ( 6)-(7 ) , based on 100,000 samples , for the four data sets , using asymmetric laplace function defined by ( 9)-(10 ) . the parameters of main interest are the estimates of the monoisotopic masses of the two overlapping peptides , m1 and m2 ; the relative abundance h2/h1 . note that , usually , instead of the relative abundance , abundances h1 and h2 of the overlapping peptides would be of interest . thus , it is of interest to verify whether the proposed models correctly estimate the relative abundance . in this respect , it is important to mention that , despite the efforts to control the experiment , it appears that , for data sets 1 and 3 , the achieved value of relative abundance h2/h1 was about 2.4 , not 3 . the value was estimated by using models for the analysis of o - labeled mass spectra [ 9 , 10 ] . first , for all of the data sets , the monoisotopic mass of the second peptide m2 is estimated at the correct peak 5th peak . this may be due to the fact that in experiments , in which o - labeling is used , a part of peptide molecules from a labeled sample do not get a complete label [ 9 , 10 ] . this , in effect , leads to the labeled sample appearing in the spectrum to be less abundant due to the amount of molecules that were incompletely labeled . thus , the downward bias observed for the estimates of the relative abundance in tables 2 and 3 may actually reflect this effect . the point estimates for isotopic ratios r are , in general , very close to the true values . taking the precision measure of the standard errors into account , the parameters that describe the shape of the peaks , that is , s , , and s , are estimated consistently for different data sets . this indicates that the peak profiles , obtained from the maldi - tof experiments , are very similar . as a comparison , to the same data sets . it should be noted that by applying this approach , based on the summary statistics , that is , the stick representation for each observed peak , the monoisotopic masses of the two peptides are not estimable . the estimated 95% confidence intervals for this parameter for the four data sets are , respectively , ( 1.9229 , 2.5406 ) , ( 0.2797 , 0.3469 ) , ( 1.9206 , 2.5261 ) , and ( 0.2826 , 0.3479 ) . they show severe efficiency loss ( as the confidence intervals are much wider ) compared with the results presented in tables 2 and 3 . for illustration purposes and simplicity , the simulation study was based on the model with a normal - density shape function . let shift be the integer of the mass difference of the two overlapping peptides , and let tilt be the mass difference after the decimal point . as a result , the mass difference of the two overlapping peptides is equal to m2 m1 = shift + tilt , or in other words , m2 = m1 + shift + tilt . it may be difficult to quantify two overlapping peptides when the mass difference between two peptides is too small , that is , either shift or tilt is very small . thus , it is of interest to investigate different settings with combinations of the two parameters . in the simulation , we chose three sets of isotopic ratios : an average one ( denoted by a ) , obtained by a poisson approximation proposed by breen et al . ; the extremely small ratios ( denoted by e1 ) ; the extremely large ratios ( denoted by e2 ) within 20001 0.5 da mass range . sets e1 and e2 are the isotopic distributions with the second isotopic variant , p2 , being the least and most abundant among all the peptides around 2001 da from the ncbi data . the other parameters were chosen as follows : ( 16)m1=2000.90 , h1=10000,=10 , s=0.08 , s=1.0015 . for each of the settings , 100 simulated data sets with random noise were generated . figures 5 and 6 show the graphical representation of the 30 settings . it can be seen that settings 13 , 57 , 916 , 18 - 19 , and 21 are difficult settings , for which the location of the second overlapping peptide is not immediately obvious . in these settings , either the second ( overlapping ) peptide is much less abundant than the first peptide ( e.g. , setting 21 ) , or the mass difference between the two peptides is very small ( e.g. , setting 2 ) . the graphical representation of the summary statistics for the important parameters is shown in figures 79 . figure 7 shows , in general , unbiased estimates for parameter m1 , except only for a few of the difficult settings , which exhibit slight bias . the point estimates of the monoisotopic mass for the second overlapping peptide m2 , shown in figure 8 , correctly represent the true mass of the peptide , except only for settings 13 , 6 , 15 , and 18 . for these settings , the 95% credible intervals , computed based on the model averaging , are very wide . the wide credible intervals are an indication of settings , for which the quantification of the overlapping peptides is difficult . for these difficult settings , the 95% credible intervals for h2/h1 , as shown in figure 9 , contain zero and thus can be viewed as another indication that the second overlapping peptide is difficult to be found . for the remaining settings , even for some of those , for which the presence of the second peptide is not clear from the data , the bayesian model averaging approach is able to estimate the monoisotopic masses of the two overlapping peptides and to correctly quantify their relative abundance . a slight bias for the estimation of h2/h1 is only observed for setting 21 . figure 10 presents , as an example , the fit of the model to the observed spectra . the figure shows that the fitted spectra correspond to the observed spectra , even for the difficult setting ( setting 3 ) . as can be seen from table 4 , settings 1 to 20 are the settings for which the monoisotopic mass difference of the two overlapping peptides is at most around one da , that is , shift = 0 or 1 . these settings can be viewed as the more difficult ones regarding their relatively small difference in the monoisotopic mass , that is , m2 m1 . table 5 gives a summary of whether or not the model is able to produce correct estimates ( + indicating correct estimation and indicating wrong estimation ) for these settings , based on the simulation study . note that poor estimates are produced when the mass difference m2 m1 or the relative abundance h2/h1 is too small . in particular , table 5 indicates that , in general , when m2 m1 0.16 , the model produces the correct parameter estimates . in order to investigate the potential influence of the misspecification of the assumed number of overlapping peptides on the parameter estimates , the simulation was repeated for settings 10 and 26 , by assuming 3 overlapping peptides ( one more than the actual number ) . the estimates of the first two peptides ( ordered according to the estimated masses ) were quite similar to the ones obtained by assuming the correct number of overlapping peptides . the abundance parameter for the third peptide was estimated only between 0.07% and 3.66% of the abundance of the second peptide . this indicates that a third peptide may be non - existent and is very likely incurred merely by noise . the bic ( bayesian information criterion ) of the models with two and three overlapping peptides confirmed the nonexistence of the third overlapping peptides . for settings 10 and 26 , the bic for the model with two overlapping peptides were both smaller ( 7196.5 and 6567.6 , resp . ) than for the model with three peptides ( 7259.8 and 6602.1 , resp . ) . hence , the two simulation studies show that our modeling approach is robust to the misspecification of the number of overlapping peptides . moreover , the bic for models with different number of overlapping peptides gives an indication of the correct number of overlapping peptides , present in the data . the quantification of the overlapping peptides is a difficult problem , because there is often a limited amount of information available in the ms data . a possible solution is to use prior information that could increase the information content of the data . for this reason , in this paper , we have considered the use of a bayesian approach to analyze high - resolution mass spectra with overlapping peptides . as compared with the existing methods [ 1 , 2 ] , our modeling approach allows for the incorporation of prior information , which should lead to more precise estimates . moreover , it avoids a multistage analysis , which poses a difficulty in , for example , estimating precision of the obtained estimates . we have presented the model for the shape representation of a mass spectrum with overlapping peptides , fitted by using the bayesian model averaging approach . we have investigated the performance of the model with applications to real - life data sets and a simulation study . the application to the real - life data yielded , in general , estimates corresponding to the true parameter values . this may be due to incomplete labeling of peptide molecules [ 9 , 10 ] . the modeling approach was compared with one of the existing approaches , proposed by breen et al . , which showed efficiency loss for the parameter of interest . the inefficiency of the parameter estimates can bring about diagnostic problems , by causing false negatives , when applied to clinical diagnostics . in the simulation study , when applying the modeling approach , we observed , in general , unbiased estimation for the parameters , with either clear or unclear separation for the overlapping peptides in the simulated ms data . moreover , for the settings , for which the quantification of the second overlapping peptide was difficult , 95% credible intervals of the parameter estimates were wider and contained mostly the true values . this indicates that the width of the 95% credible intervals correctly quantifies the uncertainty of the parameter estimates . two extra simulations were performed and showed the robustness of the model to the misspecification of the number of overlapping peptides . the feasibility of the quantification of overlapping peptides depends on the mass difference of the peptides . when bayesian model averaging approach is applied , it produces unbiased estimates for the parameters related to the overlapping peptides , when the monoisotopic mass difference is at least 0.16 da , which is roughly a half of the width of an isotopic peak , observed in an maldi - tof mass spectrum . this indicates that the two overlapping peptides can be correctly quantified by using the bayesian model averaging approach when the mass difference of the two peptides is at least a half of the width of an isotopic peak . a smaller mass difference , that is , less than a half of the isotopic peak width , would suggest a complete overlap of the peptides and would make the quantification infeasible . in summary , the proposed modeling approach offers two advantages : it produces unbiased estimates for all settings that show clear or unclear separation of the overlapping peptides in the ms data ; the model uncertainty , measured by the 95% credible intervals of the parameters , gives an indication of the separability of the overlapping peptides . although the method is focused on the application of singly charged maldi - tof mass spectrum , it can be modified to apply also for , for example , the doubly charged mass spectrum with the modification of the expression for the mean structure of the model and the prior distributions for the corresponding parameters . moreover , the proposed modeling approach , assuming unknown masses ( sequences ) of the overlapping peptides , can be modified for the application , in which the masses are known . in such case , the masses of the peptides in the model can be fixed with known values and the model simplifies . it should be noted that the validity of this approach is based on a proper - preprocessing procedure ( for details of preprocessing , refer to vaikenborg et al . ) . more specifically , it assumes that a cluster of peptide peaks is correctly found after noise filtering . this implies that , if a part of the isotopic peaks of a cluster is treated as noise generated and discarded , the method would yield biased estimation . it is also worth noting that , in the analysis , the number of overlapping peptides was assumed to be known . such estimation may be difficult by using a bayesian approach since little prior information can assist the analysis . identifying the number of overlapping peptides can be viewed as a problem of identifying the number of components of a mixture of distributions by applying a likelihood - based testing approach , or by performing a forward model selection approach . the latter approach can be done by fitting models with sequentially increasing number of components , and then by selecting the model which shows the best fit , depending on , for example , the bayesian information criterion . the feasibility of such an approach was observed from the simulation studies . to check its validity in real applications

in a high - resolution maldi - tof mass spectrum , a peptide produces multiple peaks , corresponding to the isotopic variants of the molecules . an overlap occurs when two peptides appear in the vicinity of the mass coordinate , resulting in the difficulty of quantifying the relative abundance and the exact masses of these peptides . to address the problem , two factors need to be considered : ( 1 ) the variability pertaining to the abundances of the isotopic variants ( 2 ) extra information content needed to supplement the information contained in data . we propose a bayesian model for the incorporation of prior information . such information exists , for example , for the distribution of the masses of peptides and the abundances of the isotopic variants . the model we develop allows for the correct estimation of the parameters of interest . the validity of the modeling approach is verified by a real - life case study from a controlled mass spectrometry experiment and by a simulation study .

1. Introduction 2. Shape Representation of a Mass Spectrum 3. Available Prior Information 4. Bayesian Model Formulation 5. Data analysis 6. Discussion and Conclusions

peptide - centric techniques are gaining a lot of interest for the search of new protein biomarkers , surrogate endpoints , or markers for classification of diseases . by analyzing the proteins , important information about , for example , we consider the problem of the quantification of overlapping peptides in a high - resolution matrix - assisted laser desorption and ionisation / time - of - flight ( maldi - tof ) mass spectrum ( ms ) . for a peptide of a known chemical composition it follows that , in a singly charged high - resolution mass spectrum , a peptide produces a series of peaks that are separated by one mass unit ( dalton , da ) and that correspond to different isotopic variants of the peptide . the quantification means a proper assessment of ( 1 ) the number of overlapping peptides ( components ) , ( 2 ) the monoisotopic masses of the peptides , that is , the masses of the isotopic variants that contain the most abundant isotopes of chemical elements constructing the peptides , and ( 3 ) the corresponding abundances of the peptides . this method has two limitations : ( 1 ) often no unique solution can be found for the wavelet functions to fit to the peptide profiles , and ( 2 ) greedy search is often problematic in that it can either include noise peaks as peptide peaks or discard peptide peaks , depending on the fit to the wavelet functions . suggested to model the isotopic distribution by a poisson approximation , which can also be used to identify overlapping peptides . this limits the application of the method , for example , by not allowing for the estimation of the mass locations of these peptides . especially when there is discrepancy between the true isotopic distribution and the poisson - approximated one , it would incur biased quantification for the parameters of interest , for example , the relative abundance(s ) of these peptides . the quantification of the overlapping peptides is a difficult problem , because the data may contain a very limited amount of information that can be used to distinguish between different configurations of the number , location , and abundances of the peptides , which might have led to the observed joint spectrum . a possible solution is to use prior information that could increase the information content of the data . to this aim , in the paper we propose to use a bayesian model to analyze high - resolution mass spectra with overlapping peptides . the model allows to use the prior information about , for example , the possible location of the peptides and about their isotopic distribution . this is because , in the bayesian framework , the posterior is ( combined ) information of the prior and the data , being closer to the source that contains more information . the details of the bayesian modeling approach are formulated in section 4 . in section 5 , results of an analysis of real - life data sets and a simulation study are presented . for the data representation , used for the modeling approach , one way is to base the analysis on the summarized information of the ms data by , for example , using only one data point representing one observed peak . in order to avoid the problem and retain all the information from the data , we consider the original setting of the ms data by means of the ( peak-)shape representation . assume that , for a peak cluster observed in a mass spectrum , as shown in figure 1(b ) , we have got n intensity measurements , denoted by yj ( j = 1 , , the intensity at mass coordinate xj is a sum of intensity measurements of all the isotopic peaks of the peptides that are present at that coordinate . thus , for the example shown in figure 1 , yj = h1(xj ; 1 , s ) + h2(xj ; 2 , s ) + h3(xj ; 3 , s ) , where (x ; , s ) is a suitable function capturing the shape of the peak envelope like , for example , the normal function ( either cdf or pdf ) with q and hq denoting , respectively , a mass location and an overall abundance parameter for the qth overlapping peptide , and s , a dispersion parameter . as mentioned in section 1 , the use of prior information could increase the information content of the ms data and allow for a more efficient quantification of overlapping peptides . the ncbi data can also be used to extract information about possible forms of the isotopic distribution of peptides . note that , to compute the isotopic distribution , information about the chemical composition of the peptide is needed . however , one can predict the distribution by considering the variability of the distribution of peptides with a similar monoisotopic mass . the model is applied to the isotopic ratios , which are defined as follows . let p1 , p2 , p3 , and so forth denote the probability of occurrence of , respectively , the first ( monoisotopic ) , second , third , and so forth ( with respect to the increasing mass ) , isotopic variant of a peptide , given by the isotopic distribution . thus , they give the probability of occurrence of an isotopic variant relative to the probability of the monoisotopic variant . we used the approach by fitting the following model to the logarithms of the consecutive ratios of the isotopic distributions of peptides from the ncbi data set : ( 1)log cl=k=04k(m1000)k+ , where m is the monoisotopic mass of the peptide and ~ n(0 , l ) . model ( 1 ) was applied to the logarithms of ratios cl , and not to ratios rl , because the assumptions of the model were more appropriate for the former set of ratios . the estimated coefficients of the model for isotopic ratios l = 2 to 8 are shown in table 1 . they allow to infer the form of the isotopic distribution of a peptide with monoisotopic mass m. again , this prior information can be quantified by using an appropriate prior distribution in modelling ms data . due to the fact that rl = c1c2 cl , the overestimation of consecutive ratio c1 would result in the over - estimation of all the common reference ratios r1 to rl . to circumvent the problem , the ratios can be reparameterized . recall that pl is the probability of occurrence of the lthe isotopic variant and thus i=1pl = 1 , where l is the total number of isotopic variants , observed for a peptide . hence , instead of putting a prior on r2 , we use the equality relationship in ( 2 ) and define a prior for ( 1 p1)/p1 . this is because the increase of the other ratios , as shown in ( 2 ) , would result in the shrinkage of r2 ( given p1 ) . the prior for ( 1 p1)/p1 was obtained by fitting a model with monoisotopic mass m as a covariate to the isotopic distributions of the ncbi data set . the resulting prior for the common - reference ( isotopic ) ratio rlq is lognormal , that is , ( 4)rlq~log - normal(i=1li,i=1li2 ) , where l=3, ,l , and r2q = ( 1 p1)/p1 l=3rlq . the prior for ( 1 p1)/p1 can be obtained from the estimates of the model shown in ( 3 ) . in this section , we consider a model for the peak - shape representation of a mass spectrum . for the observed intensity n ) , we assume the following model : ( 6)yj~n(e(yj),2 ) , with ( 7)e(yj)=f(h , r , m,s2,s)=q=1q l=1lhqrlq(xj;mq+(l1)s,s2 ) , where xj is the mass coordinate corresponding to intensity yj , m = ( m1 , m1 , , mq ) is a vector of monoisotopic masses of the q overlapping peptides , and s is the difference in mass locations between two neighboring isotopic peaks of the same peptide , assumed to be constant over all the isotopic peaks for all the overlapping peptides . in ( 7 ) , hq is the abundance of the qth overlapping peptide ( q = 1,2 , , parameter rlq is the lth common reference isotopic ratio for the qth peptide , and r = ( r11 , r21 , , rl1 ; r12 , r22 , , rl2 ; ; r1q , r2q , , rlq ) is a vector containing the isotopic ratios for all peptides . the function (x ; , s ) is a function of a chosen distribution , defined for the shape of the peaks . to approximate the ( underlying ) continuous mass coordinate , we chose to use the cdf , which is also believed to be a more accurate approximation of area under the curve especially when the dispersion parameter s takes very small values . to avoid numerical problems , the peak - width parameter s is constrained to be positive and not larger than 0.5 , because peaks observed in a spectrum are clearly separated from each other , with the width of a peak not larger than 1 da . the skewness parameter ( for the asymmetric laplace function ) is constrained to be smaller than one since the peak envelops are always right skewed ( at least in the maldi - tof data ) . thus , the prior for the monoisotopic mass of the qth peptide is defined as follows : ( 12)mq~n(g,m2 ) , where g = 1 , , g , with g being the number of clusters , at which the monoisotopic masses are likely to occur . for instance , assuming that the monoisotopic mass of a certain peptide can vary in the mass range of [ 1997.5,2002.5 ] da , as shown in figure 2 , then g = 5 , as there are five clusters shown in the figure . to consider all the g possible locations clusters , which can possibly contain the true value of the monoisotopic mass of the overlapping peptide , a bayesian model averaging approach can be considered . these distributions therefore need to be evaluated by numerical ( sampling ) methods , for example , a metropolis - hasting algorithm with acception - rejection rules . to investigate the performance of the proposed modeling approach , we applied the model to real - life and simulated data . a peptide mixture of tryptic digested bovine cytochrome c was purchased from lc packings and mixed with five internal standards from laser biolabs used for the calibration of the mass spectrometer . according to the data sheets of the suppliers , the mixture should contain 17 protein fragments . in the first case , three units from the unlabeled part were mixed with one unit from the labeled part , which should result in the relative abundance of 1/3 . in the second case , three units from the labeled part were mixed with one unit from the unlabeled part , what should result in the relative abundance of 3/1 . the plate was processed by a 4800 maldi - tof / tof analyzer ( applied biosystems ) mass spectrometer and yielded six spectra for the 1/3 mixture and six spectra for the 3/1 mixture . in the o labeling strategy , the labeled peptide ideally receives two o - atoms at its carboxyl terminus , which leads to a four - da mass shift of the corresponding peptide peaks when analyzed by a mass spectrometer . thus , each spectrum can be treated as containing pairs ( q = 2 ) of overlapping peptides with the difference in the monoisotopic masses equal to four units of mass difference between two neighboring isotopic peaks , that is , m2 = m1 + 4s = m1 + 4 1.0015 ( see the notation of section 4 ) . for the analysis purposes , we chose two peptides with monoisotopic masses of 1456.66 da and 1584.76 da . tables 2 and 3 show the means and the standard errors for the parameters of model ( 6)-(7 ) , based on 100,000 samples , for the four data sets , using asymmetric laplace function defined by ( 9)-(10 ) . the parameters of main interest are the estimates of the monoisotopic masses of the two overlapping peptides , m1 and m2 ; the relative abundance h2/h1 . note that , usually , instead of the relative abundance , abundances h1 and h2 of the overlapping peptides would be of interest . thus , it is of interest to verify whether the proposed models correctly estimate the relative abundance . first , for all of the data sets , the monoisotopic mass of the second peptide m2 is estimated at the correct peak 5th peak . this may be due to the fact that in experiments , in which o - labeling is used , a part of peptide molecules from a labeled sample do not get a complete label [ 9 , 10 ] . this , in effect , leads to the labeled sample appearing in the spectrum to be less abundant due to the amount of molecules that were incompletely labeled . thus , the downward bias observed for the estimates of the relative abundance in tables 2 and 3 may actually reflect this effect . taking the precision measure of the standard errors into account , the parameters that describe the shape of the peaks , that is , s , , and s , are estimated consistently for different data sets . it should be noted that by applying this approach , based on the summary statistics , that is , the stick representation for each observed peak , the monoisotopic masses of the two peptides are not estimable . let shift be the integer of the mass difference of the two overlapping peptides , and let tilt be the mass difference after the decimal point . as a result , the mass difference of the two overlapping peptides is equal to m2 m1 = shift + tilt , or in other words , m2 = m1 + shift + tilt . it may be difficult to quantify two overlapping peptides when the mass difference between two peptides is too small , that is , either shift or tilt is very small . the graphical representation of the summary statistics for the important parameters is shown in figures 79 . for the remaining settings , even for some of those , for which the presence of the second peptide is not clear from the data , the bayesian model averaging approach is able to estimate the monoisotopic masses of the two overlapping peptides and to correctly quantify their relative abundance . figure 10 presents , as an example , the fit of the model to the observed spectra . table 5 gives a summary of whether or not the model is able to produce correct estimates ( + indicating correct estimation and indicating wrong estimation ) for these settings , based on the simulation study . note that poor estimates are produced when the mass difference m2 m1 or the relative abundance h2/h1 is too small . the estimates of the first two peptides ( ordered according to the estimated masses ) were quite similar to the ones obtained by assuming the correct number of overlapping peptides . hence , the two simulation studies show that our modeling approach is robust to the misspecification of the number of overlapping peptides . moreover , the bic for models with different number of overlapping peptides gives an indication of the correct number of overlapping peptides , present in the data . the quantification of the overlapping peptides is a difficult problem , because there is often a limited amount of information available in the ms data . a possible solution is to use prior information that could increase the information content of the data . for this reason , in this paper , we have considered the use of a bayesian approach to analyze high - resolution mass spectra with overlapping peptides . as compared with the existing methods [ 1 , 2 ] , our modeling approach allows for the incorporation of prior information , which should lead to more precise estimates . moreover , it avoids a multistage analysis , which poses a difficulty in , for example , estimating precision of the obtained estimates . we have presented the model for the shape representation of a mass spectrum with overlapping peptides , fitted by using the bayesian model averaging approach . we have investigated the performance of the model with applications to real - life data sets and a simulation study . the application to the real - life data yielded , in general , estimates corresponding to the true parameter values . the modeling approach was compared with one of the existing approaches , proposed by breen et al . , which showed efficiency loss for the parameter of interest . in the simulation study , when applying the modeling approach , we observed , in general , unbiased estimation for the parameters , with either clear or unclear separation for the overlapping peptides in the simulated ms data . moreover , for the settings , for which the quantification of the second overlapping peptide was difficult , 95% credible intervals of the parameter estimates were wider and contained mostly the true values . two extra simulations were performed and showed the robustness of the model to the misspecification of the number of overlapping peptides . when bayesian model averaging approach is applied , it produces unbiased estimates for the parameters related to the overlapping peptides , when the monoisotopic mass difference is at least 0.16 da , which is roughly a half of the width of an isotopic peak , observed in an maldi - tof mass spectrum . this indicates that the two overlapping peptides can be correctly quantified by using the bayesian model averaging approach when the mass difference of the two peptides is at least a half of the width of an isotopic peak . a smaller mass difference , that is , less than a half of the isotopic peak width , would suggest a complete overlap of the peptides and would make the quantification infeasible . in summary , the proposed modeling approach offers two advantages : it produces unbiased estimates for all settings that show clear or unclear separation of the overlapping peptides in the ms data ; the model uncertainty , measured by the 95% credible intervals of the parameters , gives an indication of the separability of the overlapping peptides . although the method is focused on the application of singly charged maldi - tof mass spectrum , it can be modified to apply also for , for example , the doubly charged mass spectrum with the modification of the expression for the mean structure of the model and the prior distributions for the corresponding parameters . moreover , the proposed modeling approach , assuming unknown masses ( sequences ) of the overlapping peptides , can be modified for the application , in which the masses are known . in such case , the masses of the peptides in the model can be fixed with known values and the model simplifies . it should be noted that the validity of this approach is based on a proper - preprocessing procedure ( for details of preprocessing , refer to vaikenborg et al . ) this implies that , if a part of the isotopic peaks of a cluster is treated as noise generated and discarded , the method would yield biased estimation . the latter approach can be done by fitting models with sequentially increasing number of components , and then by selecting the model which shows the best fit , depending on , for example , the bayesian information criterion .

modular design is used for engineering complex devices in electronics , mechanics , nanotechnology and other fields . recently , biologists have begun to exploit modular parts as a way to build novel synthetic biological systems . many types of parts are required to implement diverse structural , binding and catalytic functions . here , we focus on the -helical coiled coil , which is a protein - interaction domain highly suitable for inclusion in the growing molecular parts toolkit . coiled coils are prevalent in native proteins and are useful interaction motifs due to their capacity to encode complex interaction patterns in a short protein sequence . coiled coils form a rod - like structure composed of -helices that wrap around each other with a superhelical twist . coiled - coil sequences have a characteristic motif commonly referred to as a heptad repeat , denoted as [ abcdefg]n . the a and d positions are dominated by hydrophobic residues , and are found at the core of the structure ; we refer to a and d positions as core positions in this work . in coiled - coil dimers , e and g positions are typically occupied by charged residues and form the boundary between the core and the surface of the coiled coil . the b , c , and f positions are located on the surface and are most often polar or charged . in coiled - coil notation , a prime indicates a residue on an opposing chain . for example , positions e and g are proximal and can form interhelical salt bridges in parallel coiled - coil dimers , whereas e / e and g / g pairs can interact in antiparallel coiled coils . the relationship between coiled - coil sequence and structure is incompletely understood , even after decades of study of native , mutant and de novo - designed coiled coils . this is partly due to the many topologies accessible to coiled - coil sequences . for example , coiled coils can fold into dimers , trimers , tetramers , and even higher - order oligomers . lastly , the orientations ( parallel vs antiparallel ) and axial alignments of the constituent helices can vary . the general problem of predicting detailed coiled - coil structure from sequence has not been solved , although progress has been made developing methods to predict oligomerization state from sequence , and in particular to discriminate parallel dimers from parallel trimers . they have been applied to the design of artificial transcription factors and used to manipulate cell - signaling pathways . they have also been used to build engineered crystals , and to modulate the charge - transfer properties of electronic devices . in many of these studies , modulated the conductance between two monolayers of gold using coiled - coil dimers and showed that parallel and antiparallel coiled coils differentially impacted the electronic properties of the system . coiled coils of different orientations have net molecular dipoles of different magnitude and direction , and can thereby confer different electronic properties . recently , an exciting strategy was developed to design polypeptide polyhedra based around coiled - coil dimers . used a set of parallel and antiparallel dimeric coiled coils as building blocks to engineer a nanoscale single - chain tetrahedron with coiled coils forming each edge . the design strategy involved concatenating a series of 12 sequence segments coding for different coiled - coil helices into a single chain . the artificial protein sequence was designed such that folding of the chain , driven by pairing each coiled - coil helix with its appropriate intrachain partner helix , would generate a prespecified three - dimensional structure . a crucial aspect of the design strategy was the use of coiled - coil components that were orthogonal to one another , i.e. , that had low potential to cross - interact . the designed tetrahedron was based on 4 parallel and 2 antiparallel coiled - coil dimers previously reported in the literature . as part of their work , the authors computed the number and type of coiled coils that would be needed to build different polyhedra . for example , of the 6 polyhedra considered by the authors , only an octahedron could be built without using antiparallel dimers . despite the clear benefits of having reagents that allow manipulation of orientation in a molecular assembly , most designed coiled coils adopt a parallel orientation . very few antiparallel coiled - coil dimers have been characterized or designed , and none have been tested for orthogonality . in contrast , dozens of native and synthetic parallel coiled coils have been tested for interactions and orthogonality . there are currently two databases maintained for designed coiled coils , the synzip database , and the pcomp database . currently 96% of the synzip sequences and 63% of the sequences in the pcomp database form parallel dimers . between these two databases , the biophysical properties of only one antiparallel coiled coil ( a heterodimer ) are reported . thus , designing sets of orthogonal antiparallel homodimers would expand the available coiled - coil parts in a meaningful way . because coiled - coil sequences can encode many different structures , negative design to destabilize undesired states is crucial when making peptides intended to assemble into a single topology . several negative design strategies have been used in the past that involve placing charged , beta - branched or polar asparagine residues such that they form unfavorable interactions in undesired states . a recent study relied on all three of these strategies to design a parallel homodimer , homotrimer , and homotetramer . the orientations of the helices were engineered by placing lysines at all e positions and glutamates at all g positions , which leads to electrostatic attraction in parallel assemblies but repulsion in antiparallel states . oligomerization states were specified by the differential placement of beta - branched residues in core a and d heptad positions , a strategy first discovered by harbury et al . , and by the use of asparagine residues to specify dimer formation , which was originally reported by lumb and kim . including charged residues in core a or d positions has also been observed to destabilize nondimer states . designing sets of orthogonal coiled - coil homodimers presents additional challenges related to encoding interaction specificity . this is due to the increased number of undesired , off - target states associated with forming hetero - oligomeric species . the number of possible hetero species increases dramatically as the number of designed orthogonal coiled coils grows , such that three orthogonal antiparallel homodimers have the potential to form six possible off - target parallel or antiparallel heterodimers ; other undesired structures are also possible . to design sets of orthogonal antiparallel coiled - coil dimers , we therefore turned to computational methods to keep track of the numerous desired and undesired structures in this design problem . despite the many successes of structure - based approaches for modeling and designing protein the computational costs of modeling each structure can be large , and current optimization functions used with structure - based models do not provide efficient routines for optimizing one set of states while simultaneously destabilizing many off - target states . the multistate design framework classy addresses these issues by carrying out design in protein sequence space , without the need to explicitly model all protein structures . by using a transformation of structure - based models to sequence - based models , classy addresses both the search and scoring problems of multistate design , and the method has previously been applied to design parallel coiled coils specific for binding to a target in preference to closely related off - target proteins . this paper describes our work applying classy in conjunction with the dfire statistical potential to the de novo design of sets of coiled coils consisting of three orthogonal antiparallel homodimers . we designed two sets of three proteins , and used biophysical techniques to determine the oligomerization state , helix orientation and thermal stability of structures formed by the designed sequences . some designed peptides formed trimers or higher - order assemblies , but we identified 3 peptides ( aph2 , aph3 , and aph4 ) that formed orthogonal antiparallel homodimers . in addition , we showed that these proteins homodimerize in preference to binding to aph , a previously reported antiparallel homodimer . thus , we provide evidence for four sequences that preferentially form antiparallel homodimers that can be used for protein engineering applications . as described in detail below , side chains were modeled on idealized coiled - coil backbones using rosetta and scored using difire * , a modified version of the dfire statistical potential . to construct libraries of parallel and antiparallel backbones , a set of 214 canonical coiled coils ( i.e. , left - handed coiled coils with uninterrupted heptad registers , abcdefg ) with 2 helices each longer then 20 residues were culled from the cc+ database as of august 18 , 2010 . within the parallel and antiparallel sets , seven geometrical parameters defined by crick to describe a coiled coil were fit to each structure using the cccp structure fitter . this set of backbones was then filtered to give 25 parallel and 23 antiparallel backbones with parameters within one standard deviation of the average value for each parameter . idealized versions of these 48 structures were generated using the cccp structure generator . coiled - coil sequences to be scored were modeled on each idealized backbone using the fixed - backbone packing protocol of rosetta 3.2 . the soft - potential flag and expansion of the first and second dihedral angles of the rotamer library were used , along with the side - chain minimization flag . all surface heptad positions ( b , c , and f ) were modeled as alanine . structures were scored using a modified version of dfire , a distance - dependent pairwise statistical potential based on the distance - scaled , finite ideal - gas reference state . the cutoff distance , rcut , was set to 5.8 , and interatomic energies were evaluated only between residues on opposite helices in the coiled coil . we refer to this modified version of dfire as dfire*. dfire * outperforms dfire on certain interaction prediction tests for parallel coiled coils ( v. potapov , personal communication ) . the lowest dfire * energy for each sequence over all 25 parallel or 23 antiparallel backbones was used as the parallel or antiparallel energy , respectively . two cluster - expanded functions based on dfire * were derived to score the propensity of sequences to form antiparallel and parallel coiled coils . for an outline of the protocol , see figure s1 , and for an in - depth discussion of performing cluster - expansion calculations using clever 1.0 see negron et al . in the present application , the cluster - expanded models express energy as a sum of terms corresponding to weights for single amino acids at a , d , e , and g heptad positions and pairs of amino acids at these positions . as in grigroyan et al . , only pairs of positions within the same or adjoining heptads were considered . the training data consisted of dfire * energies for a central two - heptad unit within a six - heptad structure , calculated using the scoring protocol described in the previous section for 30 000 sequences . another 8000 sequences , nonoverlapping with the training set , were generated in the same way to be used as a test set . training sequences were 42 residues ( six heptads ) long and composed of a repeating two - heptad unit . training sequences were generated randomly but with heptad - specific single - residue frequencies matching those of known coiled - coil dimers ( both parallel and antiparallel ) . once determined , cluster expansion ( ce ) weights can be used to score antiparallel and parallel coiled - coil dimers of arbitrary length . examples of parallel and antiparallel coiled coils were obtained from the filtered cc+ set and further filtered to exclude those shorter than 28 residues and those that contained non - natural amino acids . for certain sequences , three residues at the terminal ends of the two chains were removed so that the two chains fully overlapped in both the parallel and antiparallel orientations ; i.e. , the coiled coils that were modeled were blunt - ended in both orientations . the final orientation test set contained 30 antiparallel complexes ( composed of 285 heptads ) and 48 parallel complexes ( composed of 547 heptads ) . pdb ids with chain and residue numbers for the orientation test set are given in table s2 . a detailed description of how integer linear programming ( ilp ) can be applied as part of the classy multistate design method is given in negron et al . in this work , the objective function for ilp was the total energy ( e ) , given by the sum of the energies of three antiparallel homodimers ( e = e + e + e ) . all energies were obtained from either the antiparallel or parallel cluster - expanded models . the ilp solver of the ibm ilog cplex optimizer was used to minimize this objective function under a set of constraints . the constraints included energy gaps to off - target dimer states ( see figure 2a , b ) , as well as constraints on the number of polar residues allowed at a and d heptad positions ( maximum of 2 charged residues at a , and 1 lys residue at d per design sequence ) . a constraint was included on the energy gap between every antiparallel homodimer and every off - target state ( of those types considered in the calculation ) that the constituent peptide could participate in . e > , where e represents the energy of a single off - target state , of which there were several as shown in figure 2 . e represents the energy of a single antiparallel homodimer , i.e. , e , e , or e. is a user - defined specificity gap , and different values of were used as shown in figure 2c , d . a solution , consisting of three sequences , was obtained for each . two sets of design calculations were done , one including glutamate as an option at a positions ( sequence space 1 ) and one not allowing glutamate ( sequence space 2 ) . one solution was chosen manually for experimental testing from each calculation , based on predicted stabilities and specificities . synthetic genes encoding computationally designed coiled - coil sequences , and control sequences , were constructed by pcr amplification from two 258-base pair oligonucleotides and one 157-base pair oligonucleotide ( gblocks ) purchased from integrated dna technologies . low - frequency e. coli codons selected by dnaworks were manually switched with synonymous high - frequency codons . following amplification with primers to provide appropriate vector overlap , gibson cloning ( new england biolabs ) was used to clone synthetic genes into pentr vectors . the products of the gibson reactions were then recombined into pmal ( new england biolabs ) destination vectors using lr clonase ii ( invitrogen ) in 2.5 l reactions . pmal encodes mbp followed by a tev protease cleavage site ( not used ) , a gateway linker region , and a c - terminal his6 tag . the lr clonase ii reaction inserted the synthetic gene between the gateway linker region and the c - terminal his6 site . the pmal vectors were transformed into bl21 ( de3 ) cells ( agilent ) . bl21 cells were grown in liquid lb cultures ( 1 l ) at 37 c to an od600 of 0.40.6 . protein expression was then induced with 1 mm iptg for 4.55.5 h. cells were pelleted , resuspended , and then lysed by sonication . mbp - fused proteins were purified from the supernatant using ninta ( qiagen ) column purification under native conditions . the elution buffer contained 0.3 m imidazole , 20 mm tris base , and 0.5 m nacl at a ph of 7.91 . the approximate sizes of mbp - fused proteins were confirmed using protein gels with size ladders . a second set of constructs was made by amplifying from gblocks using primers encoding a cysteine either at the n - terminal or c - terminal end , as well as flanking bamhi / xhoi restriction sites . the genes were cloned by means of the bamhi / xhoi restriction sites into a modified version of the pdest17 vector . this vector encodes an n - terminal his6 tag as well as a geskeykkgsgs linker shown to improve the solubility of recombinant proteins . cysteine - containing constructs were expressed in rp3098 cells grown , induced and lysed as described above for bl21 . however , these proteins were purified from the supernatant using ninta ( qiagen ) under denaturing conditions . the elution buffer consisted of 60% acetonitrile ( hplc - grade ) and 0.1% trifluoroacetic acid ( tfa ) . ni - affinity purification was followed by reverse - phase hplc with a water / acetonitrile gradient in the presence of 0.1% tfa . concentrations of all constructs were determined using the edelhoch method , measuring uv absorbance of aromatic residues at 280 nm in 6 m guanidinium chloride . proteins were dialyzed with three changes of reference buffer ( 40 mm tris base , 150 mm nacl , ph 7.91 ) over the course of 24 h. sedimentation equilibrium runs were performed with a beckman xl - i analytical ultracentrifuge using an an-50 ti rotor at 20 c . constructs fused to mbp were spun at concentrations ranging from 4 to 40 m at 10 200 , 16 300 and 20 400 rpm . these spins were monitored either using uv absorbance at 280 nm , or with interference optics when multiple mbp constructs were mixed . for protein constructs containing cysteine , 1 mm tcep was added to the reference buffer prior to dialysis . these constructs were spun at concentrations of 20 and 40 m at 28 000 , 35 000 and 42 000 rpm and monitored using interference optics . for each speed , equilibrium was confirmed by negligible differences between the sample distributions in the cells over sequential scans . data sets for each construct were globally fit to a model for a single ideal species using the program sedphat . values for v - bar , solvent density , and viscosity were obtained from sednterp . cysteine - containing proteins in varying states of oxidation / reduction ( depending on construct ) were placed in a redox buffer ( 500 m reduced glutathione , 250 m oxidized glutathione , 40 mm tris base , 150 mm nacl , ph 7.91 ) at 20 m of each protein at room temperature . redox reactions were quenched at different time points using a drop of 6 m hydrochloric acid . the products of the reactions were then run on an analytical vydac c18 reverse - phase column with absorbance monitored at 220 nm using a linear water / acetonitrile gradient containing 0.1% tfa . equilibrium was confirmed by monitoring changes in hplc profiles as a function of time . retention times for the reduced proteins and for the oxidized states for each of the 6 cysteine - containing proteins were assigned by hplc analysis of the constructs in tbs ( 40 mm tris base , 150 mm nacl , ph 7.91 ) alone , in tbs with tcep added for an incubation time of 30 min ( to generate the fully reduced species ) , or in tbs solution left exposed to air and stirring overnight ( to generate the fully oxidized species ) . glutathione adduct peaks were assigned by the appearance , following incubation in redox buffer , of a peak with a retention time not consistent with the reduced or oxidized states of each of the six individual protein constructs . antiparallel peaks were assigned by monitoring the appearance of a peak that was only observed after mixing two constructs that encoded the same coiled coil , but with cysteine residues at opposing ends . cd spectra and thermal - denaturation curves were measured on an aviv 400 cd spectrometer . peptides were equilibrated in pbs buffer ( 137 mm nacl , 2.7 mm kcl , 10 mm na2hpo4 , 2 mm kh2po4 , ph 7.4 ) containing 1 mm of dithiothreitol ( dtt ) at 25 c for at least 1.5 h prior to measurement . measurements were made in a 1 mm quartz cuvette at a protein concentration of 20 m using the n - terminal cysteine - containing constructs . , three wavelength scans were measured and then averaged . for each wavelength scan , data were collected from 190 to 280 nm , in 1 nm steps , averaging for 5 s at each wavelength . thermal denaturation curves were generated by monitoring 222 using a 30 s averaging time , 3 min equilibration time , and temperature increments of 2.5 c from 0 to 98 c . melting temperatures , tm , were obtained by fitting the change of the cd signal over the change in temperature . the fractional helicity of each design was estimated by substituting the experimentally measured 222 into the equation ( 222 3000)/(36 000 3000 ) . computational design of orthogonal antiparallel homodimers requires an energy function capable of scoring antiparallel vs parallel dimers . to assess whether our design energy function could predict helix orientation for coiled - coil dimers of known structure we created a database of 30 antiparallel and 48 parallel dimer structures based on the cc+ database of testa et al . ; we refer to this database as the orientation test set ( see materials and methods ) . the orientation test set in this study differed from that used by apgar et al . due to its higher stringency on length , 28 residues vs 18 residues this more stringent cutoff has the effect of removing examples of short coiled - coil sequences embedded in large structures , for which the helix orientation is less likely to be determined by the sequence of the coiled - coil region alone . furthermore , sequence features of antiparallel coiled coils in the pdb are a function of their lengths ; e.g. , shorter coiled coils have a 16% higher frequency of hydrophobic residues at the g position ( table s4 ) . a modified version of dfire , dfire * , which includes only interchain energy terms , was used for scoring . the orientation test - set sequences were modeled in both parallel and antiparallel orientations using rosetta and scored using dfire * , as described in the materials and methods . the dfire * energy gap between the antiparallel and parallel state for each sequence is plotted in figure 1a . we report energies in arbitrary units ( au ) , as we have no information at this time about how predicted energies from this procedure correlate with experimental free energies . the ability of dfire * to predict orientation preference on the test set was measured using the area under the curve ( auc ) when plotting the fraction of parallel test - set sequences predicted correctly vs the fraction of antiparallel sequences predicted correctly , as a function of the score cutoff used to discriminate parallel from antiparallel sequences . as seen in figure 1b , dfire * predicts orientation preference in this test with an auc value of 0.91 ( random predictions would result in an auc of 0.5 ) . predicting coiled - coil orientation preference and testing cluster - expanded dfire*. ( a ) eap and ep are the antiparallel ( ap ) and parallel ( p ) dfire * energies for each orientation test set coiled coil . antiparallel or parallel coiled coils ( according to pdb structure ) are plotted with red crosses or black diamonds , respectively . min_gap was used to remove examples with small dfire * orientation preferences ( see text ) ; shading indicates increasing min_gap from the line of optimal separation . ( b ) the fraction of antiparallel sequences predicted correctly vs the fraction of parallel sequences predicted correctly , as the cutoff value for eap ep was changed , is plotted for dfire * and the ce model of dfire*. curves for data sets with different values of min_gap are shown for the ce model of dfire*. ( c , d ) dfire * energies vs the ce model of dfire * energies for randomly generated dimer - like test structures in the antiparallel ( c ) and parallel ( d ) states . cluster expansion ( ce ) is a computational method for generating a sequence - based scoring function that approximates energies calculated using structure - based techniques . once generated , a ce model eliminates the need for computationally costly structure building in protein design . two ce models were built to approximate dfire * energies for antiparallel and parallel coiled - coil dimers , as described in the materials and methods , and the models were used to score 8000 test sequences ( figure 1c , d ) . both models showed good correlation with dfire * , r = 0.90 , indicating that the approximation of structure - based modeling with a sequence - based function introduced relatively little error within the sequence space explored . we benchmarked the orientation prediction performance of the ce dfire * models using the orientation test set . every pair of sequences in the set was scored with the antiparallel ce model and the parallel ce model . the energy difference between the two ce models was used to predict the orientation preference of each sequence . the auc value using the ce approximation of dfire * was 0.84 ( figure 1b ) , demonstrating that the faster , yet more approximate model gave reduced performance , as expected . however , the auc value significantly improved as coiled coils with small energy gaps were removed from the orientation test set . for 44 coiled coils with the largest predicted differences in ce energy between parallel and antiparallel orientation ( greater than 0.4047 ) , the prediction performance ( 0.93 ) was similar to the performance of dfire * on the entire orientation test set . for 20 examples with predicted energy gaps greater than 0.8094 , prediction performance was perfect . this information was used to set energy gap requirements for off - target states during the sequence - design stage of classy . classy is a protein - design method that uses integer linear programming ( ilp ) to optimize a protein sequence using a ce scoring function . importantly , the method allows a user to impose numerous constraints on the designed sequence . these can include constraints on sequence composition or properties ( e.g. , total charge ) . in multistate design , it is convenient to impose a constraint on the energy of a designed sequence adopting an undesired structure , to disfavor formation of that structure . in our application , the antiparallel and parallel ce models were combined with ilp to do classy design of six - heptad antiparallel homodimers . designed antiparallel coiled coil aph is also six heptads long , and a four - heptad variant of aph had low thermal stability . on the basis of this , we reasoned that six heptads should provide ample space to include specificity elements while maintaining a folded structure . only residues at a , d , e , and g positions were designed ; these residues are thought to be most critical for interaction specificity . the b , c , and f surface positions were taken from aph , which is one of the few characterized antiparallel homodimers reported in the literature . the surface of aph mainly consists of patterned glutamine and alanine residues at b and c positions , and lysine residues at f positions . this surface design has been used for both parallel and antiparallel coiled coils , and is thought to play a minimal role in interaction specificity . we used the ce model of dfire * to design the globally best - scoring antiparallel homodimer in a sequence space without cysteine , proline , or glycine and found that the designed sequence was highly charged and contained no hydrophobic residues in any heptad position . this peptide would not be expected to fold into a coiled - coil structure . the unrealistic design sequence is not inconsistent with the good performance of dfire * and the ce model of dfire * on the orientation prediction test above . in the orientation test , in contrast , without constraints on sequence composition , optimization using the ce model of dfire * had the freedom to build a sequence entirely from charged pairs that have highly favorable ce weights . the 20 most favorable weights in the ce dfire * model are all core - to - edge ( i.e. , a or d to e or g ) , or core - to - core charge we therefore imposed constraints on the number of polar residues allowed at core heptad positions ( see materials and methods ) and restricted the design calculations to subsets of sequence space , as described below . two separate sequence spaces , sequence space 1 and sequence space 2 , were chosen to search for antiparallel homodimer sequences ( figure 2a , b ) . both sequence spaces included residues known to influence coiled - coil structural specificity through mechanisms such as electrostatic attraction / repulsion and beta - branch residue packing / clashing . sequence space 1 differed from sequence space 2 by the addition of glutamate as a choice at a positions . statistics from the coiled - coil databases we analyzed show a 3-fold frequency enrichment of glutamate in a sites of antiparallel dimers relative to parallel dimers ( table s5 ) ; this difference has also been noted by straussman et al . ( a , b ) diagram of target and off - target states included in two design calculations . colors represent distinct sequences , and colored circles indicate the n - terminus of each helix . an energetic constraint , , was enforced between the energy of each target antiparallel homodimer state ( e , e , e ) and every off - target state that peptide could participate in ( examples shown with gray dashed lines ) . the sequence space used for each design different numbers of off - target states were included for sequence space 1 ( a ) vs sequence space 2 ( b ) . ( c , d ) the total energy e = e + e + e vs is plotted for sequence space 1 ( c ) and sequence space 2 ( d ) . each value of led to a set of optimized sequences , and the gray squares mark the solutions chosen for experimental testing . to design three noninteracting coiled coils , we optimized the sum of the ce energies of three antiparallel homodimers using classy . constraints were added to allow no more than two hydrophilic residues at a positions and no more than one at d positions . this maintained the hydrophobicity of the design solutions at these positions close to that of known antiparallel dimers of lengths greater than four heptads . , all design calculations treated all three possible antiparallel heterodimer states as undesired states . without these constraints , the global energy minimum would correspond to three copies of the lowest - energy antiparallel homodimer . constraints on the off - target states were imposed as an energy gap by requiring the energy of each antiparallel homodimer to be lower than the energy of each of the off - target states that sequence could participate in , by a fixed amount ( figure 2 ) . classy design was done iteratively , by progressively increasing the energy gap that was imposed between the target antiparallel homodimers and off - target antiparallel heterodimer states . as the gap to off - target states increased , the total predicted stability of the three antiparallel homodimers decreased ( figure 2c , d ) . this type of stability - specificity trade - off has been observed previously in the case of parallel dimer design using classy . two sets of solutions , one from each of the sequence spaces , were rationally chosen based on good stability - specificity trade - offs . the designs in sequence space 1 are referred to as aphi , aphii , and aphiii . the designs in sequence space 2 are referred to as aphiv , aphv , aphvi. for each set of designed sequences , parallel and antiparallel homo- and heterodimer states were scored with the original dfire * structure - based model to predict relative energies of target and off - target structures . for the antiparallel homodimers designed in sequence space 1 , the predicted energies of all parallel and antiparallel off - target dimers were much higher than the predicted energies for the antiparallel homodimers . the smallest gap , of 0.77 au , was between the antiparallel homodimer state of aphiii and a parallel heterodimer consisting of aphiii and aphi ( figure s2a ) . the aphi antiparallel homodimer gap to this state was 1.13 au . at gaps of this magnitude , dfire * predicts the orientation preference of native sequences with an auc = 1.0 . thus , no additional states were added to the optimization protocol for sequence space 1 . for sequence space 2 , we observed that one of the parallel homodimers was predicted to be lower in energy than the corresponding antiparallel homodimer ( figure s2b ) . furthermore , other parallel homodimer states were closer in energy to the antiparallel homodimers than when design was done in sequence space 1 . to address this , we added parallel homodimer states as off - target states in the optimization protocol used for sequence space 2 , and chose a new set of solutions in that space . the final six designed sequences are shown in table 1 , with aphi , aphii and aphiii resulting from design in sequence space 1 , and aphiv , aphv and aphvi from design in sequence space 2 . the two sets of designed sequences were also scored for cross - reactivity using dfire*. predicted energies for all parallel and antiparallel heterodimers that could be formed between sets were significantly larger than predicted energies for the antiparallel homodimer states , with the smallest energy gap of 0.61 au between the antiparallel and parallel homodimer states of aphiv . the molecular weights of complexes formed by designed peptides aphi aphvi were determined using sedimentation equilibrium analytical ultracentrifugation ( see materials and methods ) . we anticipate that the aph coiled coils will be used as fusion proteins in many applications , so we did two sets of experiments : one in which the peptides were fused to maltose binding protein ( mbp ) and one in which they were not . the data for two designed peptides , aphiii and aphvi , were consistent with these peptides forming homodimers . aphi was determined to have a molecular weight greater than that expected for a dimer , and no further data were collected on this construct . single - species fits to aphii and aphiv gave molecular weights less than and greater than what was expected for a dimer , respectively . aphii and aphiv were retested at higher concentrations to stabilize higher - order states . at 20 m , aphii formed a homodimer , whereas aphiv formed a homotrimer . further experiments were carried out only on designs aphii , aphiii and aphvi , which we renamed aph2 , aph3 and aph4 , respectively ( see table 2 ) . data collected using interference optics , and a construct not fused to mbp . to determine the helix orientation in complexes formed by aph2 , aph3 , and aph4 , we performed disulfide - exchange experiments , and resolved the products of the reactions using hplc ( see materials and methods ) . key peaks are labeled in figure 3 , which shows changes in the chromatograms over time . for all three designs , starting with a combination of oxidized parallel species and/or reduced peptides , only one oxidized peak was detected at the end of 5 h , corresponding to a disulfide - linked antiparallel homodimer . on the basis of the smallest detectable peak area , we estimated a minimum 10-fold preference for forming antiparallel complexes over parallel complexes for all designs . designed peptides aph2 , aph3 , and aph4 adopt an antiparallel helix orientation . the wavy line indicates two amino acids added to the designed sequence to change peptide retention times ( aph2 = yy , aph3 = qw , aph4 = yy ) . ( b , c , d ) hplc chromatograms show the results for the disulfide - exchange reactions upon mixing equimolar amounts of n - terminal and c - terminal cysteine variants of each design sequence ( 20 m each ) . the reactions were quenched at 0 min ( red ) , 15 min ( black ) , or 5 h ( blue ) . peaks are labeled according to the scheme shown in panel a , with g indicating a glutathione adduct . the same constructs that were used to measure orientation preferences were used to determine whether the designs formed heterodimers . each design formed a disulfide cross - linked antiparallel homodimer over time , but we did not detect any disulfide bond formation between any pairs of designed peptides . each design was additionally measured for cross reactivity with the antiparallel homodimer - forming peptide aph , in a pairwise manner ( figure 5 ) . no design showed any detectable cross - reactivity with aph , in either orientation , extending the number of orthogonal antiparallel homodimers from three to four . a ) cartoon showing four cysteine - containing peptides , two for each of two designs , which were included in the disulfide - exchange cross - reactivity assay . ( b , c , d ) hplc traces for all pairwise mixtures of designed peptides after equilibration for 15 min . the blue and red traces are for reactions with equimolar amounts of n- and c - terminal cysteine variants of a single designed peptide ( 20 m each ) . the black trace is for a reaction with equimolar amounts of all four peptides in panel a ( 20 m each ) . ( b ) aph2 + aph3 , ( c ) aph2 + aph4 , ( d ) aph3 + aph4 . designed peptides aph2 , aph3 , and aph4 do not heterodimerize with aph . ( a , b , c ) hplc traces for all pairwise combinations of aph with the designed coiled coils , with experimental conditions as for figure 4 . the blue and red traces are for equimolar mixtures of n- and c - terminal cysteine variants of aph ( blue ) or aph2 , aph3 or aph4 ( red ) ( 20 m each ) . the black trace is for a mixture of four peptides , aph and the indicated design , each modified at the n- or c - terminus with a cysteine residue ( 20 m each ) . ( a ) aph + aph2 , ( b ) aph + aph3 , ( c ) aph + aph4 . to determine whether mixtures of more than two aph coiled coils formed complexes other than the expected dimers , mbp fusions of all four aph peptides were mixed at 20 or 40 m of each aph design and analyzed by sedimentation equilibrium ultracentrifugation ( as done for individual mbp fusion proteins , see materials and methods ) . the ratio of the fitted mass to the dimer mass was 0.91 , with good fit quality ( representative data in figure s3 ) , indicating that dimers formed as expected and no higher - order species were present in a mixture of all four aph fusion proteins . we measured the circular dichroism ( cd ) spectra of the three designed peptides aph2 , aph3 , and aph4 , using the n - terminal cysteine constructs in a reduced state . each construct contained 65 residues , of which 43 corresponded to the designed coiled - coil sequence ( table s3 ) . our aph construct contained 66 residues , of which 44 corresponded to the aph sequence . the cd spectra of all three designs were characteristic of coiled coils , with distinct minima at 208 and 222 nm ( figure 6a ) . the mean residue ellipticity ( mre ) of the designed peptides was similar to that of aph , which is longer by one residue in the coiled - coil region . thermal denaturation experiments established that all designs unfolded cooperatively , which is a characteristic property of coiled coils ( figure 6b ) . the thermal stabilities ( tm ) of the designs at 20 m ranged from 47.4 c for aph2 , to 59.3 c for aph4 and 78.3 c for aph3 , with aph3 being slightly less stable then aph , which had a tm of 79.3 c . , all peptides regained 95% of the original mre , and fits of refolding curves gave melting temperatures within 1.5 c of values obtained from the denaturing curves . estimating peptide helicity from the cd data using the method of morrisett et al . indicated that linkers and tags appended to the designed coiled coil contributed helical signal , as shown in figure s4 however , most of this extra signal was lost gradually with temperature in the pretransition baseline , indicating these regions are not part of the cooperative unfolding event . furthermore , these linker residues were not present in the mbp - fusion constructs used in the sedimentation equilibrium centrifugation experiments , consistent with them not being necessary for the specific interactions observed in those experiments . finally , making aph in the construct that we used for cd experiments did not change its melting temperature from the value reported in the literature for only the coiled coil . ( a ) cd spectra and ( b ) thermal denaturation curves measured at 25 c in pbs with 1 mm dtt . aph ( red ) , aph2 ( blue ) , aph3 ( green ) and aph4 ( orange ) . an expanded toolkit of coiled - coil interaction parts would be of great utility in protein engineering . many papers have reported the successful design of coiled - coil structures of diverse topologies , but apart from parallel dimers , the number of biochemically characterized complexes of any one type is limited . designing coiled coils de novo is complicated by the fact that different coiled - coil topologies have similar sequence requirements , and small sequence changes can alter coiled - coil structure . for these reasons , it is often necessary to explicitly consider competing states in the design process . treating off - target states in computational protein design can be costly , particularly when there are many such states that must be modeled . one strategy is to incorporate a design element known to strongly destabilize a set of off - target topologies , to reduce the number of off - target states that must be modeled . for instance , thomas et al . observed that the de novo design of parallel heterodimeric coiled coils composed entirely of isoleucine and leucine cores did not reliably destabilize higher - order states . but the same design strategy in the background of a single asparagine asparagine interaction , which was known from prior work to favor parallel dimer states over higher - order states , consistently gave dimeric assemblies . unfortunately , incorporating simple design elements that reliably destabilize all off - target topologies , in all sequence contexts , is not feasible . exceptions have been reported for even the most thoroughly studied coiled - coil structural specificity determinants , and for many coiled - coil topologies , the sequence - structure relationship is not well understood . of relevance for this work , there are few sequence features known to favor antiparallel over parallel helical alignments . oakley et al . showed that , in analogy to the role of asparagines favoring dimers over higher - order states , paired asparagines can be introduced at opposing a and d positions to favor an antiparallel helix alignment . charge interactions at e and g positions across the interface can impart an antiparallel vs parallel preference . placed an isoleucine at a d heptad position and an alanine residue at an opposing a heptad position to favor an antiparallel homodimer state over a parallel homodimer state in the designed sequence aph . further evidence supporting this interaction as an orientation specificity determinant was obtained via thiol - thioester exchange studies by hadley et al . although simple rules do have some utility for design , hadley et al . showed that residue residue interactions in antiparallel coiled coils can also be highly context dependent , helping explain why rules extracted from studies of model systems do not satisfactorily explain the orientations of native coiled coils . modeling off - target states explicitly and including them in the design process provides a broadly applicable mechanism for engineering specificity . in this work , we used explicit negative design to disfavor antiparallel heterodimer states by imposing energy gaps between antiparallel homo and heterodimers . most of the sequence elements in our aph designs that disfavored antiparallel heterodimerization within a design set involved charged residues predicted to participate in repulsive interactions in heterodimer states . for example , all antiparallel heterodimer states contained a - to - e and d - to - g charge charge repulsions between lysine or arginine residues . charge repulsions between glutamate residues . these core - to - edge charge charge repulsions were the most destabilizing weights available to the antiparallel ce dfire * model in the design sequence spaces chosen , with lysine at d to arginine at g being the most destabilizing . the design strategies that led to destabilization of parallel homodimers differed in sequences spaces 1 and 2 . in sequence space 1 , we allowed glutamate at a positions , and all designed sequences included this element . in fact , we identified a motif consisting of two glutamate residues at a and g , and a lysine at d with an arginine at e on the opposing helix that was present in all of the sequence space 1 designs ( figure s5 ) . interactions between residues in this motif contain the first and fourth most favorable weights available in the ce dfire * model in sequence space 1 , such that the motif is predicted to contribute strongly to antiparallel homodimer stability . interestingly , in a parallel homodimer , the residues of this motif form unfavorable interactions sufficient to provide a large energy gap between parallel and antiparallel states . certain unfavorable weights are shown in figure s5 , and this can be further demonstrated by modeling an artificial homodimer that includes the motif embedded in a poly - alanine sequence . because of the symmetry of the homodimer , this results in two copies of the motif in the structure . scoring parallel and antiparallel homodimeric structures with this sequence using dfire * revealed a significant preference of 1.64 energy units for the antiparallel state ( poly alanine alone has a preference of 0.14 energy units for the antiparallel state using this model ) . thus , in sequence space 1 , charge networks predicted to stabilize the antiparallel state led to substantial destabilization of parallel homodimers , without explicit negative design . the situation was different in sequence space 2 , which did not include glutamate residues at a positions . in this sequence space , designing antiparallel homodimers while disfavoring heterodimers did not automatically lead to large energy gaps to parallel homodimer states for all sequences ( see figure s2b ) ; it was necessary to include parallel structures as off - target states in the optimization problem . doing so led to sequences that placed more isoleucines at d heptad positions to favor antiparallel over parallel homodimers . for example , of the three sequences originally chosen in sequence space 2 , two sequences had one isoleucine residue at a d position , while one sequence had no isoleucine residues at all . after placing constraints on the energies of the parallel homodimer states , all design sequences contained one or two isoleucine residues at d heptad positions . each designed isoleucine at a d position introduced a d d isoleucine pairing across the coiled - coil interface in the parallel homodimer state . as previously mentioned , this interaction destabilizes parallel dimers . . explicit consideration of off - target states requires enumerating and modeling the relevant competing states . we successfully used this strategy to destabilize antiparallel heterodimer states in sequence spaces 1 and 2 , and to destabilize parallel homodimers when designing in sequence space 2 . but we did not explicitly model formation of higher - order assemblies , and as a result , oligomers larger than dimers were formed by designs aphi and aphiv . modeling higher - order coiled coils is challenging due to the many different topologies that are possible . each helix pair can be antiparallel or parallel , heteroassemblies can form with different stoichiometries , and the geometry of helix associations can vary in subtle ways . it is therefore difficult to include a comprehensive set of competing states and , even if such a set could be generated , the computational modeling costs for considering all possibilities explicitly would be high . one approach to disfavoring higher - order states could be to include just a small number of trimer and tetramer topologies in the calculations . adding representative off - target structures would minimally alter the computational complexity of the design framework , yet might lead to broader destabilization of additional higher - order states . indeed , our study provided an example where specificity was obtained against states that were not explicitly modeled , possibly due to constraints on specificity against related states . the design solutions from sequence space 1 were predicted not to form heterodimers with design solutions from sequence space 2 , despite these interactions not being explicitly constrained during optimization . we hypothesize that this occurred because the consideration of many off - target dimer states gave rise to interfaces with charge patterns low in symmetry , as well as hydrophobic cores with unique geometries due to the placements of beta - branched residues in the core . as a result , the probability of cross - reacting with another sequence to form dimers was low . considering just a few higher - order states may also have the effect of reducing or removing design features known to favor higher - order states generally . for example , isoleucines at d heptad positions are known to favor parallel trimer and tetramer states in preference to parallel dimer states . yet isoleucines at d heptad positions also favor antiparallel dimers over parallel dimers , and were included in many of our designs for this reason , as discussed above ( also see table 1 ) . interestingly , in native coiled coils isoleucines are approximately 4-fold more common in antiparallel dimers than in parallel dimers ( table s5 ) . isoleucines at d heptad positions that were included in the design to favor antiparallel dimers might have promoted the formation of higher - order assemblies , which were not treated in the model . a constraint to disfavor just a few trimers or tetramers might be sufficient to limit the use of this sequence element , or to drive inclusion of compensating elements that are poorly accommodated in higher - order assemblies . a significant obstacle to including even a few higher - order states in design is the small amount of structural data available for coiled - coil trimers and tetramers of a specific toplogy . benchmarking the predictive power of models using experimental data is important for assessing performance , and is useful for setting meaningful energy cutoffs in design calculations . however , very few known structures of higher - order states of any specific topology passed our orientation test set filters of 50% sequence identity and > 27 residues ( 0 antiparallel trimers , 6 antiparallel tetramers , and 9 parallel tetramers in the august 18 , 2010 cc+ database ) . for these reasons , we did not benchmark dfire * on the problem of predicting oligomerization state , and we did not attempt to use it for this purpose . we examined the structure - prediction power of other methods when applied to our antiparallel coiled coils . logicoil is a computational predictor trained on coiled - coil sequences in the cc+ database to discriminate parallel dimers , antiparallel dimers , trimers and tetramers . for aph2 , aph3 , and aph4 , logicoil makes predictions based on a single - chain sequence , without information about interchain interactions that are crucial design elements in the aph designs . additionally , the aph sequences are de novo designed sequences , with intrachain pairwise frequencies that may not resemble those in native sequences . ccbuilder is a new web - based application that generates coiled - coil structures and can be used for predicting coiled - coil topology . ccbuilder can model parallel dimers , antiparallel dimers , parallel trimers , and parallel tetramers , and the program computes the stability of coiled - coil complexes using two energy functions : rosetta and bude . both energy functions , used with default settings provided by the web site , correctly predict that aph2 , aph3 , and aph4 favor the antiparallel dimer state over the parallel dimer state . when higher - order states are considered , both energy functions predict that aph2 and aph4 favor the trimer state , and rosetta also predicts that aph3 will form a trimer . better methods for oligomerization state prediction are needed and , if developed , could be incorporated into our design framework . the rankings of the thermal stabilities ( figure 6 ) are not predicted well by dfire*. dfire * instead predicts that aph3 is the most stable complex , followed by aph2 , aph4 , and aph . these predicted ranking are consistent with the favorable weights that the ce of dfire * assigns between core and edge positions . but the relative thermal stabilities of the aph coiled coils appear to be related to the number of charged residues in the central two heptads of the designed coiled coils ( figure 7 ) . aph2 , which has the greatest number of charged residues in the central two heptads , is the least stable . in contrast , both aph and aph3 contain no charged residues in the central two heptads and are the most thermally stable . this is consistent with many studies showing coiled - coil destabilization by polar residues in the core . it should be noted that ccbuilder accurately predicts the thermal stability rankings using rosetta or bude scores , if all structures are scored as antiparallel dimers . helical - wheel diagrams of aph , aph2 , aph3 , and aph4 as antiparallel homodimers . positively and negatively charged amino acids are shown in blue and red , respectively , with noncharged polar residues in orange and hydrophobic residues in gray . the new aph designs have many desirable properties for synthetic biology and materials science . first , the surface residues of all aph designs were engineered to be passive and may provide useful positions for adding novel functions or modulating stability . the designed structures also provide users with a range of thermal stabilities , and it may be possible to tune the dimer stabilities using mutations of the surface residues , as needed . finally , the designs are orthogonal to each other when used in pairwise or higher - order combinations . proteins with this property have been sought for many applications in synthetic biology and are thought to be one of the limiting reagents slowing progress in this field . it should also be noted that heterodimers involving the aph proteins could be included as off - target states in future design studies using the classy framework , allowing for the extension of this set . in conclusion , the antiparallel homodimer sequences represent a significant expansion to the coiled - coil toolkit , which is currently dominated by parallel dimers , and thus may find application in many molecular engineering projects .

molecular engineering of protein assemblies , including the fabrication of nanostructures and synthetic signaling pathways , relies on the availability of modular parts that can be combined to give different structures and functions . currently , a limited number of well - characterized protein interaction components are available . coiled - coil interaction modules have been demonstrated to be useful for biomolecular design , and many parallel homodimers and heterodimers are available in the coiled - coil toolkit . in this work , we sought to design a set of orthogonal antiparallel homodimeric coiled coils using a computational approach . there are very few antiparallel homodimers described in the literature , and none have been measured for cross - reactivity . we tested the ability of the distance - dependent statistical potential dfire to predict orientation preferences for coiled - coil dimers of known structure . the dfire model was then combined with the classy multistate protein design framework to engineer sets of three orthogonal antiparallel homodimeric coiled coils . experimental measurements confirmed the successful design of three peptides that preferentially formed antiparallel homodimers that , furthermore , did not interact with one additional previously reported antiparallel homodimer . two designed peptides that formed higher - order structures suggest how future design protocols could be improved . the successful designs represent a significant expansion of the existing protein - interaction toolbox for molecular engineers .

Introduction Materials and Methods Results Discussion

here , we focus on the -helical coiled coil , which is a protein - interaction domain highly suitable for inclusion in the growing molecular parts toolkit . the a and d positions are dominated by hydrophobic residues , and are found at the core of the structure ; we refer to a and d positions as core positions in this work . in coiled - coil dimers , e and g positions are typically occupied by charged residues and form the boundary between the core and the surface of the coiled coil . for example , positions e and g are proximal and can form interhelical salt bridges in parallel coiled - coil dimers , whereas e / e and g / g pairs can interact in antiparallel coiled coils . the relationship between coiled - coil sequence and structure is incompletely understood , even after decades of study of native , mutant and de novo - designed coiled coils . for example , coiled coils can fold into dimers , trimers , tetramers , and even higher - order oligomers . the general problem of predicting detailed coiled - coil structure from sequence has not been solved , although progress has been made developing methods to predict oligomerization state from sequence , and in particular to discriminate parallel dimers from parallel trimers . in many of these studies , modulated the conductance between two monolayers of gold using coiled - coil dimers and showed that parallel and antiparallel coiled coils differentially impacted the electronic properties of the system . recently , an exciting strategy was developed to design polypeptide polyhedra based around coiled - coil dimers . used a set of parallel and antiparallel dimeric coiled coils as building blocks to engineer a nanoscale single - chain tetrahedron with coiled coils forming each edge . a crucial aspect of the design strategy was the use of coiled - coil components that were orthogonal to one another , i.e. the designed tetrahedron was based on 4 parallel and 2 antiparallel coiled - coil dimers previously reported in the literature . very few antiparallel coiled - coil dimers have been characterized or designed , and none have been tested for orthogonality . in contrast , dozens of native and synthetic parallel coiled coils have been tested for interactions and orthogonality . there are currently two databases maintained for designed coiled coils , the synzip database , and the pcomp database . thus , designing sets of orthogonal antiparallel homodimers would expand the available coiled - coil parts in a meaningful way . because coiled - coil sequences can encode many different structures , negative design to destabilize undesired states is crucial when making peptides intended to assemble into a single topology . a recent study relied on all three of these strategies to design a parallel homodimer , homotrimer , and homotetramer . designing sets of orthogonal coiled - coil homodimers presents additional challenges related to encoding interaction specificity . the number of possible hetero species increases dramatically as the number of designed orthogonal coiled coils grows , such that three orthogonal antiparallel homodimers have the potential to form six possible off - target parallel or antiparallel heterodimers ; other undesired structures are also possible . to design sets of orthogonal antiparallel coiled - coil dimers , we therefore turned to computational methods to keep track of the numerous desired and undesired structures in this design problem . despite the many successes of structure - based approaches for modeling and designing protein the computational costs of modeling each structure can be large , and current optimization functions used with structure - based models do not provide efficient routines for optimizing one set of states while simultaneously destabilizing many off - target states . by using a transformation of structure - based models to sequence - based models , classy addresses both the search and scoring problems of multistate design , and the method has previously been applied to design parallel coiled coils specific for binding to a target in preference to closely related off - target proteins . this paper describes our work applying classy in conjunction with the dfire statistical potential to the de novo design of sets of coiled coils consisting of three orthogonal antiparallel homodimers . we designed two sets of three proteins , and used biophysical techniques to determine the oligomerization state , helix orientation and thermal stability of structures formed by the designed sequences . some designed peptides formed trimers or higher - order assemblies , but we identified 3 peptides ( aph2 , aph3 , and aph4 ) that formed orthogonal antiparallel homodimers . in addition , we showed that these proteins homodimerize in preference to binding to aph , a previously reported antiparallel homodimer . thus , we provide evidence for four sequences that preferentially form antiparallel homodimers that can be used for protein engineering applications . as described in detail below , side chains were modeled on idealized coiled - coil backbones using rosetta and scored using difire * , a modified version of the dfire statistical potential . to construct libraries of parallel and antiparallel backbones , a set of 214 canonical coiled coils ( i.e. this set of backbones was then filtered to give 25 parallel and 23 antiparallel backbones with parameters within one standard deviation of the average value for each parameter . the soft - potential flag and expansion of the first and second dihedral angles of the rotamer library were used , along with the side - chain minimization flag . structures were scored using a modified version of dfire , a distance - dependent pairwise statistical potential based on the distance - scaled , finite ideal - gas reference state . the cutoff distance , rcut , was set to 5.8 , and interatomic energies were evaluated only between residues on opposite helices in the coiled coil . training sequences were generated randomly but with heptad - specific single - residue frequencies matching those of known coiled - coil dimers ( both parallel and antiparallel ) . once determined , cluster expansion ( ce ) weights can be used to score antiparallel and parallel coiled - coil dimers of arbitrary length . a detailed description of how integer linear programming ( ilp ) can be applied as part of the classy multistate design method is given in negron et al . in this work , the objective function for ilp was the total energy ( e ) , given by the sum of the energies of three antiparallel homodimers ( e = e + e + e ) . the ilp solver of the ibm ilog cplex optimizer was used to minimize this objective function under a set of constraints . the constraints included energy gaps to off - target dimer states ( see figure 2a , b ) , as well as constraints on the number of polar residues allowed at a and d heptad positions ( maximum of 2 charged residues at a , and 1 lys residue at d per design sequence ) . a constraint was included on the energy gap between every antiparallel homodimer and every off - target state ( of those types considered in the calculation ) that the constituent peptide could participate in . synthetic genes encoding computationally designed coiled - coil sequences , and control sequences , were constructed by pcr amplification from two 258-base pair oligonucleotides and one 157-base pair oligonucleotide ( gblocks ) purchased from integrated dna technologies . computational design of orthogonal antiparallel homodimers requires an energy function capable of scoring antiparallel vs parallel dimers . to assess whether our design energy function could predict helix orientation for coiled - coil dimers of known structure we created a database of 30 antiparallel and 48 parallel dimer structures based on the cc+ database of testa et al . due to its higher stringency on length , 28 residues vs 18 residues this more stringent cutoff has the effect of removing examples of short coiled - coil sequences embedded in large structures , for which the helix orientation is less likely to be determined by the sequence of the coiled - coil region alone . the ability of dfire * to predict orientation preference on the test set was measured using the area under the curve ( auc ) when plotting the fraction of parallel test - set sequences predicted correctly vs the fraction of antiparallel sequences predicted correctly , as a function of the score cutoff used to discriminate parallel from antiparallel sequences . two ce models were built to approximate dfire * energies for antiparallel and parallel coiled - coil dimers , as described in the materials and methods , and the models were used to score 8000 test sequences ( figure 1c , d ) . for 44 coiled coils with the largest predicted differences in ce energy between parallel and antiparallel orientation ( greater than 0.4047 ) , the prediction performance ( 0.93 ) was similar to the performance of dfire * on the entire orientation test set . the b , c , and f surface positions were taken from aph , which is one of the few characterized antiparallel homodimers reported in the literature . , a or d to e or g ) , or core - to - core charge we therefore imposed constraints on the number of polar residues allowed at core heptad positions ( see materials and methods ) and restricted the design calculations to subsets of sequence space , as described below . statistics from the coiled - coil databases we analyzed show a 3-fold frequency enrichment of glutamate in a sites of antiparallel dimers relative to parallel dimers ( table s5 ) ; this difference has also been noted by straussman et al . each value of led to a set of optimized sequences , and the gray squares mark the solutions chosen for experimental testing . to design three noninteracting coiled coils , we optimized the sum of the ce energies of three antiparallel homodimers using classy . constraints on the off - target states were imposed as an energy gap by requiring the energy of each antiparallel homodimer to be lower than the energy of each of the off - target states that sequence could participate in , by a fixed amount ( figure 2 ) . for sequence space 2 , we observed that one of the parallel homodimers was predicted to be lower in energy than the corresponding antiparallel homodimer ( figure s2b ) . to address this , we added parallel homodimer states as off - target states in the optimization protocol used for sequence space 2 , and chose a new set of solutions in that space . the two sets of designed sequences were also scored for cross - reactivity using dfire*. on the basis of the smallest detectable peak area , we estimated a minimum 10-fold preference for forming antiparallel complexes over parallel complexes for all designs . each design formed a disulfide cross - linked antiparallel homodimer over time , but we did not detect any disulfide bond formation between any pairs of designed peptides . each design was additionally measured for cross reactivity with the antiparallel homodimer - forming peptide aph , in a pairwise manner ( figure 5 ) . no design showed any detectable cross - reactivity with aph , in either orientation , extending the number of orthogonal antiparallel homodimers from three to four . a ) cartoon showing four cysteine - containing peptides , two for each of two designs , which were included in the disulfide - exchange cross - reactivity assay . the ratio of the fitted mass to the dimer mass was 0.91 , with good fit quality ( representative data in figure s3 ) , indicating that dimers formed as expected and no higher - order species were present in a mixture of all four aph fusion proteins . the mean residue ellipticity ( mre ) of the designed peptides was similar to that of aph , which is longer by one residue in the coiled - coil region . finally , making aph in the construct that we used for cd experiments did not change its melting temperature from the value reported in the literature for only the coiled coil . an expanded toolkit of coiled - coil interaction parts would be of great utility in protein engineering . many papers have reported the successful design of coiled - coil structures of diverse topologies , but apart from parallel dimers , the number of biochemically characterized complexes of any one type is limited . designing coiled coils de novo is complicated by the fact that different coiled - coil topologies have similar sequence requirements , and small sequence changes can alter coiled - coil structure . observed that the de novo design of parallel heterodimeric coiled coils composed entirely of isoleucine and leucine cores did not reliably destabilize higher - order states . but the same design strategy in the background of a single asparagine asparagine interaction , which was known from prior work to favor parallel dimer states over higher - order states , consistently gave dimeric assemblies . exceptions have been reported for even the most thoroughly studied coiled - coil structural specificity determinants , and for many coiled - coil topologies , the sequence - structure relationship is not well understood . of relevance for this work , there are few sequence features known to favor antiparallel over parallel helical alignments . showed that , in analogy to the role of asparagines favoring dimers over higher - order states , paired asparagines can be introduced at opposing a and d positions to favor an antiparallel helix alignment . in this work , we used explicit negative design to disfavor antiparallel heterodimer states by imposing energy gaps between antiparallel homo and heterodimers . in fact , we identified a motif consisting of two glutamate residues at a and g , and a lysine at d with an arginine at e on the opposing helix that was present in all of the sequence space 1 designs ( figure s5 ) . interactions between residues in this motif contain the first and fourth most favorable weights available in the ce dfire * model in sequence space 1 , such that the motif is predicted to contribute strongly to antiparallel homodimer stability . in this sequence space , designing antiparallel homodimers while disfavoring heterodimers did not automatically lead to large energy gaps to parallel homodimer states for all sequences ( see figure s2b ) ; it was necessary to include parallel structures as off - target states in the optimization problem . each designed isoleucine at a d position introduced a d d isoleucine pairing across the coiled - coil interface in the parallel homodimer state . but we did not explicitly model formation of higher - order assemblies , and as a result , oligomers larger than dimers were formed by designs aphi and aphiv . modeling higher - order coiled coils is challenging due to the many different topologies that are possible . it is therefore difficult to include a comprehensive set of competing states and , even if such a set could be generated , the computational modeling costs for considering all possibilities explicitly would be high . one approach to disfavoring higher - order states could be to include just a small number of trimer and tetramer topologies in the calculations . adding representative off - target structures would minimally alter the computational complexity of the design framework , yet might lead to broader destabilization of additional higher - order states . isoleucines at d heptad positions that were included in the design to favor antiparallel dimers might have promoted the formation of higher - order assemblies , which were not treated in the model . a significant obstacle to including even a few higher - order states in design is the small amount of structural data available for coiled - coil trimers and tetramers of a specific toplogy . however , very few known structures of higher - order states of any specific topology passed our orientation test set filters of 50% sequence identity and > 27 residues ( 0 antiparallel trimers , 6 antiparallel tetramers , and 9 parallel tetramers in the august 18 , 2010 cc+ database ) . for these reasons , we did not benchmark dfire * on the problem of predicting oligomerization state , and we did not attempt to use it for this purpose . logicoil is a computational predictor trained on coiled - coil sequences in the cc+ database to discriminate parallel dimers , antiparallel dimers , trimers and tetramers . ccbuilder is a new web - based application that generates coiled - coil structures and can be used for predicting coiled - coil topology . ccbuilder can model parallel dimers , antiparallel dimers , parallel trimers , and parallel tetramers , and the program computes the stability of coiled - coil complexes using two energy functions : rosetta and bude . but the relative thermal stabilities of the aph coiled coils appear to be related to the number of charged residues in the central two heptads of the designed coiled coils ( figure 7 ) . proteins with this property have been sought for many applications in synthetic biology and are thought to be one of the limiting reagents slowing progress in this field . it should also be noted that heterodimers involving the aph proteins could be included as off - target states in future design studies using the classy framework , allowing for the extension of this set . in conclusion , the antiparallel homodimer sequences represent a significant expansion to the coiled - coil toolkit , which is currently dominated by parallel dimers , and thus may find application in many molecular engineering projects .

education in the broad and complex field of biology needs to be founded on some core concepts ( klymkowsky , 2010 ) . cells are the basic units of life ; hence , how cells work and the molecular mechanisms involved in cell physiology are an essential part of the curricula of all undergraduate programs in the biological sciences . as we understand them , cells are incredibly sophisticated , self - sufficient , microscopic machines . despite their complexity , cells are governed by the same principles that apply to all physicochemical systems . specifically , they handle energy in the same way that macroscopic machines do . therefore , basic concepts of thermodynamics are essential to an understanding of the molecular mechanisms underlying all cell functions ( klymkowsky , 2010 ) . although a qualitative understanding would be sufficient for most biology students , the relevant thermodynamic principles are not the standard ones that apply to simple thermal engines . therefore , several nonstandard concepts need to be taught to explain why these very ordered systems proliferate actively all over our planet despite their apparent disregard of the second law of thermodynamics . they think about proteins ( antibodies , receptors , enzymes ) , lipids ( phospholipids , cholesterol , triglycerides ) , and other biological molecules as real entities . in contrast , entropy , enthalpy , and free energy are abstract concepts that students can learn to express in an equation and use to get a result without really grasping the important concepts . and as soon as they forget the formula , they are likely to forget most of the underlying thermodynamic principles , except that energy is somehow important . students would almost certainly incorporate these concepts more readily if they had an intuitive idea of the molecular basis of the laws of thermodynamics . however , the formal means of connecting molecules and thermodynamics is statistical mechanics , which is mathematically challenging for many biology students , especially as it applies to nonequilibrium situations . the goal is to teach the thermodynamic concepts necessary for understanding how cells handle energy , while keeping mathematics to a level accessible to the students . a useful strategy for this is to work with a simple macroscopic system that can be readily visualized by the students and that behaves as a set of molecules exchanging energy during a process . the idea presented here is an extension of the boxes used in the cell biology textbook the world of the cell ( becker et al . , 2008 ) . the analogy was first developed by harold f. blum in the book time 's arrow and evolution ( 1968 ) . the strategy is to picture a box containing small balls that are in constant movement due to a stream of air blowing from below ( figure 1 ) . this device is analogous to the lottery ball picker machines frequently used for random selection of numbers in several television shows . the collisions of balls against the box walls and against one another are not elastic ; hence , if the airstream is turned off , the balls will rapidly lose energy and come to rest on the floor of the box . in the four boxes in figure 1 , the stream of air is blowing with increasing strength from left to right . in this system , we can think of balls as molecules having different levels of energy following a boltzmann distribution . the airstream , which provides the energy that propels the balls upward in the box , is equivalent to the absolute temperature in the boltzmann distribution . the higher the temperature , the more kinetic energy the molecules in the system will have . the important concept is that the number of molecules at different energy levels ( i.e. , different distances from the floor ) follows a boltzmann distribution . students can easily and intuitively visualize balls with different energy levels without any mathematical deduction . the influence of the temperature in this distribution is also very intuitive . to reinforce the box image , a simulation was programmed in visual basic as a macro in an excel worksheet ( supplemental material ) . a movie generated with this simulation shows that the distribution of the balls in the box is dynamic and changes with temperature ( see movie s1 in the supplemental material ) . two hundred balls were distributed in a two - dimensional box according to the following rules : for each ball , the x position was randomly selected within the limits of the box . the y coordinate ( energy ) was randomly selected as y = r t ln(rnd ) , where r is 1.986 cal k mol , t is the absolute temperature in degrees kelvin , and rnd is a random number in the 01 range . the figure was generated using a simulation programmed in visual basic as a macro in an excel worksheet ( see supplemental material ) . now , if two sectors of the box are considered , the number of molecules in each sector depends on the area of each sector and on the floor level of the sector . these two factors accurately correspond to the entropy and the enthalpy difference between the two sectors ( figure 2 ) . in this macroscopic system , also , the influence of temperature on the distribution of molecules can be intuitively inferred . in other words , students should be able to grasp the concept of free energy based on the distribution of balls in boxes with different shapes . two hundred balls were distributed in two - dimensional boxes divided into two sectors according to the following rules and considering an absolute temperature of 310 k. for each ball initially in sector i ( or sector ii ) , the y coordinate ( energy ) was randomly selected as y = hi r t ln(rnd ) , where hi is the floor level for the ball 's sector ( 0 for sector i and h for sector ii ) and rnd is a random number in the 01 range . if the height of the y coordinate was higher than the energy step necessary to pass to the other sector , the x coordinate was randomly selected within the limits of the entire box , such that the wider sector received proportionally more balls . if the y coordinate was lower than the energy step , the x coordinate was randomly selected within the limits of the sector where the ball was located initially . the left sector in box ( b ) has lower entropy than the right sector ( s = r ln(0.666/0.333 ) = 1.3766 cal k mol ) . the right sector in box ( c ) has lower enthalpy than the left sector ( h = 1 kcal mol ) . the number of balls in each sector is shown at the tops of the boxes . the figure was generated using a simulation programmed in visual basic as a macro in an excel worksheet ( see supplemental material ) . notice that the box analogy is quantitatively correct for a single - molecule reaction ( e.g. , 1-propanol 2-propanol in figure 3a ) . for this reaction , the change in standard molar enthalpy ( h ) and entropy ( s ) are known ( 3.63 kcal mol and 3.85 cal mol k , respectively [ stretton , 2005 ] ) . the box in figure 3a was built considering h as the difference in floor levels . the size of both compartments in the box was calculated from the relationship between entropy and probability . teaching thermodynamic concepts with a box representing a real chemical reaction . the difference in floor levels corresponds to the change of standard molar enthalpy of the reaction ( h = 3.63 kcal mol ) . the width of the right and left sectors corresponds to the s ( 3.85 cal mol k ) and was calculated as explained in the section molecular thermodynamics in a box . two hundred balls were distributed in this box following the rules described in figure 2 , and the process was repeated until equilibrium was reached . ( b ) using this box , simulations with 2500 balls were started with 2499 balls in sector i and one ball in sector ii , or one ball in sector i and the rest in sector ii , or 50% of the balls in each sector . the balls were redistributed 100 times ; at each step , the number of balls in the 1-p and 2-p sectors of the previous step was used . inset , the 05% range was enlarged to show that all three conditions converge to 1.57% , corresponding to keq = 62.79 . ( c ) the numbers of balls in the two sectors , as shown in ( b ) , were used to estimate the free energy of the system at different steps ( see the section molecular thermodynamics in a box for details in the calculation ) . notice that g approaches 0 independent of the initial conditions used . ( d ) the table shows the absolute temperature , total number of balls , and g calculated from the known temperature and the changes in entropy and enthalpy of the reaction . simulations with these parameters were run , and keq , g , and g were estimated by averaging the values of the last 60 steps ( where the simulations had reached equilibrium ) . s = r ln(p2/p1 ) , where r is the gas constant ( 1.986 cal mol k ) and p1 and p2 are the relative width of the right and left compartments , respectively ( notice that p1 + p2 = 1 ) . hence , knowing s , the size of the left compartment can be calculated as : p1 = 1/[1 + exp(s/r ) ] , and p2 = 1 p1 ( in figure 3a , p1 = 0.875 and p2 = 0.125 ) . g depends only on the shape of the box and the absolute temperature ( t ) , and it is calculated as g c = h ts,where c stands for calculated ( figure 3d ) . g = g + [ r t ln(m2/m1 ) ] , where m2 and m1 are the number of balls in the right and left compartments , respectively . when students start simulations with this box , g can be negative or positive at the beginning , according to the number of balls initially allocated in each sector ( figure 3 , b and c ) . as the simulation progresses , g approaches 0 and the system reaches equilibrium ( the number of balls moving forward equals the number of balls moving backward ; figure 3c ) . at this stage , m2/m1 fluctuates around the equilibrium constant ( keq ; inset in figure 3b ) . calculated value ( figure 3d ) : where e stands for experimental . students can use the simulation to quantitatively explore the effect of temperature on the equilibrium constant and g ( figure 3d ) . the stochastic nature of the laws of thermodynamics is also evident in all these simulations and can be visualized by changing the number of balls in the box . the fluctuation of g at equilibrium increases as the number of molecules in the simulation decreases ( figure 3d ) . a barrier can be built between the two sectors ( figure 4a ) , such that only balls jumping higher than the barrier can pass to the other sector . students can be prompted to start simulations with all of the balls in one sector and to follow the kinetics of transfer to the other compartment in boxes having different activation energies . this strategy is a useful means of introducing the concept of reaction rate and the influence of activation energy . it also shows that the activation energy does not affect the equilibrium constant ( figure 4b and movie s2 in the supplemental material ) . teaching chemical reaction kinetics with a box . ( a ) the box represents the glucose-6-phosphate ( g6p ) fructose-6-phosphate ( f6p ) reaction with activation energy = 1 kcal mol . the box was drawn assuming that the s for the reaction is close to 0 and h = 0.4 kcal mol . two hundred balls were distributed in this box following the rules described in figure 2 , and the process was repeated until equilibrium was reached ( t = 298 k ) . ( b ) using this box , simulations with different activation energies were initiated with all the balls in the g6p sector ( hence , initial rates correspond to the forward reaction ) . the balls were redistributed 100 times ; at each step , the number of balls in the g6p and f6p sectors of the previous step was used . ( c ) simulations at different temperatures , using the same box and conditions described in ( b ) with an activation energy = 1 kcal mol . the red curve was adjusted to the simulation values for t = 473 k , modeling the reaction in copasi and using the parameter estimation module ( hoops et al . , 2006 ) . ( d ) simulations shown in ( c ) were used to estimate kinetic constants for the forward and backward reactions , as described in ( c ) . these constants were used in an arrhenius plot to estimate activation energies for the forward and backward reactions . notice that the estimated activation energies are close to those shown for the box in ( a ) : 1 kcal mol above the 0.4 kcal mol of the h step for the g6p f6p reaction and 1 kcal mol for the backward reaction . ( e ) a table that compares the equilibrium constants calculated from the free energy of the reaction with the equilibrium constants obtained from the simulations in ( c ) , averaging the last 20 values and from the kinetic constants of the reaction estimated in ( c ) . students can quantitatively explore the effect of temperature on the reaction kinetics using the simulation ( figure 4c and movie s3 in the supplemental material ) . the changes in the proportion of molecules in the two compartments as the simulation progresses follow reversible , first - order reaction kinetics . the kinetic constant for the forward reaction is obtained graphically from the initial rates in simulations , as shown in figure 4b . for example , an a b reaction was modeled in copasi ( www.copasi.org ) , and the parameter estimation routine was used to adjust the values obtained in the simulation to the differential equation dm1/dt = ( k1 m1 ) + ( k2 m2 ) , where k1 and k2 are the forward and backward kinetic constants , respectively ; t is the time variable measured in steps ( figure 4 , c and e ) . students can use the kinetic constants for the forward and backward reactions at different temperatures to calculate the activation energy in an arrhenius plot ( figure 4d ) or to calculate the equilibrium constant of the reaction ( figure 4e ) . the thermodynamic concepts introduced with boxes in the previous sections are sufficient for describing most physicochemical systems , but some additional concepts are required for understanding cell function . the molecular level of understanding is never lost , so nonequilibrium conditions are easily visualized . students can readily understand the necessity of driving an energy - requiring reaction ( this is a reaction with a positive g value ) by coupling it to a reaction that releases energy . for example , if the synthesis of glucose-6-phosphate from glucose and inorganic phosphate with a g = + 3.3 kcal mol is modeled in the box simulation , it is evident that very little product can be obtained , and this can not be changed by manipulating the activation energy of the reaction ( figure 5a ) . on the other hand , the box of the synthesis of glucose-6-phosphate using glucose and atp , with a g = 3.7 kcal mol , has a clear tendency to form the product ( figure 5b ) . an enzyme lowering the activation energy of this reaction would control the synthesis of glucose-6-phosphate . students can play with a three - compartment box representing the two reactions and can analyze the glucose metabolism in cells having different sets of enzymes for catalyzing these reactions ( figure 5c ) . the key concept that enzymes function by decreasing activation energy as a means of guiding molecules through the required metabolic pathways can be visualized and incorporated as a foundational concept in cell biology by using boxes with several compartments isolated by large energy barriers . boxes qualitatively representing the reaction of glucose phosphorylation from ( a ) inorganic phosphate or ( b ) atp are shown . ( c ) a three - compartment box symbolizing the basic strategy for the synthesis of glucose-6-phosphate ( g6p ) in the cell . according to clausius ' interpretation of the second law of thermodynamics , isolated systems spontaneously result in an increase in entropy , which is generally associated with disorder . most people find it hard to understand that this law is compatible with the existence of cells , which are highly ordered systems . the explanation generally used is that every spontaneous process results in an increase in total entropy that is , the entropy of the universe and that thermodynamics therefore does not deny the possibility that a part of the system may become highly ordered if the rest of the system undergoes an increase of entropy that compensates for the local decrease in the ordered sector . although valid , this statement tends not to satisfy students , who would like thermodynamics to provide a more convincing framework explaining why living organisms are so abundant in our world . the relationship between information and energy is key to understanding biological systems ( schneider , 2010 ) . maxwell 's thought experiment is a convenient way to introduce information in a thermodynamic context ( maroney , 2009 ) . starting with a box with two identical sectors having initially the same number of balls , the system will progress to an uneven distribution if a demon can decrease the energy barrier only when balls are coming from the left compartment . this paradox is solved by assuming that the demon has information : it could distinguish molecules coming from the left from molecules coming from the right . the energy required to obtain and use the information preserves the validity of the second law ( schneider , 2010 ) . the system has moved to the higher probability state in the presence of a demon with the necessary information . students can learn about the power and limitations of a demon to manipulate the entropy of a two - compartment box by exploring the excellent interactive simulation modeled by criado and lpez ( 2004 ) . in this simulation , the physical properties of the demon and the information that it handles can be controlled . instructors can use dna synthesis to introduce the central role of information in cell biology . building a dna strand from deoxynucleoside triphosphates however , students are well aware that the cell must synthesize a very specific dna that is , a dna molecule with a unique sequence of nucleotides . the total number of different combinations for a small piece of dna having 600 nucleotides is 4 = 10 . the representation for the synthesis of dna should be a box like the one shown in figure 6 . the left sector represents the 10 1 incorrect combinations , and the one on the right represents the single correct combination ( dna * ) . the cell uses a dna polymerase , which behaves as a maxwell 's demon ( operating under nonequilibrium conditions ) , to solve this problem and synthesize the dna strand with the required nucleotide combination . the addition of a nucleotide to the growing strand has high activation energy ; hence , it will not occur unless an enzyme can decrease this barrier . the dna polymerase will only catalyze the incorporation of a new nucleotide if it matches the template strand , which contains the information used by the demon . so , from the 10 possible combinations , the polymerase will only decrease the activation energy for the synthesis of the complementary strand . the synthesis of all other possible chains is prevented by the large activation energy . in brief , the dna polymerase reads the information coded in the template strand as a means of decreasing the activation energy of only the complementary deoxynucleotide to be incorporated into the growing strand . the student can identify real molecules that behave as maxwell 's demons , limiting the possible outcomes of a process and maintaining the high degree of organization required for the cell . this three - compartment box introduces a maxwell 's demon that , by using the information in the template strand , decreases the activation energy only when the correct deoxynucleotide ( dntp ) is incorporated into the enzymatic complex . the information limits the outcome of the reaction to a unique nucleotide sequence ( dna * ) . the discontinuous lines in the box floor symbolize the large entropy differences between random dna sequences or free deoxynucleotides and the unique dna strand synthesized . balls in a box can in principle be used in different courses and as a means of intuitively introducing thermodynamic principles to students at different levels . as an example , we present the results of using this approach in an introductory bioenergetics class for first - year students at the school of medicine of the national university of cuyo , mendoza , argentina . the design of the tests and the results reported were approved by the medical education committee of the school of medicine . the data collection of past examinations was performed according to the regulations of the school of medicine . the pretest was a voluntary activity for students , who were informed that the test was part of this research , and the posttest was part of the regular activities of the course . , to be admitted into the medical school , a student must attend a course and pass a chemistry exam that includes the topics of chemical equilibrium and reaction rates . since only about 15% of the students are selected ( 120 out of 800 applicants ) , the students are well - trained in these subjects . the class on bioenergetics is part of a 16-wk intensive course in biochemistry and cell biology . the 60-min class is complemented by a small - group exercise ( 20 students ) that requires about 30 min to be completed . the material provided to the students is included here as supplemental material ( bioenergetics ; see also box 1 ) . the results shown were collected from three final examinations ( 20092011 ) , each of which included a question about bioenergetics that was one of a total of 50 questions . as shown in figure 7 , moreover , bioenergetics was not a particularly difficult subject for the students . when the 50 questions were ordered according to the average score obtained , thermodynamics ranked in the 18th , 10th , and 15th place for the 2009 , 2010 , and 2011 exams , respectively . scores for bioenergetics questions in final examinations of a biochemistry and cell biology course for medical students . ( showed that the student understood the concepts required for the item ) , wrong ( failed to show that the student understood the concepts required for the item ) , on the right track ( showed that the students understood some concepts but not all that were required for the item ) , or no answer are the percentages in each category in the 2009 , 2010 , and 2011 exams ( 122 , 127 , and 134 students , respectively ) . english translations of the questions and an example for the classification of answers for one specific question ( 2011-box ) can be found in the supplemental material . box 1 . suggested design for a bioenergetics course employing balls in a box introduce thermodynamic laws based on two intuitive concepts that are familiar to most students : energy and probability.present boxes as a way to represent a set of molecules having energies distributed according to a boltzmann distribution . divide the box in two and explain where enthalpy and entropy are represented in the shape of the box . students can use the simulation to assess the effect of temperature and box shape on the final distribution of balls in the two sectorsintroduce the concepts of standard free energy , free energy , and equilibrium constant using boxes filled with different numbers of balls in each sector and allowing the molecules to reach equilibrium . this simulation is a very useful means of experimentally testing these concepts.introduce reaction kinetics by assessing the rate of transfer of balls between the two sectors . use the simulation for experimentally testing many aspects of these concepts.at this point , present the special challenges that energy and probability represent for biological systems . use boxes with multiple compartments to show how enzymes guide the flow of reactions along the metabolic pathways by lowering the activation energies . multicompartment box simulations have not yet been programmed ; however , the use of boxes as a means of qualitatively representing different pathways is still useful.finally , introduce the role of information in living organisms by using the classical two - compartment thought experiment of maxwell . use dna synthesis as a means of introducing information concepts to a biological system . dna polymerase is an enzyme that students know well , and it is easy to show how it works as a maxwell demon that uses the information in the template strand to guide the synthesis of a unique dna molecule . introduce thermodynamic laws based on two intuitive concepts that are familiar to most students : energy and probability . present boxes as a way to represent a set of molecules having energies distributed according to a boltzmann distribution . divide the box in two and explain where enthalpy and entropy are represented in the shape of the box . students can use the simulation to assess the effect of temperature and box shape on the final distribution of balls in the two sectors introduce the concepts of standard free energy , free energy , and equilibrium constant using boxes filled with different numbers of balls in each sector and allowing the molecules to reach equilibrium . introduce reaction kinetics by assessing the rate of transfer of balls between the two sectors . the representation of activation energy as a barrier between the sectors is very intuitive . introduce the concept of enzymes as regulators of the activation energy . use the simulation for experimentally testing many aspects of these concepts . at this point , use boxes with multiple compartments to show how enzymes guide the flow of reactions along the metabolic pathways by lowering the activation energies . multicompartment box simulations have not yet been programmed ; however , the use of boxes as a means of qualitatively representing different pathways is still useful . finally , introduce the role of information in living organisms by using the classical two - compartment thought experiment of maxwell . use dna synthesis as a means of introducing information concepts to a biological system . dna polymerase is an enzyme that students know well , and it is easy to show how it works as a maxwell demon that uses the information in the template strand to guide the synthesis of a unique dna molecule . to assess whether the balls in a box approach could improve understanding of a subject the student had previously learned , a pre / posttest was administered that was limited to chemical equilibrium and reaction rates . it is worth noting that the bioenergetics class was not focused on these subjects , since it covered broader aspects of thermodynamics in biological systems . for the test , similar questions were asked before and after the class as part of a weekly quiz ( see supplemental material ) . as shown in figure 8 , most of the students improved their understanding , even in concepts that they had studied previously using other approaches . on average , they performed better in five out of eight questions , and they improved their overall score in the quiz ( data not shown ) . however , in one aspect , the box representation was less helpful than the methods that had been used previously ( figure 8d ) . in the box , the effect of a rise of temperature in an endothermic or exothermic reaction is inferred by imaging the number of balls in each compartment when the temperature is modified . however , this concept was taught in the chemistry course with a still simpler ( although less flexible ) approach , in which heat is considered a reactant or a product for endothermic or exothermic reactions , respectively . the result of this pre / postassessment will be used to improve the bioenergetics class in future years by incorporating previous concepts learned with different approaches . in conclusion , even for a single class , which is not the best scenario to introduce an innovative representation for complex processes , the balls in a box approach was an effective method to teach critical concepts about thermodynamics . pre / posttest to evaluate the box method as a means of teaching the topics of chemical equilibrium and reaction rate . similar questions were asked before and after the bioenergetics class as part of the weekly quiz . the difference of pre / posttest answers for each question was compared using a wilcoxon signed - rank test for paired observations ( * , p < 0.05 ; * * , p < 0.01 ) . thermodynamics is a fundamental discipline that enables us to understand how energy is handled by living organisms . however , what students need to learn is by no means trivial ; what is needed is a conceptual understanding of how nonequilibrium systems at the micron scale are maintained in pseudo - steady - state conditions by very sophisticated processes handling large amounts of energy and information . many of the concepts are better understood by considering that the laws of thermodynamics are based on the stochastic behavior of large sets of molecules . students ( and most biologists at any level , for that matter ) are used to thinking about molecules as the major players in most events in cellular and molecular biology . boxes are useful as a means of applying thermodynamics to molecules without using statistical mechanics . hence , it is a good way for students to incorporate concepts of thermodynamics into their molecular world . with boxes , the basic concepts of entropy , enthalpy , free energy , standard free energy , and activation energy can be taught while maintaining a connection with the behavior of individual molecules . a very nice simulation of balls interacting in a box that keeps track of the speed and kinetic energy of individual molecules is freely available ( lemaster , 2011 ) . this simulation shows a more realistic view of molecules moving at different temperatures and can be used to complement the introduction of the boxes mimicking entropy / enthalpy differences . moreover , the boxes represent a very intuitive means of introducing nonequilibrium states and maxwell 's demons . students are familiar with enzymatic complexes that are perfect examples of maxwell 's demons ( e.g. , dna polymerase , rna polymerase , ribosomes ) and know much about how these demons work . students can then easily grasp the concept of how information is used to guide a process to a final outcome that a priori would be predicted to be very unlikely . in conclusion , we propose boxes as a very valuable strategy , especially when an intuitive framework of thermodynamics for biology must be taught in a short time . for more extended courses in bioenergetics , boxes may be useful tools to introduce some concepts and to simulate simple reactions , but then equations and real metabolic processes should be emphasized to ensure that students genuinely understand cell physiology . after all , balls bouncing in a box do not really represent all the complexity of the biochemical reactions required to maintain a cell .

thermodynamic principles are basic to an understanding of the complex fluxes of energy and information required to keep cells alive . these microscopic machines are nonequilibrium systems at the micron scale that are maintained in pseudo - steady - state conditions by very sophisticated processes . therefore , several nonstandard concepts need to be taught to rationalize why these very ordered systems proliferate actively all over our planet in seeming contradiction to the second law of thermodynamics . we propose a model consisting of boxes with different shapes that contain small balls that are in constant motion due to a stream of air blowing from below . this is a simple macroscopic system that can be easily visualized by students and that can be understood as mimicking the behavior of a set of molecules exchanging energy . with such boxes , the basic concepts of entropy , enthalpy , and free energy can be taught while reinforcing a molecular understanding of the concepts and stressing the stochastic nature of the thermodynamic laws . in addition , time - related concepts , such as reaction rates and activation energy , can be readily visualized . moreover , the boxes provide an intuitive way to introduce the role in cellular organization of information and maxwell 's demons operating under nonequilibrium conditions .

INTRODUCTION MOLECULAR THERMODYNAMICS IN A BOX CHEMICAL REACTION KINETICS IN A BOX THERMODYNAMICS FOR CELL BIOLOGY IN A BOX USE OF BOXES IN AN UNDERGRADUATE COURSE CONCLUSIONS Supplementary Material

education in the broad and complex field of biology needs to be founded on some core concepts ( klymkowsky , 2010 ) . cells are the basic units of life ; hence , how cells work and the molecular mechanisms involved in cell physiology are an essential part of the curricula of all undergraduate programs in the biological sciences . as we understand them , cells are incredibly sophisticated , self - sufficient , microscopic machines . therefore , basic concepts of thermodynamics are essential to an understanding of the molecular mechanisms underlying all cell functions ( klymkowsky , 2010 ) . although a qualitative understanding would be sufficient for most biology students , the relevant thermodynamic principles are not the standard ones that apply to simple thermal engines . therefore , several nonstandard concepts need to be taught to explain why these very ordered systems proliferate actively all over our planet despite their apparent disregard of the second law of thermodynamics . in contrast , entropy , enthalpy , and free energy are abstract concepts that students can learn to express in an equation and use to get a result without really grasping the important concepts . and as soon as they forget the formula , they are likely to forget most of the underlying thermodynamic principles , except that energy is somehow important . students would almost certainly incorporate these concepts more readily if they had an intuitive idea of the molecular basis of the laws of thermodynamics . the goal is to teach the thermodynamic concepts necessary for understanding how cells handle energy , while keeping mathematics to a level accessible to the students . a useful strategy for this is to work with a simple macroscopic system that can be readily visualized by the students and that behaves as a set of molecules exchanging energy during a process . the idea presented here is an extension of the boxes used in the cell biology textbook the world of the cell ( becker et al . the strategy is to picture a box containing small balls that are in constant movement due to a stream of air blowing from below ( figure 1 ) . the collisions of balls against the box walls and against one another are not elastic ; hence , if the airstream is turned off , the balls will rapidly lose energy and come to rest on the floor of the box . in the four boxes in figure 1 , the stream of air is blowing with increasing strength from left to right . in this system , we can think of balls as molecules having different levels of energy following a boltzmann distribution . the important concept is that the number of molecules at different energy levels ( i.e. the influence of the temperature in this distribution is also very intuitive . a movie generated with this simulation shows that the distribution of the balls in the box is dynamic and changes with temperature ( see movie s1 in the supplemental material ) . two hundred balls were distributed in a two - dimensional box according to the following rules : for each ball , the x position was randomly selected within the limits of the box . the y coordinate ( energy ) was randomly selected as y = r t ln(rnd ) , where r is 1.986 cal k mol , t is the absolute temperature in degrees kelvin , and rnd is a random number in the 01 range . now , if two sectors of the box are considered , the number of molecules in each sector depends on the area of each sector and on the floor level of the sector . in this macroscopic system , also , the influence of temperature on the distribution of molecules can be intuitively inferred . in other words , students should be able to grasp the concept of free energy based on the distribution of balls in boxes with different shapes . two hundred balls were distributed in two - dimensional boxes divided into two sectors according to the following rules and considering an absolute temperature of 310 k. for each ball initially in sector i ( or sector ii ) , the y coordinate ( energy ) was randomly selected as y = hi r t ln(rnd ) , where hi is the floor level for the ball 's sector ( 0 for sector i and h for sector ii ) and rnd is a random number in the 01 range . if the height of the y coordinate was higher than the energy step necessary to pass to the other sector , the x coordinate was randomly selected within the limits of the entire box , such that the wider sector received proportionally more balls . if the y coordinate was lower than the energy step , the x coordinate was randomly selected within the limits of the sector where the ball was located initially . the number of balls in each sector is shown at the tops of the boxes . for this reaction , the change in standard molar enthalpy ( h ) and entropy ( s ) are known ( 3.63 kcal mol and 3.85 cal mol k , respectively [ stretton , 2005 ] ) . the difference in floor levels corresponds to the change of standard molar enthalpy of the reaction ( h = 3.63 kcal mol ) . the width of the right and left sectors corresponds to the s ( 3.85 cal mol k ) and was calculated as explained in the section molecular thermodynamics in a box . the balls were redistributed 100 times ; at each step , the number of balls in the 1-p and 2-p sectors of the previous step was used . ( c ) the numbers of balls in the two sectors , as shown in ( b ) , were used to estimate the free energy of the system at different steps ( see the section molecular thermodynamics in a box for details in the calculation ) . ( d ) the table shows the absolute temperature , total number of balls , and g calculated from the known temperature and the changes in entropy and enthalpy of the reaction . simulations with these parameters were run , and keq , g , and g were estimated by averaging the values of the last 60 steps ( where the simulations had reached equilibrium ) . hence , knowing s , the size of the left compartment can be calculated as : p1 = 1/[1 + exp(s/r ) ] , and p2 = 1 p1 ( in figure 3a , p1 = 0.875 and p2 = 0.125 ) . g depends only on the shape of the box and the absolute temperature ( t ) , and it is calculated as g c = h ts,where c stands for calculated ( figure 3d ) . when students start simulations with this box , g can be negative or positive at the beginning , according to the number of balls initially allocated in each sector ( figure 3 , b and c ) . the stochastic nature of the laws of thermodynamics is also evident in all these simulations and can be visualized by changing the number of balls in the box . the fluctuation of g at equilibrium increases as the number of molecules in the simulation decreases ( figure 3d ) . a barrier can be built between the two sectors ( figure 4a ) , such that only balls jumping higher than the barrier can pass to the other sector . students can be prompted to start simulations with all of the balls in one sector and to follow the kinetics of transfer to the other compartment in boxes having different activation energies . this strategy is a useful means of introducing the concept of reaction rate and the influence of activation energy . ( b ) using this box , simulations with different activation energies were initiated with all the balls in the g6p sector ( hence , initial rates correspond to the forward reaction ) . the balls were redistributed 100 times ; at each step , the number of balls in the g6p and f6p sectors of the previous step was used . ( c ) simulations at different temperatures , using the same box and conditions described in ( b ) with an activation energy = 1 kcal mol . the red curve was adjusted to the simulation values for t = 473 k , modeling the reaction in copasi and using the parameter estimation module ( hoops et al . notice that the estimated activation energies are close to those shown for the box in ( a ) : 1 kcal mol above the 0.4 kcal mol of the h step for the g6p f6p reaction and 1 kcal mol for the backward reaction . ( e ) a table that compares the equilibrium constants calculated from the free energy of the reaction with the equilibrium constants obtained from the simulations in ( c ) , averaging the last 20 values and from the kinetic constants of the reaction estimated in ( c ) . for example , an a b reaction was modeled in copasi ( www.copasi.org ) , and the parameter estimation routine was used to adjust the values obtained in the simulation to the differential equation dm1/dt = ( k1 m1 ) + ( k2 m2 ) , where k1 and k2 are the forward and backward kinetic constants , respectively ; t is the time variable measured in steps ( figure 4 , c and e ) . students can use the kinetic constants for the forward and backward reactions at different temperatures to calculate the activation energy in an arrhenius plot ( figure 4d ) or to calculate the equilibrium constant of the reaction ( figure 4e ) . the molecular level of understanding is never lost , so nonequilibrium conditions are easily visualized . students can readily understand the necessity of driving an energy - requiring reaction ( this is a reaction with a positive g value ) by coupling it to a reaction that releases energy . for example , if the synthesis of glucose-6-phosphate from glucose and inorganic phosphate with a g = + 3.3 kcal mol is modeled in the box simulation , it is evident that very little product can be obtained , and this can not be changed by manipulating the activation energy of the reaction ( figure 5a ) . on the other hand , the box of the synthesis of glucose-6-phosphate using glucose and atp , with a g = 3.7 kcal mol , has a clear tendency to form the product ( figure 5b ) . the key concept that enzymes function by decreasing activation energy as a means of guiding molecules through the required metabolic pathways can be visualized and incorporated as a foundational concept in cell biology by using boxes with several compartments isolated by large energy barriers . according to clausius ' interpretation of the second law of thermodynamics , isolated systems spontaneously result in an increase in entropy , which is generally associated with disorder . the explanation generally used is that every spontaneous process results in an increase in total entropy that is , the entropy of the universe and that thermodynamics therefore does not deny the possibility that a part of the system may become highly ordered if the rest of the system undergoes an increase of entropy that compensates for the local decrease in the ordered sector . the relationship between information and energy is key to understanding biological systems ( schneider , 2010 ) . maxwell 's thought experiment is a convenient way to introduce information in a thermodynamic context ( maroney , 2009 ) . starting with a box with two identical sectors having initially the same number of balls , the system will progress to an uneven distribution if a demon can decrease the energy barrier only when balls are coming from the left compartment . the energy required to obtain and use the information preserves the validity of the second law ( schneider , 2010 ) . the system has moved to the higher probability state in the presence of a demon with the necessary information . in this simulation , the physical properties of the demon and the information that it handles can be controlled . instructors can use dna synthesis to introduce the central role of information in cell biology . the left sector represents the 10 1 incorrect combinations , and the one on the right represents the single correct combination ( dna * ) . the cell uses a dna polymerase , which behaves as a maxwell 's demon ( operating under nonequilibrium conditions ) , to solve this problem and synthesize the dna strand with the required nucleotide combination . the addition of a nucleotide to the growing strand has high activation energy ; hence , it will not occur unless an enzyme can decrease this barrier . so , from the 10 possible combinations , the polymerase will only decrease the activation energy for the synthesis of the complementary strand . in brief , the dna polymerase reads the information coded in the template strand as a means of decreasing the activation energy of only the complementary deoxynucleotide to be incorporated into the growing strand . the student can identify real molecules that behave as maxwell 's demons , limiting the possible outcomes of a process and maintaining the high degree of organization required for the cell . this three - compartment box introduces a maxwell 's demon that , by using the information in the template strand , decreases the activation energy only when the correct deoxynucleotide ( dntp ) is incorporated into the enzymatic complex . the information limits the outcome of the reaction to a unique nucleotide sequence ( dna * ) . as an example , we present the results of using this approach in an introductory bioenergetics class for first - year students at the school of medicine of the national university of cuyo , mendoza , argentina . the data collection of past examinations was performed according to the regulations of the school of medicine . the pretest was a voluntary activity for students , who were informed that the test was part of this research , and the posttest was part of the regular activities of the course . , to be admitted into the medical school , a student must attend a course and pass a chemistry exam that includes the topics of chemical equilibrium and reaction rates . since only about 15% of the students are selected ( 120 out of 800 applicants ) , the students are well - trained in these subjects . the class on bioenergetics is part of a 16-wk intensive course in biochemistry and cell biology . the results shown were collected from three final examinations ( 20092011 ) , each of which included a question about bioenergetics that was one of a total of 50 questions . as shown in figure 7 , moreover , bioenergetics was not a particularly difficult subject for the students . when the 50 questions were ordered according to the average score obtained , thermodynamics ranked in the 18th , 10th , and 15th place for the 2009 , 2010 , and 2011 exams , respectively . ( showed that the student understood the concepts required for the item ) , wrong ( failed to show that the student understood the concepts required for the item ) , on the right track ( showed that the students understood some concepts but not all that were required for the item ) , or no answer are the percentages in each category in the 2009 , 2010 , and 2011 exams ( 122 , 127 , and 134 students , respectively ) . english translations of the questions and an example for the classification of answers for one specific question ( 2011-box ) can be found in the supplemental material . suggested design for a bioenergetics course employing balls in a box introduce thermodynamic laws based on two intuitive concepts that are familiar to most students : energy and probability.present boxes as a way to represent a set of molecules having energies distributed according to a boltzmann distribution . students can use the simulation to assess the effect of temperature and box shape on the final distribution of balls in the two sectorsintroduce the concepts of standard free energy , free energy , and equilibrium constant using boxes filled with different numbers of balls in each sector and allowing the molecules to reach equilibrium . multicompartment box simulations have not yet been programmed ; however , the use of boxes as a means of qualitatively representing different pathways is still useful.finally , introduce the role of information in living organisms by using the classical two - compartment thought experiment of maxwell . use dna synthesis as a means of introducing information concepts to a biological system . dna polymerase is an enzyme that students know well , and it is easy to show how it works as a maxwell demon that uses the information in the template strand to guide the synthesis of a unique dna molecule . introduce thermodynamic laws based on two intuitive concepts that are familiar to most students : energy and probability . present boxes as a way to represent a set of molecules having energies distributed according to a boltzmann distribution . divide the box in two and explain where enthalpy and entropy are represented in the shape of the box . students can use the simulation to assess the effect of temperature and box shape on the final distribution of balls in the two sectors introduce the concepts of standard free energy , free energy , and equilibrium constant using boxes filled with different numbers of balls in each sector and allowing the molecules to reach equilibrium . introduce the concept of enzymes as regulators of the activation energy . multicompartment box simulations have not yet been programmed ; however , the use of boxes as a means of qualitatively representing different pathways is still useful . finally , introduce the role of information in living organisms by using the classical two - compartment thought experiment of maxwell . use dna synthesis as a means of introducing information concepts to a biological system . dna polymerase is an enzyme that students know well , and it is easy to show how it works as a maxwell demon that uses the information in the template strand to guide the synthesis of a unique dna molecule . to assess whether the balls in a box approach could improve understanding of a subject the student had previously learned , a pre / posttest was administered that was limited to chemical equilibrium and reaction rates . as shown in figure 8 , most of the students improved their understanding , even in concepts that they had studied previously using other approaches . however , in one aspect , the box representation was less helpful than the methods that had been used previously ( figure 8d ) . in the box , the effect of a rise of temperature in an endothermic or exothermic reaction is inferred by imaging the number of balls in each compartment when the temperature is modified . the result of this pre / postassessment will be used to improve the bioenergetics class in future years by incorporating previous concepts learned with different approaches . in conclusion , even for a single class , which is not the best scenario to introduce an innovative representation for complex processes , the balls in a box approach was an effective method to teach critical concepts about thermodynamics . thermodynamics is a fundamental discipline that enables us to understand how energy is handled by living organisms . however , what students need to learn is by no means trivial ; what is needed is a conceptual understanding of how nonequilibrium systems at the micron scale are maintained in pseudo - steady - state conditions by very sophisticated processes handling large amounts of energy and information . many of the concepts are better understood by considering that the laws of thermodynamics are based on the stochastic behavior of large sets of molecules . students ( and most biologists at any level , for that matter ) are used to thinking about molecules as the major players in most events in cellular and molecular biology . hence , it is a good way for students to incorporate concepts of thermodynamics into their molecular world . with boxes , the basic concepts of entropy , enthalpy , free energy , standard free energy , and activation energy can be taught while maintaining a connection with the behavior of individual molecules . this simulation shows a more realistic view of molecules moving at different temperatures and can be used to complement the introduction of the boxes mimicking entropy / enthalpy differences . moreover , the boxes represent a very intuitive means of introducing nonequilibrium states and maxwell 's demons . students are familiar with enzymatic complexes that are perfect examples of maxwell 's demons ( e.g. students can then easily grasp the concept of how information is used to guide a process to a final outcome that a priori would be predicted to be very unlikely . in conclusion , we propose boxes as a very valuable strategy , especially when an intuitive framework of thermodynamics for biology must be taught in a short time . for more extended courses in bioenergetics , boxes may be useful tools to introduce some concepts and to simulate simple reactions , but then equations and real metabolic processes should be emphasized to ensure that students genuinely understand cell physiology . after all , balls bouncing in a box do not really represent all the complexity of the biochemical reactions required to maintain a cell .

recently , dna chip or microarray technology has been developed that allows researchers to measure the expression levels of thousands of genes simultaneously over different time points , different experimental conditions or different tissue samples . it is based on the hybridization of dna or rna molecules with a library of complementary strands fixed on a solid surface . oligonucleotide chips contain thousands of features with gene - specific sequences about 25 bases long . these oligos are then hybridized with labeled probe derived from a given tissue or cell line . complex mrna probes are reverse transcribed to cdna and labeled with red or green fluorescent dyes . this technique is often called the spotted array or cdna array . in both methods , thousands of mrna concentrations can be measured in parallel , potentially revealing complex gene regulatory networks . one important application of the microarray gene expression data in medicine is to study the relationship between tissue phenotypes and gene expression profiles on the whole - genome scale . the phenotype could be several different types of cancers [ 1 , 2 , 3 ] , responses of cell lines to different chemical compounds , or time to tumor recurrence after treatment . for binary phenotypes such as two different types of cancers , it has been suggested that gene expression may provide the additional information needed to improve cancer classification and diagnosis . for continuous phenotypes such as drug sensitivity , the problem of interest is to relate gene expression patterns to sensitivity to drugs and , therefore , aid in the process of drug discovery and provide a rationale for selection of therapy on the basis of the molecular characteristics of a patient 's tumor . from the statistical point of view , the challenge is that the microarray gene expression data are often measured with a great deal of noise , and that the sample size of tissues or cell lines , denoted by n , is usually very small compared to the number of genes in expression arrays , denoted by p. this results in the ' large p , small n ' problem . most current approaches to dealing with this problem first select genes that can best separate tissues of different types by performing univariate analysis . the expression levels of these genes are then combined linearly in a weighted way to form compound covariates . these covariates are then used in the standard regression models for model fitting and prediction . proposed a bayesian binary regression approach using the singular value decomposition to first reduce the dimension of the variable space ( p ) to the number of samples ( n ) . they called the resulting linear combination of the expression levels of all the genes the expression of the ' supergenes ' . all these approaches reduce the variable space by making one or several linear combinations of the expression levels of some or all of the genes . linear combination may not , however , be the best way of reducing the dimension of the variable space . another popular approach to analyzing gene expression data is to use clustering methods to simultaneously cluster both samples and genes in order to determine some clusters of genes that are mostly correlated with some clusters of samples . examples of such an application include analysis of gene expression data and drug response for the 60 human cancer cell lines of the national cancer institute ( nci60 data ) , and analysis of cancerous and normal colon tissues . however , the clustering approach is purely exploratory and requires an external similarity measure . methods that can be used to assess the significance of the clustering results are needed . the rasch model ( rm ) and its extensions [ 6 , 7 ] are an important staple of psychological research and are used in other fields such as sociology , educational testing and medicine . the idea of the rm is that one can indirectly infer a person 's position on a latent trait from his / her responses to a set of well - chosen items . for example , the rm has been used to infer the quality of life of cancer patients from their answers to a well - designed questionnaire , or to measure disability from activities of daily living . for these applications , data are usually given in a matrix , with rows being individuals and columns being responses to a set of items . microarray gene expression data are also given in a transposable matrix form with rows being genes and columns being samples , and vice versa . the rm can therefore be used to explain the observed patterns over different columns . here we propose two different formulations of the polytomous rm for analysis of microarray gene expression data . the first formulation treats samples as ' persons ' and genes as ' items ' . the idea is to infer several latent factors associated with a given sample on the basis of its expression profile over many genes . we combine a model - based clustering method with the rm to define a small set of latent factors associated with samples . for a given sample , we assume that genes in the same cluster determine one latent factor associated with this sample , and use the rm to estimate this latent factor for each gene cluster . the rational of this approach is that genes of similar function yield similar expression patterns in microarray hybridization experiments [ 11 , 12 , 13 ] . co - regulated genes may share similar expression profiles , maybe involved in related functions or regulated by common regulatory elements . therefore , if genes are clustered together , it is impossible from a statistical point of view to differentiate one gene from the other . in this case , consideration of genes in the same cluster can potentially reduce noise associated with a single gene . the second formulation is to treat genes as ' persons ' and samples as ' items ' . the idea is to infer several latent factors associated with each gene based on its expression levels across samples from different tissue or cell types . this formulation provides simple summary statistics for genes based on their expression profiles over samples , and helps to identify genes that are more likely be over- or underexpressed within samples of the same type or between samples of different types . we apply our proposed methods to the analysis of the leukemia dataset and the nci60 dataset and conclude with discussion of our method . the rm was originally proposed as an item - response theory model in the psychological test or attitude scale . the idea is that the use of a test or scale presupposes that one can indirectly infer a person 's position on a latent trait from his / her responses to a set of well - chosen items . let zij be the response of individual i to the item j , where the response can take one from m + 1 possible ordinal categories , 0 , ... , m. one version of the rm , which we use in this paper , called the partial credit model , assumes the probability of response h , as for i = 1 , ... , i , j = 1 , ... , j , and h = 0 , 1 , ... , m , where jl is the item - specific parameter , which expresses the attractiveness of the respective level l of item j. i is the person parameter that expresses the latent factor of the ith person that is measured by the j items . it is easy to verify that the probability of the response is monotonous in both person and item parameters . for example , for m = 3 , figure 1 plots the rasch probabilities as a function of the value of the latent factor ( a ) for two sets of item - specific values . it can be seen from these plots that for a given item , persons with larger value tend to have greater probability of expressing high scores , and for a given person , the response probabilities are different for items with different values . to make the model ( 1 ) identifiable , the following constraints are required example of rasch probabilities as a function of the value of the latent factor for an item with four different response categories for two different sets of item - specific parameters ( a ) = ( 0.3,0.5,-0.5,0 ) and ( b ) = ( -0.3,-0.5,0.5,0 ) . therefore , there is a total of jm - 1 unconstraint item - specific parameters . the item parameters can be estimated based on the conditional likelihood , given minimal sufficient statistics for the person parameters . for a given person , the minimal sufficient statistic is the sum of the category weights corresponding to the observed responses . after the parameters are estimated , the person parameters can then be estimated by maximizing the likelihood function . details on the conditional likelihood estimation of the item parameters can be found in anderson . typical microarray data consist of expression levels for a large number of genes on a relatively small number of samples . let xij be the gene expression level of the jth gene in the ith sample , for i = 1 , ... , , n is usually much smaller than p. for spotted arrays , to moderate the influence of gene expression ratios above and below one , we may apply the natural log transform to all the red to green ratios . upregulated genes thus have a positive log expression ratio , whereas downregulated genes have a negative log expression ratio . in addition , for the ith sample , we have observed phenotype yi , which could be a binary indicator such as two different types of cancer , a continuous measurement such as drug - response activity or censored survival time such as time to tumor recurrence . to apply the rm to the gene expression data xij , we first need to discretize the gene expression levels xij into zij , which takes value from 0 , ... , m , for i = 1 , ... , n , j = 1 , ... , p. in practice , we can use the quantiles or the quantiles within quantiles as cut - off points for discretization . because this approach uses only ranks rather than the actual expression levels , there may be slight loss of information . however , in return , we gain a valid analysis with robustness to the outliers . our goal is to infer several latent factors associated with each sample based on its gene expression profile , and relate these latent factors to the observed phenotypes . using the terms of the rm , we treat each gene as an ' item ' , each tissue sample or cell line as a ' person ' , and treat the expression level as the response of a given tissue to a given gene . the unidimensional rm may not , however , hold for the complete set of genes generated by microarrays . here we assume that genes with similar expressions determine one latent factor , and that the rm holds for each set of genes with similar expression profiles . to identify genes with similar expression profiles over samples , we first use the model - based clustering method of fraley and raftery to cluster p genes into k clusters based on their gene expression profiles over n samples . note that the cluster - step is performed based on the observed continuous gene expression data , not the discretized gene expression patterns . for a given sample , using expression profiles of genes in a given cluster , we estimate a latent factor by fitting a rm . these latent factors are then used in a regression analysis to study the relationship between the gene expressions and the phenotype . the method allows to investigate whether a cluster of genes with similar expression profiles is related to the observed phenotypes , and can also be used for future prediction by estimating the latent factors using the maximum likelihood estimation . cluster analysis , based on multivariate normal mixture models [ 10 , 17 ] , has been used for clustering various types of biological , zoological , financial and industrial data . let xj = { x1j , ... , xnj } be the n - dimensional vector of the jth gene expression over n samples . we assume that the gene expression values of p genes , x1 , ... , xp , arise from a mixture of k n - dimensional gaussian distribution with density where the k is the probability that a gene belongs to the kth cluster , and n(x|k,k ) denotes the density function of the multivariate normal distribution with mean k and variance - covariance matrix k possible parameterization of the covariance matrix is discussed in fraley and raftery . note that if we assume a simple covariance structure , k = i , where i is the identity matrix , and is the variance , then the model - based clustering method becomes the k - means clustering method . treating clustering as a mixture model problem allows us to use the em algorithm to estimate the probability of a given gene belonging to each of the k clusters , and to estimate the corresponding mean vector and covariance matrix for each cluster . one advantage of this approach is that it allows us to obtain an estimate of the number of gene clusters . following fraley and raftery , we propose to use the bayesian inference criterion ( bic ) for selecting the number of gene clusters . bic is defined as bic(k ) = 2l(k ) - nk log p , where l(k ) is the maximized log - likelihood , nk is the number of independent parameters to be estimated in the k - cluster model and p is the sample size ( number of the genes ) as the first step of our approach , we cluster genes into k clusters using the model - based clustering method described above , where the number of gene clusters k is determined by maximizing the bic scores . let ck denote the genes in cluster k , and pk denote the number of genes in this cluster , for k = 1 , ... , k. we fit a rm model as in equation ( 1 ) for genes in each of the k clusters respectively , treating samples as ' persons ' and genes as ' items ' . to fit the rm ( equation 1 ) for genes in the kth cluster , we let i be the sample index , and j be the gene index , for i = 1 , ... , n , and j = 1 , ... , pk , and let i = ik in model ( 1 ) be the latent factor for the ith sample which is determined by the genes in the kth cluster , and jl be the gene - specific parameter for the jth gene . the rm assumes that the variation of the gene expression patterns observed over different samples is due to a latent factor , and it provides a probabilistic model to describe the gene expression pattern for a given sample . let ik be the maximum likelihood estimate of the latent factor for the ith sample determined by the genes in the kth cluster . let i = ( i1 , ... , ik ) be the vector of the estimated latent factors based on the gene expression profiles for the ith sample . in general , we can relate the phenotype yi for the ith sample to the estimated of latent factors i by a regression model , for i = 1 , ... , n , where is the vector of regression parameters , i , is the error term , and the actual model of the regression function ( f ) and the distribution of the error depend on type of the phenotype . if the phenotype is a continuous variable , linear regression can be used , if it is a binary variable , logistic or probit regression can be used , and if the phenotype is survival time , the cox regression model can be used . one advantage of the proposed method is that we can model the interactions between the latent factors in the standard way of modeling interactions in the regression models . as only the estimated and not the observed latent factors are available in the regression model ( equation 3 ) , the variance of the estimate of the parameter has to be corrected . we propose to use a two - step bootstrap resampling procedure to estimate the variance of the estimate of the parameter in the regression model . first , within the kth gene cluster , we resample genes and re - estimate the ik parameter by fitting the rm ( 1 ) . second , for a given set of estimated parameters , we resample the n samples from ( yi , i ) , for i = 1 , ... , n , and fit the regression model ( equation 3 ) to obtain a new estimate of . we can then estimate the variance of with these resampled estimates . for a new sample with gene expression xnew = ( xnew,1, ... ,xnew , p ) , these p genes are first divided into k clusters according to the clustering result . for a gene j in cluster k , we first discretize its expression level into one of the m + 1 categories using the cut - off points used in the discretization - step , denoted by znew , j . we can then estimate the corresponding latent factor k by maximizing the following likelihood function , where jh is the estimated gene - specific parameter for gene j in the kth cluster based on the training sample . using the estimated vector of the latent factors , the regression model ( 3 ) the second formulation of the rm for gene expression data is to treat genes as ' persons ' , and samples as ' items ' . assume that we have gene expression data of p different genes , indexed by i , over nk samples of the kth sample type , indexed by j , for k = 1 , ... , k. note that the indices i and j are used differently from the previous sections . we are interested in identifying genes that are expressed differently among these different sample types . here for gene i , we can estimate a latent factor ik based on its gene expression profile over nk samples from the kth sample type by fitting the rm ( equation 1 ) , for k = 1 , ... , k. in this formulation of the rm , we treat genes as ' persons ' , treat samples as ' items ' , and treat each gene 's expression level over samples as the responses . in rm ( 1 ) , i = p , and j = nk , i = ik , which is the latent factor for the ith gene determined by the samples of the kth type , and jl is the sample - specific parameter . this model assumes that the variation of gene expression patterns across different samples among different genes is due to several gene - specific latent factors . here the latent factor ik can be interpreted as some quantities related to the transcription factors of the ith gene which determine the gene expression levels in samples of the kth sample type . for a given tissue or cell line type k , genes with larger estimated latent factor ( ik ) tend to have higher expression levels than those with smaller estimated latent factor . for a given sample type k , the estimated latent factors , ( 1k } , ... , pk ) , provide a nice way to order genes based on their expression levels over a small number of samples of sample type k , and to identify genes that are relatively over- or underexpressed in the kth sample type . in addition , by comparing the estimated latent factors associated with genes across different sample types , we can identify genes that are differentially expressed among different tissue or cell line types . acute leukemias can broadly be divided into two classes , acute myeloid leukemia ( aml ) and acute lymphoblastic leukemia ( all ) , that originate , respectively , from cells of myeloid or lymphoid origin . the two diseases appear identical under the microscope . however , correct diagnosis is critical , as they respond best to different treatment regimens . golub et al . used a set of 38 leukemia samples including 11 aml and 27 all as training samples set , and used an additional 34 samples ( 14 aml , 20 all ) as a test set for testing their proposed method for class prediction . in our analysis , we combined both the training and the test datasets into a dataset of 72 samples ( 25 aml and 47 all ) . for each of the 72 samples , the gene expression data were extracted from affymetrix expression arrays . we first selected the subset of 3,571 genes based on an initial processing adopted by the authors of the leukemia study . the expressions summarized are the log ( base 10 ) values of the actual expression levels following this initial filtering and transformation . we then select 50 genes that are mostly over - expressed in aml , and 50 genes that are mostly overexpressed in all by using the wilcoxon rank sums test . this simple rule of selecting a smaller set of genes are also used in golub et al . using slightly different tests . as expected , the model - based clustering method assuming the common covariance matrix clusters these 100 genes into two clusters , with 50 genes in each cluster . the left plot of figure 2 shows the gene expression levels of these 100 genes for the 72 leukemia samples . clearly , these 100 genes are highly differentially expressed between the two types of the leukemia samples . ( a ) log ( base 10 ) of gene expression levels of 100 genes chosen using the wilcox rank sum tests for the leukemias dataset . the first 47 samples along the x - axis are all , the next 25 samples are aml . ( b ) plot of two latent factors estimated using the rasch model for all 72 samples based on their gene expression profiles over 100 genes selected . given the 100 genes selected , methods such as principal component analysis , partial least - square regression or composite covariate predictor can be used to further reduce the gene dimension to two or three dimensions by taking linear combinations of the gene expression levels . instead of taking linear combination of gene expression levels , we first discretize the expression levels of all the 100 genes over 72 samples into four categories using the quantiles as the cut - off points . therefore , for each gene , their expression level can take one of four possible values of 0 , 1 , 2 and 3 . the same analysis was also done by discretizing gene expression levels into eight categories , the results were essentially the same . in the following fitting two rms to these discretized gene expression levels , we estimate two latent factors for each sample ; one latent factor is determined by gene expression profiles of 50 genes in one cluster , the other is determined by the gene expression profiles of 50 genes in another cluster . the right panel of figure 2 shows the estimated values of these two latent factors for all the 72 samples . this plot shows that the two leukemia types are well separated by these two latent factors , with no overlap , except that two leukemia samples , one from all group and the other from aml group , are close to each other in this two - dimensional space . discriminant analysis using these two latent factors would expect to perform very well in classification . we performed a leave - one - out cross validation analysis to estimate the misclassification rate . specifically , we leave one sample out , and first estimate the sample - specific latent factors ik for the ith gene for k = 1,2 and gene - specific parameter jl using the remaining samples . we then estimate the latent factors of the left - out sample by maximizing the likelihood function ( equation 4 ) . fisher 's linear discriminant analysis using the estimated latent factors was then used to classify the left - out sample . the above procedure was applied to each of the 72 samples , and resulted in a misclassification rate of 2/72 = 3% . we use this example to demonstrate that two latent factors carry most of the information of the gene expression levels of the 100 genes . in order to study the difference of the gene expression profiles between the all and aml samples , we fit two rms treating genes as ' persons ' . the first model uses the all samples as ' items ' , and the second uses the aml samples as ' items ' . therefore , for each gene , we obtain two latent factors , one based on the gene expression profiles of all samples , the other based on the gene expression profiles of aml samples . figure 3 plots the estimated latent factors for each gene together with the 99% point - wise confidence intervals . from these two plots , we conclude that the gene expression levels of most of the genes ( genes with 99% confidence intervals containing zero ) are not significantly different in both aml and all samples . for the all samples , 189 genes expressed at lower level and 164 genes expressed at higher level compared to the rest of the 3,753 genes . for the aml samples , 92 genes were expressed at lower level and 94 genes at higher level compared to the rest of the 3,920 genes . ( a ) estimated latent factor and its 99% confidence interval for each gene based on its expression profile over the all samples . ( b ) estimated latent factor and its 99% confidence interval for each gene based on its expression profile over the aml samples . for each plot , genes between the two vertical lines are those whose expression levels are not significantly different . for a given leukemia type , genes with 99% confidence interval of the estimated latent factor not including zero show significantly different expression from those genes with 99% confidence interval of the estimated latent factor including zero . in order to see the difference of gene expression between all and aml samples , genes in the upper left quadrant tend to have higher gene expression level in aml , but lower expression level in all . on the other hand , genes in the lower right quadrant tend to higher gene expression level in all , but lower expression level in aml . the logarithm ( base 10 ) of the gene expression levels of these genes are plotted on the right panel in figure 4 . further examination indicates that all the 100 genes identified by the wilcoxon rank - sum test are included in these genes . genes in the upper left quadrant tend to be overexpressed in the aml samples , but underexpressed in the all samples , and genes in the lower right quadrant tend to be overexpressed in the all samples , but underexpressed in the all samples . ( b ) log ( base 10 ) of gene expression levels for genes differentially expressed between all and aml samples . reported the use of cdna microarrays to assess gene expression profiles in a set of 60 human cancer cell lines that have been characterized pharmacologically by treatment with more than 70,000 different drug agents , one at time and independently . this dataset offers us a unique opportunity to relate variations in gene expression to the molecular pharmacology of cancer . the nci60 set includes cell lines derived from cancers of colorectal ( co , seven cell lines ) , renal ( re , eight cell lines ) , breast ( br , eight cell lines ) , ovarian ( ov , six cell lines ) , prostate ( pr , two cell lines ) , lung ( lc , nine cell lines ) and central nervous system ( cns , six cell lines ) origin , as well as leukemias ( le , six cell lines ) and melanomas ( me , eight cell lines ) . in this analysis , we consider only the 90 drug subsets whose mechanisms of action is putatively understood , and their activity data are available from the web . we used the 1,376 gene subset along with 40 individually assessed targets for the present analysis . they applied the clustering methods to cluster cell lines basing on both gene expression profiles , and the drug expression profiles . the phenotype of interest is chemother - apeutic susceptibility , as measured by -log gi50 , where gi50 measures the dose needed to cause 50% growth inhibition . we first cluster the 1,476 genes using the model - based clustering method described previously using the original data of the log - ratios . the bic scores in the upper left panel of figure 5 indicate that there are four gene clusters , with 307 , 312 , 323 and 474 genes in each cluster , respectively . as a comparison the dendrogram shown in the upper right plot of figure 5 also indicates four gene clusters . for each cell line , a latent factor is estimated using the rm , based on the gene expression levels of the genes in each of the four clusters . to fit the rm , we discretize the gene expression levels into four categories using the quartiles . the same analysis was also done with eight catagories , and the results are the same . the lower left plot of figure 5 shows the levels of these four latent factors sorted by cancer types . in general , cell lines with the same origin tend to have similar levels of the latent factors ; therefore , these factors can be used for discriminating among the nine different cell lines . however , for the third latent factor , the cell lines mda - mb-435 ( derived from the pleural effusion of a patient with breast cancer ) and its erb / b2 transfectant mda - n have similar levels to those of latent factors estimated for the melanoma cell lines . to verify the utilities of these latent factors in clustering cell lines , we performed the hierarchical clustering analysis based on these four factors ( see lower right plot in figure 5 ) . hierarchical clustering analysis using all the genes also resulted in clustering these two cell lines with melanomas . in general , cell lines of the same origin are clustered together on the basis of the four latent factors estimated with the rm . the clustering result of the cell lines using these four factors are similar to the clusters obtained using all the genes ( see ) . ( a ) bic scores as a function of the number of clusters for the nci60 dataset . the cancer indexes are sorted by cancer types as cns , br , re , lc , me , pr , ov , co , le ( see text for abbreviations ) . ( d ) dendrogram showing hierarchical clustering of cell lines based on four latent factors estimated by using the rasch model . genes over- and underexpressed in breast - cancer cell line each of the 60 cell lines is now characterized by four different latent factors , where each latent factor is estimated based on the expression profiles of the genes in each of the four clusters . it would be interesting to relate these four latent factors to the drug activity patterns as measured by -log gi50 across the 60 cell lines . for a given drug , we first performed a simple linear regression analysis treating the drug activity as response variable and using one of the four latent factors as a predictor , and obtained the parameter estimate of in the following model : drug activity = + x latent factor . the left panel of figure 6 shows the estimated value together with point - wise 99% confidence interval for each of the 90 drugs using one of the latent factors as a predictor . the variance of the regression parameter and the 99% confidence interval was estimated using the bootstrap procedure , where 50 resamples of genes in each cluster and 50 resamples of samples were used . for each latent factor , greater positive parameter estimate implies that higher gene expression level in a given gene cluster corresponds to a higher drug activity . for a given latent factor , drugs with 99% confidence interval of the estimated parameter not including zero are those whose activities are related to genes which determine this latent factor . ( a ) parameter estimate and the bootstrap 99% confidence interval of simple linear regression parameter for each of the 90 drugs and for each latent factor . for a given latent factor , drugs with 99% confidence interval of the estimated parameter not including zero are those whose activities are related to genes which determine this latent factor . ( b ) parameter estimates ( for each drug , four regression coefficients for four latent factors ) of multiple linear regression for each of the 90 drugs . in order to relate the drug activity of a given drug to all the four latent factors , we performed multiple linear regression analysis where drug activity for a given cell line was treated as a response variable , and the four latent factors were treated as the predictors . the right plot of figure 6 shows the estimates of the parameters in the multiple regression model for each of the 90 drugs . this plot can be used for selecting drugs that are related to gene expression profiles . for example , only drugs with at least one large parameter estimate are important for further study , as only for these drugs , their activity levels are related to gene expression profiles . it is also interesting to identify genes that are over- or under - expressed relative to other genes for a given cell line type . using the rm , we treat genes as ' persons ' and cell line samples as different ' items ' , and estimate the latent factor for each gene based on its expression profiles over all the samples of a given cell line type . figure 7 shows the estimated latent factor for each gene based on the gene expression profiles over each of nine different cell lines . clearly , the gene expression profiles are different across different cell line types . estimated latent factor for each gene and each cell line type . genes are in the same order across different cell lines to show that genes have different values of the latent factors , and therefore , different expression profiles across different cell line types , we can also infer which genes are over- or underexpressed compared to other genes based on the estimated latent factors . figure 8 shows the estimated latent factor and the point - wise 99% confidence interval for each gene for each of the nine cell line types . for a given cell line , genes with 99% confidence interval of the estimated latent factor not including zero show significantly different expression from those genes with 99% confidence interval of the estimated latent factor including zero . for example , for breast cancer cell line , the method identified 15 genes or expressed sequence tags ( ests ) that are relatively overexpressed ( the estimated latent factor is greater than zero , and is significant at the 0.01 level ) and 23 genes or ests that are relatively underexpressed ( estimated latent factor is less than zero , and is significant at the 0.01 level ) in the breast cancer cell lines . table 1 lists the known genes . interestingly , we note that genes that are overexpressed include p53 , and genes that are underexpressed include that for tumor - associated antigen co-029 . on the basis of our analysis , genes that are over- or underexpressed in other types of cell line can be similarly identified . estimated latent factor and the associated point - wise 99% confidence interval for each gene and each cell line type . for cell line type , the genes are sorted by the estimated value of the latent factor . for a given cell line , genes with 99% confidence interval of the estimated latent factor not including zero show significantly different expression from those with 99% confidence interval of the estimated latent factor including zero . the rm was originally proposed as an item - response theory model in the psychological test or attitude scale . the idea is that the use of a test or scale presupposes that one can indirectly infer a person 's position on a latent trait from his / her responses to a set of well - chosen items . let zij be the response of individual i to the item j , where the response can take one from m + 1 possible ordinal categories , 0 , ... , m. one version of the rm , which we use in this paper , called the partial credit model , assumes the probability of response h , as for i = 1 , ... , i , j = 1 , ... , j , and h = 0 , 1 , ... , m , where jl is the item - specific parameter , which expresses the attractiveness of the respective level l of item j. i is the person parameter that expresses the latent factor of the ith person that is measured by the j items . it is easy to verify that the probability of the response is monotonous in both person and item parameters . for example , for m = 3 , figure 1 plots the rasch probabilities as a function of the value of the latent factor ( a ) for two sets of item - specific values . it can be seen from these plots that for a given item , persons with larger value tend to have greater probability of expressing high scores , and for a given person , the response probabilities are different for items with different values . to make the model ( 1 ) identifiable , the following constraints are required example of rasch probabilities as a function of the value of the latent factor for an item with four different response categories for two different sets of item - specific parameters ( a ) = ( 0.3,0.5,-0.5,0 ) and ( b ) = ( -0.3,-0.5,0.5,0 ) . therefore , there is a total of jm - 1 unconstraint item - specific parameters . the item parameters can be estimated based on the conditional likelihood , given minimal sufficient statistics for the person parameters . for a given person , the minimal sufficient statistic is the sum of the category weights corresponding to the observed responses . after the parameters are estimated , the person parameters can then be estimated by maximizing the likelihood function . details on the conditional likelihood estimation of the item parameters can be found in anderson . typical microarray data consist of expression levels for a large number of genes on a relatively small number of samples . let xij be the gene expression level of the jth gene in the ith sample , for i = 1 , ... , , n is usually much smaller than p. for spotted arrays , to moderate the influence of gene expression ratios above and below one , we may apply the natural log transform to all the red to green ratios . upregulated genes thus have a positive log expression ratio , whereas downregulated genes have a negative log expression ratio . or xij might be the expression level from an oligonucleotide array . in addition , for the ith sample , we have observed phenotype yi , which could be a binary indicator such as two different types of cancer , a continuous measurement such as drug - response activity or censored survival time such as time to tumor recurrence . to apply the rm to the gene expression data xij , we first need to discretize the gene expression levels xij into zij , which takes value from 0 , ... , m , for i = 1 , ... , n , j = 1 , ... , p. in practice , we can use the quantiles or the quantiles within quantiles as cut - off points for discretization . because this approach uses only ranks rather than the actual expression levels , there may be slight loss of information . however , in return , we gain a valid analysis with robustness to the outliers . our goal is to infer several latent factors associated with each sample based on its gene expression profile , and relate these latent factors to the observed phenotypes . using the terms of the rm , we treat each gene as an ' item ' , each tissue sample or cell line as a ' person ' , and treat the expression level as the response of a given tissue to a given gene . the unidimensional rm may not , however , hold for the complete set of genes generated by microarrays . here we assume that genes with similar expressions determine one latent factor , and that the rm holds for each set of genes with similar expression profiles . to identify genes with similar expression profiles over samples , we first use the model - based clustering method of fraley and raftery to cluster p genes into k clusters based on their gene expression profiles over n samples . note that the cluster - step is performed based on the observed continuous gene expression data , not the discretized gene expression patterns . for a given sample , using expression profiles of genes in a given cluster , we estimate a latent factor by fitting a rm . these latent factors are then used in a regression analysis to study the relationship between the gene expressions and the phenotype . the method allows to investigate whether a cluster of genes with similar expression profiles is related to the observed phenotypes , and can also be used for future prediction by estimating the latent factors using the maximum likelihood estimation . cluster analysis , based on multivariate normal mixture models [ 10 , 17 ] , has been used for clustering various types of biological , zoological , financial and industrial data . let xj = { x1j , ... , xnj } be the n - dimensional vector of the jth gene expression over n samples . we assume that the gene expression values of p genes , x1 , ... , xp , arise from a mixture of k n - dimensional gaussian distribution with density where the k is the probability that a gene belongs to the kth cluster , and n(x|k,k ) denotes the density function of the multivariate normal distribution with mean k and variance - covariance matrix k possible parameterization of the covariance matrix is discussed in fraley and raftery . note that if we assume a simple covariance structure , k = i , where i is the identity matrix , and is the variance , then the model - based clustering method becomes the k - means clustering method . treating clustering as a mixture model problem allows us to use the em algorithm to estimate the probability of a given gene belonging to each of the k clusters , and to estimate the corresponding mean vector and covariance matrix for each cluster . one advantage of this approach is that it allows us to obtain an estimate of the number of gene clusters . following fraley and raftery , we propose to use the bayesian inference criterion ( bic ) for selecting the number of gene clusters . bic is defined as bic(k ) = 2l(k ) - nk log p , where l(k ) is the maximized log - likelihood , nk is the number of independent parameters to be estimated in the k - cluster model and p is the sample size ( number of the genes ) . as the first step of our approach , we cluster genes into k clusters using the model - based clustering method described above , where the number of gene clusters k is determined by maximizing the bic scores . let ck denote the genes in cluster k , and pk denote the number of genes in this cluster , for k = 1 , ... , k. we fit a rm model as in equation ( 1 ) for genes in each of the k clusters respectively , treating samples as ' persons ' and genes as ' items ' . to fit the rm ( equation 1 ) for genes in the kth cluster , we let i be the sample index , and j be the gene index , for i = 1 , ... , n , and j = 1 , ... , pk , and let i = ik in model ( 1 ) be the latent factor for the ith sample which is determined by the genes in the kth cluster , and jl be the gene - specific parameter for the jth gene . the rm assumes that the variation of the gene expression patterns observed over different samples is due to a latent factor , and it provides a probabilistic model to describe the gene expression pattern for a given sample . let ik be the maximum likelihood estimate of the latent factor for the ith sample determined by the genes in the kth cluster . let i = ( i1 , ... , ik ) be the vector of the estimated latent factors based on the gene expression profiles for the ith sample . in general , we can relate the phenotype yi for the ith sample to the estimated of latent factors i by a regression model , for i = 1 , ... , n , where is the vector of regression parameters , i , is the error term , and the actual model of the regression function ( f ) and the distribution of the error depend on type of the phenotype . if the phenotype is a continuous variable , linear regression can be used , if it is a binary variable , logistic or probit regression can be used , and if the phenotype is survival time , the cox regression model can be used . one advantage of the proposed method is that we can model the interactions between the latent factors in the standard way of modeling interactions in the regression models . as only the estimated and not the observed latent factors are available in the regression model ( equation 3 ) , the variance of the estimate of the parameter has to be corrected . we propose to use a two - step bootstrap resampling procedure to estimate the variance of the estimate of the parameter in the regression model . first , within the kth gene cluster , we resample genes and re - estimate the ik parameter by fitting the rm ( 1 ) . second , for a given set of estimated parameters , we resample the n samples from ( yi , i ) , for i = 1 , ... , n , and fit the regression model ( equation 3 ) to obtain a new estimate of . we can then estimate the variance of with these resampled estimates . for a new sample with gene expression xnew = ( xnew,1, ... ,xnew , p ) , these p genes are first divided into k clusters according to the clustering result . for a gene j in cluster k , we first discretize its expression level into one of the m + 1 categories using the cut - off points used in the discretization - step , denoted by znew , j . we can then estimate the corresponding latent factor k by maximizing the following likelihood function , where jh is the estimated gene - specific parameter for gene j in the kth cluster based on the training sample . using the estimated vector of the latent factors , the regression model ( 3 ) our goal is to infer several latent factors associated with each sample based on its gene expression profile , and relate these latent factors to the observed phenotypes . using the terms of the rm , we treat each gene as an ' item ' , each tissue sample or cell line as a ' person ' , and treat the expression level as the response of a given tissue to a given gene . the unidimensional rm may not , however , hold for the complete set of genes generated by microarrays . here we assume that genes with similar expressions determine one latent factor , and that the rm holds for each set of genes with similar expression profiles . to identify genes with similar expression profiles over samples , we first use the model - based clustering method of fraley and raftery to cluster p genes into k clusters based on their gene expression profiles over n samples . note that the cluster - step is performed based on the observed continuous gene expression data , not the discretized gene expression patterns . for a given sample , using expression profiles of genes in a given cluster , we estimate a latent factor by fitting a rm . these latent factors are then used in a regression analysis to study the relationship between the gene expressions and the phenotype . the method allows to investigate whether a cluster of genes with similar expression profiles is related to the observed phenotypes , and can also be used for future prediction by estimating the latent factors using the maximum likelihood estimation . cluster analysis , based on multivariate normal mixture models [ 10 , 17 ] , has been used for clustering various types of biological , zoological , financial and industrial data . let xj = { x1j , ... , xnj } be the n - dimensional vector of the jth gene expression over n samples . we assume that the gene expression values of p genes , x1 , ... , xp , arise from a mixture of k n - dimensional gaussian distribution with density where the k is the probability that a gene belongs to the kth cluster , and n(x|k,k ) denotes the density function of the multivariate normal distribution with mean k and variance - covariance matrix k possible parameterization of the covariance matrix is discussed in fraley and raftery . note that if we assume a simple covariance structure , k = i , where i is the identity matrix , and is the variance , then the model - based clustering method becomes the k - means clustering method . treating clustering as a mixture model problem allows us to use the em algorithm to estimate the probability of a given gene belonging to each of the k clusters , and to estimate the corresponding mean vector and covariance matrix for each cluster . one advantage of this approach is that it allows us to obtain an estimate of the number of gene clusters . following fraley and raftery , we propose to use the bayesian inference criterion ( bic ) for selecting the number of gene clusters . bic is defined as bic(k ) = 2l(k ) - nk log p , where l(k ) is the maximized log - likelihood , nk is the number of independent parameters to be estimated in the k - cluster model and p is the sample size ( number of the genes ) . as the first step of our approach , we cluster genes into k clusters using the model - based clustering method described above , where the number of gene clusters k is determined by maximizing the bic scores . let ck denote the genes in cluster k , and pk denote the number of genes in this cluster , for k = 1 , ... , k. we fit a rm model as in equation ( 1 ) for genes in each of the k clusters respectively , treating samples as ' persons ' and genes as ' items ' . to fit the rm ( equation 1 ) for genes in the kth cluster , we let i be the sample index , and j be the gene index , for i = 1 , ... n , and j = 1 , ... , pk , and let i = ik in model ( 1 ) be the latent factor for the ith sample which is determined by the genes in the kth cluster , and jl be the gene - specific parameter for the jth gene . the rm assumes that the variation of the gene expression patterns observed over different samples is due to a latent factor , and it provides a probabilistic model to describe the gene expression pattern for a given sample . let ik be the maximum likelihood estimate of the latent factor for the ith sample determined by the genes in the kth cluster . let i = ( i1 , ... , ik ) be the vector of the estimated latent factors based on the gene expression profiles for the ith sample . in general , we can relate the phenotype yi for the ith sample to the estimated of latent factors i by a regression model , for i = 1 , ... , n , where is the vector of regression parameters , i , is the error term , and the actual model of the regression function ( f ) and the distribution of the error depend on type of the phenotype . if the phenotype is a continuous variable , linear regression can be used , if it is a binary variable , logistic or probit regression can be used , and if the phenotype is survival time , the cox regression model can be used . alternatively , the generalized linear model can be used . one advantage of the proposed method is that we can model the interactions between the latent factors in the standard way of modeling interactions in the regression models . as only the estimated and not the observed latent factors are available in the regression model ( equation 3 ) , the variance of the estimate of the parameter has to be corrected . we propose to use a two - step bootstrap resampling procedure to estimate the variance of the estimate of the parameter in the regression model . first , within the kth gene cluster , we resample genes and re - estimate the ik parameter by fitting the rm ( 1 ) . second , for a given set of estimated parameters , we resample the n samples from ( yi , i ) , for i = 1 , ... , n , and fit the regression model ( equation 3 ) to obtain a new estimate of . we can then estimate the variance of with these resampled estimates . ,xnew , p ) , these p genes are first divided into k clusters according to the clustering result . for a gene j in cluster k , we first discretize its expression level into one of the m + 1 categories using the cut - off points used in the discretization - step , denoted by znew , j . we can then estimate the corresponding latent factor k by maximizing the following likelihood function , where jh is the estimated gene - specific parameter for gene j in the kth cluster based on the training sample . using the estimated vector of the latent factors , the regression model ( 3 ) the second formulation of the rm for gene expression data is to treat genes as ' persons ' , and samples as ' items ' . assume that we have gene expression data of p different genes , indexed by i , over nk samples of the kth sample type , indexed by j , for k = 1 , ... , k. note that the indices i and j are used differently from the previous sections . we are interested in identifying genes that are expressed differently among these different sample types . here for gene i , we can estimate a latent factor ik based on its gene expression profile over nk samples from the kth sample type by fitting the rm ( equation 1 ) , for k = 1 , ... , k. in this formulation of the rm , we treat genes as ' persons ' , treat samples as ' items ' , and treat each gene 's expression level over samples as the responses . in rm ( 1 ) , i = p , and j = nk , i = ik , which is the latent factor for the ith gene determined by the samples of the kth type , and jl is the sample - specific parameter . this model assumes that the variation of gene expression patterns across different samples among different genes is due to several gene - specific latent factors . here the latent factor ik can be interpreted as some quantities related to the transcription factors of the ith gene which determine the gene expression levels in samples of the kth sample type . for a given tissue or cell line type k , genes with larger estimated latent factor ( ik ) tend to have higher expression levels than those with smaller estimated latent factor . for a given sample type k , the estimated latent factors , ( 1k } , ... , pk ) , provide a nice way to order genes based on their expression levels over a small number of samples of sample type k , and to identify genes that are relatively over- or underexpressed in the kth sample type . in addition , by comparing the estimated latent factors associated with genes across different sample types , we can identify genes that are differentially expressed among different tissue or cell line types . acute leukemias can broadly be divided into two classes , acute myeloid leukemia ( aml ) and acute lymphoblastic leukemia ( all ) , that originate , respectively , from cells of myeloid or lymphoid origin . the two diseases appear identical under the microscope . however , correct diagnosis is critical , as they respond best to different treatment regimens . golub et al . used a set of 38 leukemia samples including 11 aml and 27 all as training samples set , and used an additional 34 samples ( 14 aml , 20 all ) as a test set for testing their proposed method for class prediction . in our analysis , we combined both the training and the test datasets into a dataset of 72 samples ( 25 aml and 47 all ) . for each of the 72 samples , we first selected the subset of 3,571 genes based on an initial processing adopted by the authors of the leukemia study . the expressions summarized are the log ( base 10 ) values of the actual expression levels following this initial filtering and transformation . we then select 50 genes that are mostly over - expressed in aml , and 50 genes that are mostly overexpressed in all by using the wilcoxon rank sums test . this simple rule of selecting a smaller set of genes are also used in golub et al . using slightly different tests . as expected , the model - based clustering method assuming the common covariance matrix clusters these 100 genes into two clusters , with 50 genes in each cluster . the left plot of figure 2 shows the gene expression levels of these 100 genes for the 72 leukemia samples . clearly , these 100 genes are highly differentially expressed between the two types of the leukemia samples . ( a ) log ( base 10 ) of gene expression levels of 100 genes chosen using the wilcox rank sum tests for the leukemias dataset . the first 47 samples along the x - axis are all , the next 25 samples are aml . ( b ) plot of two latent factors estimated using the rasch model for all 72 samples based on their gene expression profiles over 100 genes selected . given the 100 genes selected , methods such as principal component analysis , partial least - square regression or composite covariate predictor can be used to further reduce the gene dimension to two or three dimensions by taking linear combinations of the gene expression levels . instead of taking linear combination of gene expression levels , we first discretize the expression levels of all the 100 genes over 72 samples into four categories using the quantiles as the cut - off points . therefore , for each gene , their expression level can take one of four possible values of 0 , 1 , 2 and 3 . the same analysis was also done by discretizing gene expression levels into eight categories , the results were essentially the same . in the following , we only present the results using four categories . fitting two rms to these discretized gene expression levels , we estimate two latent factors for each sample ; one latent factor is determined by gene expression profiles of 50 genes in one cluster , the other is determined by the gene expression profiles of 50 genes in another cluster . the right panel of figure 2 shows the estimated values of these two latent factors for all the 72 samples . this plot shows that the two leukemia types are well separated by these two latent factors , with no overlap , except that two leukemia samples , one from all group and the other from aml group , are close to each other in this two - dimensional space . discriminant analysis using these two latent factors would expect to perform very well in classification . we performed a leave - one - out cross validation analysis to estimate the misclassification rate . specifically , we leave one sample out , and first estimate the sample - specific latent factors ik for the ith gene for k = 1,2 and gene - specific parameter jl using the remaining samples . we then estimate the latent factors of the left - out sample by maximizing the likelihood function ( equation 4 ) . fisher 's linear discriminant analysis using the estimated latent factors was then used to classify the left - out sample . the above procedure was applied to each of the 72 samples , and resulted in a misclassification rate of 2/72 = 3% . we use this example to demonstrate that two latent factors carry most of the information of the gene expression levels of the 100 genes . in order to study the difference of the gene expression profiles between the all and aml samples , we fit two rms treating genes as ' persons ' . the first model uses the all samples as ' items ' , and the second uses the aml samples as ' items ' . therefore , for each gene , we obtain two latent factors , one based on the gene expression profiles of all samples , the other based on the gene expression profiles of aml samples . figure 3 plots the estimated latent factors for each gene together with the 99% point - wise confidence intervals . from these two plots , we conclude that the gene expression levels of most of the genes ( genes with 99% confidence intervals containing zero ) are not significantly different in both aml and all samples . for the all samples , 189 genes expressed at lower level and 164 genes expressed at higher level compared to the rest of the 3,753 genes . for the aml samples , 92 genes were expressed at lower level and 94 genes at higher level compared to the rest of the 3,920 genes . ( a ) estimated latent factor and its 99% confidence interval for each gene based on its expression profile over the all samples . ( b ) estimated latent factor and its 99% confidence interval for each gene based on its expression profile over the aml samples . for each plot , genes between the two vertical lines are those whose expression levels are not significantly different . for a given leukemia type , genes with 99% confidence interval of the estimated latent factor not including zero show significantly different expression from those genes with 99% confidence interval of the estimated latent factor including zero . in order to see the difference of gene expression between all and aml samples , genes in the upper left quadrant tend to have higher gene expression level in aml , but lower expression level in all . on the other hand , genes in the lower right quadrant tend to higher gene expression level in all , but lower expression level in aml . the logarithm ( base 10 ) of the gene expression levels of these genes are plotted on the right panel in figure 4 . further examination indicates that all the 100 genes identified by the wilcoxon rank - sum test are included in these genes . genes in the upper left quadrant tend to be overexpressed in the aml samples , but underexpressed in the all samples , and genes in the lower right quadrant tend to be overexpressed in the all samples , but underexpressed in the all samples . ( b ) log ( base 10 ) of gene expression levels for genes differentially expressed between all and aml samples . acute leukemias can broadly be divided into two classes , acute myeloid leukemia ( aml ) and acute lymphoblastic leukemia ( all ) , that originate , respectively , from cells of myeloid or lymphoid origin . the two diseases appear identical under the microscope . however , correct diagnosis is critical , as they respond best to different treatment regimens . golub et al . used a set of 38 leukemia samples including 11 aml and 27 all as training samples set , and used an additional 34 samples ( 14 aml , 20 all ) as a test set for testing their proposed method for class prediction . in our analysis , we combined both the training and the test datasets into a dataset of 72 samples ( 25 aml and 47 all ) . for each of the 72 samples , we first selected the subset of 3,571 genes based on an initial processing adopted by the authors of the leukemia study . the expressions summarized are the log ( base 10 ) values of the actual expression levels following this initial filtering and transformation . we then select 50 genes that are mostly over - expressed in aml , and 50 genes that are mostly overexpressed in all by using the wilcoxon rank sums test . this simple rule of selecting a smaller set of genes are also used in golub et al . using slightly different tests . as expected , the model - based clustering method assuming the common covariance matrix clusters these 100 genes into two clusters , with 50 genes in each cluster . the left plot of figure 2 shows the gene expression levels of these 100 genes for the 72 leukemia samples . clearly , these 100 genes are highly differentially expressed between the two types of the leukemia samples . ( a ) log ( base 10 ) of gene expression levels of 100 genes chosen using the wilcox rank sum tests for the leukemias dataset . the first 47 samples along the x - axis are all , the next 25 samples are aml . ( b ) plot of two latent factors estimated using the rasch model for all 72 samples based on their gene expression profiles over 100 genes selected . given the 100 genes selected , methods such as principal component analysis , partial least - square regression or composite covariate predictor can be used to further reduce the gene dimension to two or three dimensions by taking linear combinations of the gene expression levels . instead of taking linear combination of gene expression levels , we first discretize the expression levels of all the 100 genes over 72 samples into four categories using the quantiles as the cut - off points . therefore , for each gene , their expression level can take one of four possible values of 0 , 1 , 2 and 3 . the same analysis was also done by discretizing gene expression levels into eight categories , the results were essentially the same . in the following , we only present the results using four categories . fitting two rms to these discretized gene expression levels , we estimate two latent factors for each sample ; one latent factor is determined by gene expression profiles of 50 genes in one cluster , the other is determined by the gene expression profiles of 50 genes in another cluster . the right panel of figure 2 shows the estimated values of these two latent factors for all the 72 samples . this plot shows that the two leukemia types are well separated by these two latent factors , with no overlap , except that two leukemia samples , one from all group and the other from aml group , are close to each other in this two - dimensional space . discriminant analysis using these two latent factors would expect to perform very well in classification . we performed a leave - one - out cross validation analysis to estimate the misclassification rate . specifically , we leave one sample out , and first estimate the sample - specific latent factors ik for the ith gene for k = 1,2 and gene - specific parameter jl using the remaining samples . we then estimate the latent factors of the left - out sample by maximizing the likelihood function ( equation 4 ) . fisher 's linear discriminant analysis using the estimated latent factors was then used to classify the left - out sample . the above procedure was applied to each of the 72 samples , and resulted in a misclassification rate of 2/72 = 3% . we use this example to demonstrate that two latent factors carry most of the information of the gene expression levels of the 100 genes . in order to study the difference of the gene expression profiles between the all and aml samples , we fit two rms treating genes as ' persons ' . the first model uses the all samples as ' items ' , and the second uses the aml samples as ' items ' . therefore , for each gene , we obtain two latent factors , one based on the gene expression profiles of all samples , the other based on the gene expression profiles of aml samples . figure 3 plots the estimated latent factors for each gene together with the 99% point - wise confidence intervals . from these two plots , we conclude that the gene expression levels of most of the genes ( genes with 99% confidence intervals containing zero ) are not significantly different in both aml and all samples . for the all samples , 189 genes expressed at lower level and 164 genes expressed at higher level compared to the rest of the 3,753 genes . for the aml samples , 92 genes were expressed at lower level and 94 genes at higher level compared to the rest of the 3,920 genes . ( a ) estimated latent factor and its 99% confidence interval for each gene based on its expression profile over the all samples . ( b ) estimated latent factor and its 99% confidence interval for each gene based on its expression profile over the aml samples . for each plot , genes between the two vertical lines are those whose expression levels are not significantly different . for a given leukemia type , genes with 99% confidence interval of the estimated latent factor not including zero show significantly different expression from those genes with 99% confidence interval of the estimated latent factor including zero . in order to see the difference of gene expression between all and aml samples , genes in the upper left quadrant tend to have higher gene expression level in aml , but lower expression level in all . on the other hand , genes in the lower right quadrant tend to higher gene expression level in all , but lower expression level in aml . the logarithm ( base 10 ) of the gene expression levels of these genes are plotted on the right panel in figure 4 . further examination indicates that all the 100 genes identified by the wilcoxon rank - sum test are included in these genes . genes in the upper left quadrant tend to be overexpressed in the aml samples , but underexpressed in the all samples , and genes in the lower right quadrant tend to be overexpressed in the all samples , but underexpressed in the all samples . ( b ) log ( base 10 ) of gene expression levels for genes differentially expressed between all and aml samples . scherf et al . reported the use of cdna microarrays to assess gene expression profiles in a set of 60 human cancer cell lines that have been characterized pharmacologically by treatment with more than 70,000 different drug agents , one at time and independently . this dataset offers us a unique opportunity to relate variations in gene expression to the molecular pharmacology of cancer . the nci60 set includes cell lines derived from cancers of colorectal ( co , seven cell lines ) , renal ( re , eight cell lines ) , breast ( br , eight cell lines ) , ovarian ( ov , six cell lines ) , prostate ( pr , two cell lines ) , lung ( lc , nine cell lines ) and central nervous system ( cns , six cell lines ) origin , as well as leukemias ( le , six cell lines ) and melanomas ( me , eight cell lines ) . in this analysis , we consider only the 90 drug subsets whose mechanisms of action is putatively understood , and their activity data are available from the web . we used the 1,376 gene subset along with 40 individually assessed targets for the present analysis . they applied the clustering methods to cluster cell lines basing on both gene expression profiles , and the drug expression profiles . the phenotype of interest is chemother - apeutic susceptibility , as measured by -log gi50 , where gi50 measures the dose needed to cause 50% growth inhibition . we first cluster the 1,476 genes using the model - based clustering method described previously using the original data of the log - ratios . the bic scores in the upper left panel of figure 5 indicate that there are four gene clusters , with 307 , 312 , 323 and 474 genes in each cluster , respectively . as a comparison the dendrogram shown in the upper right plot of figure 5 also indicates four gene clusters . for each cell line , a latent factor is estimated using the rm , based on the gene expression levels of the genes in each of the four clusters . to fit the rm , we discretize the gene expression levels into four categories using the quartiles . the same analysis was also done with eight catagories , and the results are the same . the lower left plot of figure 5 shows the levels of these four latent factors sorted by cancer types . in general , cell lines with the same origin tend to have similar levels of the latent factors ; therefore , these factors can be used for discriminating among the nine different cell lines . however , for the third latent factor , the cell lines mda - mb-435 ( derived from the pleural effusion of a patient with breast cancer ) and its erb / b2 transfectant mda - n have similar levels to those of latent factors estimated for the melanoma cell lines . to verify the utilities of these latent factors in clustering cell lines , we performed the hierarchical clustering analysis based on these four factors ( see lower right plot in figure 5 ) . hierarchical clustering analysis using all the genes also resulted in clustering these two cell lines with melanomas . in general , cell lines of the same origin are clustered together on the basis of the four latent factors estimated with the rm . the clustering result of the cell lines using these four factors are similar to the clusters obtained using all the genes ( see ) . ( a ) bic scores as a function of the number of clusters for the nci60 dataset . the cancer indexes are sorted by cancer types as cns , br , re , lc , me , pr , ov , co , le ( see text for abbreviations ) . ( d ) dendrogram showing hierarchical clustering of cell lines based on four latent factors estimated by using the rasch model . genes over- and underexpressed in breast - cancer cell line each of the 60 cell lines is now characterized by four different latent factors , where each latent factor is estimated based on the expression profiles of the genes in each of the four clusters . it would be interesting to relate these four latent factors to the drug activity patterns as measured by -log gi50 across the 60 cell lines . for a given drug , we first performed a simple linear regression analysis treating the drug activity as response variable and using one of the four latent factors as a predictor , and obtained the parameter estimate of in the following model : drug activity = + x latent factor . the left panel of figure 6 shows the estimated value together with point - wise 99% confidence interval for each of the 90 drugs using one of the latent factors as a predictor . the variance of the regression parameter and the 99% confidence interval was estimated using the bootstrap procedure , where 50 resamples of genes in each cluster and 50 resamples of samples were used . for each latent factor , greater positive parameter estimate implies that higher gene expression level in a given gene cluster corresponds to a higher drug activity . for a given latent factor , drugs with 99% confidence interval of the estimated parameter not including zero are those whose activities are related to genes which determine this latent factor . ( a ) parameter estimate and the bootstrap 99% confidence interval of simple linear regression parameter for each of the 90 drugs and for each latent factor . for a given latent factor , drugs with 99% confidence interval of the estimated parameter not including zero are those whose activities are related to genes which determine this latent factor . ( b ) parameter estimates ( for each drug , four regression coefficients for four latent factors ) of multiple linear regression for each of the 90 drugs . in order to relate the drug activity of a given drug to all the four latent factors , we performed multiple linear regression analysis where drug activity for a given cell line was treated as a response variable , and the four latent factors were treated as the predictors . the right plot of figure 6 shows the estimates of the parameters in the multiple regression model for each of the 90 drugs . this plot can be used for selecting drugs that are related to gene expression profiles . for example , only drugs with at least one large parameter estimate are important for further study , as only for these drugs , their activity levels are related to gene expression profiles . it is also interesting to identify genes that are over- or under - expressed relative to other genes for a given cell line type . using the rm , we treat genes as ' persons ' and cell line samples as different ' items ' , and estimate the latent factor for each gene based on its expression profiles over all the samples of a given cell line type . figure 7 shows the estimated latent factor for each gene based on the gene expression profiles over each of nine different cell lines . clearly , the gene expression profiles are different across different cell line types . estimated latent factor for each gene and each cell line type . genes are in the same order across different cell lines to show that genes have different values of the latent factors , and therefore , different expression profiles across different cell line types . , we can also infer which genes are over- or underexpressed compared to other genes based on the estimated latent factors . figure 8 shows the estimated latent factor and the point - wise 99% confidence interval for each gene for each of the nine cell line types . for a given cell line , genes with 99% confidence interval of the estimated latent factor not including zero show significantly different expression from those genes with 99% confidence interval of the estimated latent factor including zero . for example , for breast cancer cell line , the method identified 15 genes or expressed sequence tags ( ests ) that are relatively overexpressed ( the estimated latent factor is greater than zero , and is significant at the 0.01 level ) and 23 genes or ests that are relatively underexpressed ( estimated latent factor is less than zero , and is significant at the 0.01 level ) in the breast cancer cell lines . interestingly , we note that genes that are overexpressed include p53 , and genes that are underexpressed include that for tumor - associated antigen co-029 . on the basis of our analysis , genes that are over- or underexpressed in other types of cell line can be similarly identified . estimated latent factor and the associated point - wise 99% confidence interval for each gene and each cell line type . for cell line type , the genes are sorted by the estimated value of the latent factor . for a given cell line , genes with 99% confidence interval of the estimated latent factor not including zero show significantly different expression from those with 99% confidence interval of the estimated latent factor including zero . scherf et al . reported the use of cdna microarrays to assess gene expression profiles in a set of 60 human cancer cell lines that have been characterized pharmacologically by treatment with more than 70,000 different drug agents , one at time and independently . this dataset offers us a unique opportunity to relate variations in gene expression to the molecular pharmacology of cancer . the nci60 set includes cell lines derived from cancers of colorectal ( co , seven cell lines ) , renal ( re , eight cell lines ) , breast ( br , eight cell lines ) , ovarian ( ov , six cell lines ) , prostate ( pr , two cell lines ) , lung ( lc , nine cell lines ) and central nervous system ( cns , six cell lines ) origin , as well as leukemias ( le , six cell lines ) and melanomas ( me , eight cell lines ) . in this analysis , we consider only the 90 drug subsets whose mechanisms of action is putatively understood , and their activity data are available from the web . we used the 1,376 gene subset along with 40 individually assessed targets for the present analysis . they applied the clustering methods to cluster cell lines basing on both gene expression profiles , and the drug expression profiles . the phenotype of interest is chemother - apeutic susceptibility , as measured by -log gi50 , where gi50 measures the dose needed to cause 50% growth inhibition . we first cluster the 1,476 genes using the model - based clustering method described previously using the original data of the log - ratios . the bic scores in the upper left panel of figure 5 indicate that there are four gene clusters , with 307 , 312 , 323 and 474 genes in each cluster , respectively . as a comparison the dendrogram shown in the upper right plot of figure 5 also indicates four gene clusters . for each cell line , a latent factor is estimated using the rm , based on the gene expression levels of the genes in each of the four clusters . to fit the rm , we discretize the gene expression levels into four categories using the quartiles . the same analysis was also done with eight catagories , and the results are the same . the lower left plot of figure 5 shows the levels of these four latent factors sorted by cancer types . in general , cell lines with the same origin tend to have similar levels of the latent factors ; therefore , these factors can be used for discriminating among the nine different cell lines . however , for the third latent factor , the cell lines mda - mb-435 ( derived from the pleural effusion of a patient with breast cancer ) and its erb / b2 transfectant mda - n have similar levels to those of latent factors estimated for the melanoma cell lines . to verify the utilities of these latent factors in clustering cell lines , we performed the hierarchical clustering analysis based on these four factors ( see lower right plot in figure 5 ) . hierarchical clustering analysis using all the genes also resulted in clustering these two cell lines with melanomas . in general , cell lines of the same origin are clustered together on the basis of the four latent factors estimated with the rm . the clustering result of the cell lines using these four factors are similar to the clusters obtained using all the genes ( see ) . ( a ) bic scores as a function of the number of clusters for the nci60 dataset . the cancer indexes are sorted by cancer types as cns , br , re , lc , me , pr , ov , co , le ( see text for abbreviations ) . ( d ) dendrogram showing hierarchical clustering of cell lines based on four latent factors estimated by using the rasch model . genes over- and underexpressed in breast - cancer cell line each of the 60 cell lines is now characterized by four different latent factors , where each latent factor is estimated based on the expression profiles of the genes in each of the four clusters . it would be interesting to relate these four latent factors to the drug activity patterns as measured by -log gi50 across the 60 cell lines . for a given drug , we first performed a simple linear regression analysis treating the drug activity as response variable and using one of the four latent factors as a predictor , and obtained the parameter estimate of in the following model : drug activity = + x latent factor . the left panel of figure 6 shows the estimated value together with point - wise 99% confidence interval for each of the 90 drugs using one of the latent factors as a predictor . the variance of the regression parameter and the 99% confidence interval was estimated using the bootstrap procedure , where 50 resamples of genes in each cluster and 50 resamples of samples were used . for each latent factor , greater positive parameter estimate implies that higher gene expression level in a given gene cluster corresponds to a higher drug activity . for a given latent factor , drugs with 99% confidence interval of the estimated parameter not including zero are those whose activities are related to genes which determine this latent factor . ( a ) parameter estimate and the bootstrap 99% confidence interval of simple linear regression parameter for each of the 90 drugs and for each latent factor . for a given latent factor , drugs with 99% confidence interval of the estimated parameter not including zero are those whose activities are related to genes which determine this latent factor . ( b ) parameter estimates ( for each drug , four regression coefficients for four latent factors ) of multiple linear regression for each of the 90 drugs . in order to relate the drug activity of a given drug to all the four latent factors , we performed multiple linear regression analysis where drug activity for a given cell line was treated as a response variable , and the four latent factors were treated as the predictors . the right plot of figure 6 shows the estimates of the parameters in the multiple regression model for each of the 90 drugs . this plot can be used for selecting drugs that are related to gene expression profiles . for example , only drugs with at least one large parameter estimate are important for further study , as only for these drugs , their activity levels are related to gene expression profiles . it is also interesting to identify genes that are over- or under - expressed relative to other genes for a given cell line type . using the rm , we treat genes as ' persons ' and cell line samples as different ' items ' , and estimate the latent factor for each gene based on its expression profiles over all the samples of a given cell line type . figure 7 shows the estimated latent factor for each gene based on the gene expression profiles over each of nine different cell lines . clearly , the gene expression profiles are different across different cell line types . estimated latent factor for each gene and each cell line type . genes are in the same order across different cell lines to show that genes have different values of the latent factors , and therefore , different expression profiles across different cell line types , we can also infer which genes are over- or underexpressed compared to other genes based on the estimated latent factors . figure 8 shows the estimated latent factor and the point - wise 99% confidence interval for each gene for each of the nine cell line types . for a given cell line , genes with 99% confidence interval of the estimated latent factor not including zero show significantly different expression from those genes with 99% confidence interval of the estimated latent factor including zero . for example , for breast cancer cell line , the method identified 15 genes or expressed sequence tags ( ests ) that are relatively overexpressed ( the estimated latent factor is greater than zero , and is significant at the 0.01 level ) and 23 genes or ests that are relatively underexpressed ( estimated latent factor is less than zero , and is significant at the 0.01 level ) in the breast cancer cell lines . interestingly , we note that genes that are overexpressed include p53 , and genes that are underexpressed include that for tumor - associated antigen co-029 . on the basis of our analysis , genes that are over- or underexpressed in other types of cell line can be similarly identified . estimated latent factor and the associated point - wise 99% confidence interval for each gene and each cell line type . for cell line type , the genes are sorted by the estimated value of the latent factor . for a given cell line , genes with 99% confidence interval of the estimated latent factor not including zero show significantly different expression from those with 99% confidence interval of the estimated latent factor including zero . we have described two different formulations of the rm for relating gene expression data to phenotypes . the first formulation can be used for cancer class prediction , and for identifying clusters of genes with similar expression profiles that are related to drug responses . the method is based on a combination of clustering analysis , the rm and the regression analysis . the second formulation can be used for identifying differentially expressed genes from different types of sample . we applied this method to a publicly available leukemia dataset to demonstrate the application of the proposed method for class prediction . we also applied this method to an analysis of the nci60 data to show that the method can also be applied to other phenotypes . these models are used to reduce the complexity of the raw data and offer a certain degree of simplification . in contrast to most of the currently available methods for gene expression data , such as principal component analysis , the model used here provides a non - linear method for dimension reduction . second , compared with traditional density - based models , the method is more robust to outliers , as it uses ranks rather than actual expression levels . there is a long sequence of steps in the laboratory as well as in the image analysis before a single number is produced for an expression level , and there are many potential sources of error . methods that use ranks rather than the original measured gene expression data are also advocated by a tsodikov , a szabo and d jones ( unpublished data ) and park et al . . third , in contrast to clustering methods for simultaneously clustering both genes and samples , the model - based approach allows formal estimates of the variance and therefore facilitates formal tests of null hypotheses and assignments of confidence intervals . first , in order to apply the rm , the gene expression levels are dis - cretized . certainly , additional information is present in the level of gene expression , but normalization or scaling errors across subjects or slides make it difficult to determine the precision of these numbers . we believe that discretization provides a reasonably unbiased approach for dealing with this type of data . for both the leukemia and nci60 datasets , we fitted the rms by discretizing the gene expression levels into both four and eight categories , and obtained essentially the same conclusions . of course , the more categories we use , the closer are the discretized data to the real continuous data . second , this approach assumes that genes can be clustered into several subgroups based on their expression profiles over samples , we can consider all the possible clusters of genes in a step - wise regression analysis as proposed by hastie et al . . third , we used the bootstrap resampling procedure to estimate the variance of the regression parameter after we have clustered genes into several classes . this procedure does not account for possible variability associated with the clustering step , and therefore , the bootstrap variance estimates are likely to be underestimated . in our proposed method , the clustering , the rasch modeling and the regression analysis are done separately . important research for the future is to take a joint likelihood approach that can combine all three steps to obtain better estimates of the number of clusters , the latent factors and the regression parameters . this kind of mixture rm provides a natural framework for unifying statistical inference and clustering . we are currently carrying out research in this direction . in conclusion , we demonstrate here the potential application of the rms in analysis of gene expression data . rms provide a probability model for describing gene expression profiles measured over different samples or over different times . we are currently exploring various other formulations of the microarray gene expression problems in the framework of the rms , including class discovery in cases of hidden taxonomies based on the estimated latent factors using the rm . this research is supported in part by an nih grant ( es09911 ) and a uc davis health system research award grant .

backgroundwe propose two different formulations of the rasch statistical models to the problem of relating gene expression profiles to the phenotypes . one formulation allows us to investigate whether a cluster of genes with similar expression profiles is related to the observed phenotypes ; this model can also be used for future prediction . the other formulation provides an alternative way of identifying genes that are over- or underexpressed from their expression levels in tissue or cell samples of a given tissue or cell type.resultswe illustrate the methods on available datasets of a classification of acute leukemias and of 60 cancer cell lines . for tumor classification , the results are comparable to those previously obtained . for the cancer cell lines dataset , we found four clusters of genes that are related to drug response for many of the 90 drugs that we considered . in addition , for each type of cell line , we identified genes that are over- or underexpressed relative to other genes.conclusionsthe cluster - rasch model provides a probabilistic model for describing gene expression patterns across samples and can be used to relate gene expression profiles to phenotypes .

Background Results The Rasch model (RM) Relating gene expression profiles to phenotypes Outline of the approach Model-based clustering analysis Rasch model and regression analysis Prediction RM for latent factors associated with genes Analysis of the leukemia dataset Classification using cluster-RM Summary of gene expression profiles Analysis of NC160 dataset Relating gene expression profiles to drug activities Identifying genes differentially expressed in different cell lines Discussion Acknowledgements

these oligos are then hybridized with labeled probe derived from a given tissue or cell line . for continuous phenotypes such as drug sensitivity , the problem of interest is to relate gene expression patterns to sensitivity to drugs and , therefore , aid in the process of drug discovery and provide a rationale for selection of therapy on the basis of the molecular characteristics of a patient 's tumor . from the statistical point of view , the challenge is that the microarray gene expression data are often measured with a great deal of noise , and that the sample size of tissues or cell lines , denoted by n , is usually very small compared to the number of genes in expression arrays , denoted by p. this results in the ' large p , small n ' problem . another popular approach to analyzing gene expression data is to use clustering methods to simultaneously cluster both samples and genes in order to determine some clusters of genes that are mostly correlated with some clusters of samples . examples of such an application include analysis of gene expression data and drug response for the 60 human cancer cell lines of the national cancer institute ( nci60 data ) , and analysis of cancerous and normal colon tissues . methods that can be used to assess the significance of the clustering results are needed . here we propose two different formulations of the polytomous rm for analysis of microarray gene expression data . this formulation provides simple summary statistics for genes based on their expression profiles over samples , and helps to identify genes that are more likely be over- or underexpressed within samples of the same type or between samples of different types . for a given person , the minimal sufficient statistic is the sum of the category weights corresponding to the observed responses . in addition , for the ith sample , we have observed phenotype yi , which could be a binary indicator such as two different types of cancer , a continuous measurement such as drug - response activity or censored survival time such as time to tumor recurrence . to apply the rm to the gene expression data xij , we first need to discretize the gene expression levels xij into zij , which takes value from 0 , ... , m , for i = 1 , ... , n , j = 1 , ... , p. in practice , we can use the quantiles or the quantiles within quantiles as cut - off points for discretization . using the terms of the rm , we treat each gene as an ' item ' , each tissue sample or cell line as a ' person ' , and treat the expression level as the response of a given tissue to a given gene . here we assume that genes with similar expressions determine one latent factor , and that the rm holds for each set of genes with similar expression profiles . to identify genes with similar expression profiles over samples , we first use the model - based clustering method of fraley and raftery to cluster p genes into k clusters based on their gene expression profiles over n samples . the method allows to investigate whether a cluster of genes with similar expression profiles is related to the observed phenotypes , and can also be used for future prediction by estimating the latent factors using the maximum likelihood estimation . treating clustering as a mixture model problem allows us to use the em algorithm to estimate the probability of a given gene belonging to each of the k clusters , and to estimate the corresponding mean vector and covariance matrix for each cluster . the rm assumes that the variation of the gene expression patterns observed over different samples is due to a latent factor , and it provides a probabilistic model to describe the gene expression pattern for a given sample . in general , we can relate the phenotype yi for the ith sample to the estimated of latent factors i by a regression model , for i = 1 , ... , n , where is the vector of regression parameters , i , is the error term , and the actual model of the regression function ( f ) and the distribution of the error depend on type of the phenotype . assume that we have gene expression data of p different genes , indexed by i , over nk samples of the kth sample type , indexed by j , for k = 1 , ... , k. note that the indices i and j are used differently from the previous sections . here the latent factor ik can be interpreted as some quantities related to the transcription factors of the ith gene which determine the gene expression levels in samples of the kth sample type . for a given tissue or cell line type k , genes with larger estimated latent factor ( ik ) tend to have higher expression levels than those with smaller estimated latent factor . for a given sample type k , the estimated latent factors , ( 1k } , ... , pk ) , provide a nice way to order genes based on their expression levels over a small number of samples of sample type k , and to identify genes that are relatively over- or underexpressed in the kth sample type . in addition , by comparing the estimated latent factors associated with genes across different sample types , we can identify genes that are differentially expressed among different tissue or cell line types . given the 100 genes selected , methods such as principal component analysis , partial least - square regression or composite covariate predictor can be used to further reduce the gene dimension to two or three dimensions by taking linear combinations of the gene expression levels . in the following fitting two rms to these discretized gene expression levels , we estimate two latent factors for each sample ; one latent factor is determined by gene expression profiles of 50 genes in one cluster , the other is determined by the gene expression profiles of 50 genes in another cluster . therefore , for each gene , we obtain two latent factors , one based on the gene expression profiles of all samples , the other based on the gene expression profiles of aml samples . from these two plots , we conclude that the gene expression levels of most of the genes ( genes with 99% confidence intervals containing zero ) are not significantly different in both aml and all samples . for each cell line , a latent factor is estimated using the rm , based on the gene expression levels of the genes in each of the four clusters . however , for the third latent factor , the cell lines mda - mb-435 ( derived from the pleural effusion of a patient with breast cancer ) and its erb / b2 transfectant mda - n have similar levels to those of latent factors estimated for the melanoma cell lines . genes over- and underexpressed in breast - cancer cell line each of the 60 cell lines is now characterized by four different latent factors , where each latent factor is estimated based on the expression profiles of the genes in each of the four clusters . in order to relate the drug activity of a given drug to all the four latent factors , we performed multiple linear regression analysis where drug activity for a given cell line was treated as a response variable , and the four latent factors were treated as the predictors . this plot can be used for selecting drugs that are related to gene expression profiles . it is also interesting to identify genes that are over- or under - expressed relative to other genes for a given cell line type . using the rm , we treat genes as ' persons ' and cell line samples as different ' items ' , and estimate the latent factor for each gene based on its expression profiles over all the samples of a given cell line type . genes are in the same order across different cell lines to show that genes have different values of the latent factors , and therefore , different expression profiles across different cell line types , we can also infer which genes are over- or underexpressed compared to other genes based on the estimated latent factors . for example , for breast cancer cell line , the method identified 15 genes or expressed sequence tags ( ests ) that are relatively overexpressed ( the estimated latent factor is greater than zero , and is significant at the 0.01 level ) and 23 genes or ests that are relatively underexpressed ( estimated latent factor is less than zero , and is significant at the 0.01 level ) in the breast cancer cell lines . on the basis of our analysis , genes that are over- or underexpressed in other types of cell line can be similarly identified . for a given cell line , genes with 99% confidence interval of the estimated latent factor not including zero show significantly different expression from those with 99% confidence interval of the estimated latent factor including zero . in addition , for the ith sample , we have observed phenotype yi , which could be a binary indicator such as two different types of cancer , a continuous measurement such as drug - response activity or censored survival time such as time to tumor recurrence . using the terms of the rm , we treat each gene as an ' item ' , each tissue sample or cell line as a ' person ' , and treat the expression level as the response of a given tissue to a given gene . here we assume that genes with similar expressions determine one latent factor , and that the rm holds for each set of genes with similar expression profiles . to identify genes with similar expression profiles over samples , we first use the model - based clustering method of fraley and raftery to cluster p genes into k clusters based on their gene expression profiles over n samples . the method allows to investigate whether a cluster of genes with similar expression profiles is related to the observed phenotypes , and can also be used for future prediction by estimating the latent factors using the maximum likelihood estimation . treating clustering as a mixture model problem allows us to use the em algorithm to estimate the probability of a given gene belonging to each of the k clusters , and to estimate the corresponding mean vector and covariance matrix for each cluster . the rm assumes that the variation of the gene expression patterns observed over different samples is due to a latent factor , and it provides a probabilistic model to describe the gene expression pattern for a given sample . in general , we can relate the phenotype yi for the ith sample to the estimated of latent factors i by a regression model , for i = 1 , ... , n , where is the vector of regression parameters , i , is the error term , and the actual model of the regression function ( f ) and the distribution of the error depend on type of the phenotype . using the estimated vector of the latent factors , the regression model ( 3 ) our goal is to infer several latent factors associated with each sample based on its gene expression profile , and relate these latent factors to the observed phenotypes . using the terms of the rm , we treat each gene as an ' item ' , each tissue sample or cell line as a ' person ' , and treat the expression level as the response of a given tissue to a given gene . here we assume that genes with similar expressions determine one latent factor , and that the rm holds for each set of genes with similar expression profiles . to identify genes with similar expression profiles over samples , we first use the model - based clustering method of fraley and raftery to cluster p genes into k clusters based on their gene expression profiles over n samples . the method allows to investigate whether a cluster of genes with similar expression profiles is related to the observed phenotypes , and can also be used for future prediction by estimating the latent factors using the maximum likelihood estimation . treating clustering as a mixture model problem allows us to use the em algorithm to estimate the probability of a given gene belonging to each of the k clusters , and to estimate the corresponding mean vector and covariance matrix for each cluster . the rm assumes that the variation of the gene expression patterns observed over different samples is due to a latent factor , and it provides a probabilistic model to describe the gene expression pattern for a given sample . in general , we can relate the phenotype yi for the ith sample to the estimated of latent factors i by a regression model , for i = 1 , ... , n , where is the vector of regression parameters , i , is the error term , and the actual model of the regression function ( f ) and the distribution of the error depend on type of the phenotype . assume that we have gene expression data of p different genes , indexed by i , over nk samples of the kth sample type , indexed by j , for k = 1 , ... , k. note that the indices i and j are used differently from the previous sections . here the latent factor ik can be interpreted as some quantities related to the transcription factors of the ith gene which determine the gene expression levels in samples of the kth sample type . for a given tissue or cell line type k , genes with larger estimated latent factor ( ik ) tend to have higher expression levels than those with smaller estimated latent factor . for a given sample type k , the estimated latent factors , ( 1k } , ... , pk ) , provide a nice way to order genes based on their expression levels over a small number of samples of sample type k , and to identify genes that are relatively over- or underexpressed in the kth sample type . in addition , by comparing the estimated latent factors associated with genes across different sample types , we can identify genes that are differentially expressed among different tissue or cell line types . given the 100 genes selected , methods such as principal component analysis , partial least - square regression or composite covariate predictor can be used to further reduce the gene dimension to two or three dimensions by taking linear combinations of the gene expression levels . fitting two rms to these discretized gene expression levels , we estimate two latent factors for each sample ; one latent factor is determined by gene expression profiles of 50 genes in one cluster , the other is determined by the gene expression profiles of 50 genes in another cluster . therefore , for each gene , we obtain two latent factors , one based on the gene expression profiles of all samples , the other based on the gene expression profiles of aml samples . from these two plots , we conclude that the gene expression levels of most of the genes ( genes with 99% confidence intervals containing zero ) are not significantly different in both aml and all samples . given the 100 genes selected , methods such as principal component analysis , partial least - square regression or composite covariate predictor can be used to further reduce the gene dimension to two or three dimensions by taking linear combinations of the gene expression levels . fitting two rms to these discretized gene expression levels , we estimate two latent factors for each sample ; one latent factor is determined by gene expression profiles of 50 genes in one cluster , the other is determined by the gene expression profiles of 50 genes in another cluster . therefore , for each gene , we obtain two latent factors , one based on the gene expression profiles of all samples , the other based on the gene expression profiles of aml samples . for each cell line , a latent factor is estimated using the rm , based on the gene expression levels of the genes in each of the four clusters . in general , cell lines with the same origin tend to have similar levels of the latent factors ; therefore , these factors can be used for discriminating among the nine different cell lines . however , for the third latent factor , the cell lines mda - mb-435 ( derived from the pleural effusion of a patient with breast cancer ) and its erb / b2 transfectant mda - n have similar levels to those of latent factors estimated for the melanoma cell lines . genes over- and underexpressed in breast - cancer cell line each of the 60 cell lines is now characterized by four different latent factors , where each latent factor is estimated based on the expression profiles of the genes in each of the four clusters . in order to relate the drug activity of a given drug to all the four latent factors , we performed multiple linear regression analysis where drug activity for a given cell line was treated as a response variable , and the four latent factors were treated as the predictors . the right plot of figure 6 shows the estimates of the parameters in the multiple regression model for each of the 90 drugs . this plot can be used for selecting drugs that are related to gene expression profiles . it is also interesting to identify genes that are over- or under - expressed relative to other genes for a given cell line type . using the rm , we treat genes as ' persons ' and cell line samples as different ' items ' , and estimate the latent factor for each gene based on its expression profiles over all the samples of a given cell line type . , we can also infer which genes are over- or underexpressed compared to other genes based on the estimated latent factors . for a given cell line , genes with 99% confidence interval of the estimated latent factor not including zero show significantly different expression from those genes with 99% confidence interval of the estimated latent factor including zero . for example , for breast cancer cell line , the method identified 15 genes or expressed sequence tags ( ests ) that are relatively overexpressed ( the estimated latent factor is greater than zero , and is significant at the 0.01 level ) and 23 genes or ests that are relatively underexpressed ( estimated latent factor is less than zero , and is significant at the 0.01 level ) in the breast cancer cell lines . on the basis of our analysis , genes that are over- or underexpressed in other types of cell line can be similarly identified . for each cell line , a latent factor is estimated using the rm , based on the gene expression levels of the genes in each of the four clusters . however , for the third latent factor , the cell lines mda - mb-435 ( derived from the pleural effusion of a patient with breast cancer ) and its erb / b2 transfectant mda - n have similar levels to those of latent factors estimated for the melanoma cell lines . genes over- and underexpressed in breast - cancer cell line each of the 60 cell lines is now characterized by four different latent factors , where each latent factor is estimated based on the expression profiles of the genes in each of the four clusters . the right plot of figure 6 shows the estimates of the parameters in the multiple regression model for each of the 90 drugs . this plot can be used for selecting drugs that are related to gene expression profiles . it is also interesting to identify genes that are over- or under - expressed relative to other genes for a given cell line type . using the rm , we treat genes as ' persons ' and cell line samples as different ' items ' , and estimate the latent factor for each gene based on its expression profiles over all the samples of a given cell line type . genes are in the same order across different cell lines to show that genes have different values of the latent factors , and therefore , different expression profiles across different cell line types , we can also infer which genes are over- or underexpressed compared to other genes based on the estimated latent factors . for example , for breast cancer cell line , the method identified 15 genes or expressed sequence tags ( ests ) that are relatively overexpressed ( the estimated latent factor is greater than zero , and is significant at the 0.01 level ) and 23 genes or ests that are relatively underexpressed ( estimated latent factor is less than zero , and is significant at the 0.01 level ) in the breast cancer cell lines . on the basis of our analysis , genes that are over- or underexpressed in other types of cell line can be similarly identified . for a given cell line , genes with 99% confidence interval of the estimated latent factor not including zero show significantly different expression from those with 99% confidence interval of the estimated latent factor including zero . we have described two different formulations of the rm for relating gene expression data to phenotypes . the first formulation can be used for cancer class prediction , and for identifying clusters of genes with similar expression profiles that are related to drug responses . second , this approach assumes that genes can be clustered into several subgroups based on their expression profiles over samples , we can consider all the possible clusters of genes in a step - wise regression analysis as proposed by hastie et al . rms provide a probability model for describing gene expression profiles measured over different samples or over different times .

human papillomavirus ( hpv ) is the main cause of cervical cancer , the third most common cancer in women . there is , however , growing evidence linking genital hpv infection to other anogenital cancers ( anus , vulva , vagina , and penis ) and head and neck cancers in both men and women [ 311 ] . hpv is carried by both females and males and can spread with high ( up to 0,6 per act for hpv16 ) transmission probability , and most ( up to 7080% ) people will get infected during their lifetime . thus , hpv can be characterised as a ubiquitous , sexually transmitted infection ( sti ) causing significant disease burden in both sexes , but especially in women with up to 6 - 7% lifetime risk of developing cervical cancer in latin america . of the estimated 530000 annual cases of cervical cancer , its standardised incidence ratio ( world , 100 ) is substantially higher in the developing countries ( 116 ) than in the developed countries ( 60 ) . it is important to note that cervical cancer is the most common cancer in women in most parts of africa , central america , southern asia , and melanesia . it is also the most important cause of years of life lost in latin america and the caribbean , and among cancers in the populous regions of sub - saharan africa and south - central asia . furthermore , largely due to changes in sexual risk taking behaviour and ( in some countries ) dynamic state of epidemics by high - risk ( hr ) hpv types [ 15 , 16 ] , the number of cervical cancer cases has been predicted to rapidly increase ( up to 90% ) by 2020 in developing countries if no intervention is implemented [ 17 , 18 ] . it has been established that the hpv attributable proportion in cancers of the anus , vagina , penis , vulva , oropharynx , and oral cavity is 25% or higher [ 8 , 11 , 1921 ] . there are clear signs that the incidence of most hpv - related cancers is increasing . the incidence rates of anal cancer in scotland and england have nearly doubled in both women and men from 1986 to 2003 . increase in the incidence of anal cancer is also reported in australia . in the developed countries , also the incidence of hpv - related head and neck cancer is rapidly increasing especially in males and in the younger birth cohorts [ 5 , 6 , 911 , 24 ] . in australia , netherlands , sweden , and usa , the incidence of hpv - related tonsillar cancer has rapidly increased during recent years [ 7 , 23 , 25 , 26 ] . most of the genital infections with hrhpv type(s ) are asymptomatic and heal without treatment . the risk of cervical cancer increases as the hrhpv infection persists . over 70% of cervical cancers are attributed to hpv types 16 and 18 and approximately 20% to hpv types 31 , 33 , 35 , 45 , 52 , and 58 [ 3 , 27 ] . precancerous cervical lesions usually appear within 5 years in individuals with an established persistent infection with hpv types 16 and 18 . a majority of other hpv - related cancers are also attributable to hpv types 16 and 18 . for example , it is estimated that 24% of cancers in the mouth are associated with hpv and 95% of these cancers are attributable to hpv types 16 and 18 . over 80% of anal cancers are associated with hpv and 92% of these cancers are attributable to hpv types 16 and 18 . thus , it can be assumed that targeting hpv types 16 and 18 and a remarkable proportion of phylogenetically related hrhpv types ( 31 , 33 , 45 ) by prophylactic vaccination [ 2830 ] would play a significant role in preventing all hpv - related cancers . prevention of hrhpv infections could decrease the incidence of numerous cancers in both sexes [ 5 , 31 ] . in healthy men , hpv infection in the genital tract alike other stis , hpv transmits more easily from men to women than from women to men . male circumcision and use of condoms prevent the spreading of hrhpv infections , which probably explains the low incidence of cervical cancer in countries such as israel where circumcision is widespread [ 3 , 33 ] . racial differences have been reported showing that african - americans are less likely to have hpv - positive head and neck ( oropharyngeal ) cancers [ 34 , 35 ] . reasons for the recent increase of oropharyngeal hpv - related cancers , especially in younger male birth cohorts in the developed countries , are not clear , but changes in sexual behaviour , efficiency of hpv transmission through oral sex , and lack of protective immunity from hrhpv infections of the genital mucosae have been suggested [ 6 , 36 ] . various studies indicate that a high number of lifetime sexual partners , tobacco smoking , parity , oral contraceptive use , and coinfections with chlamydia trachomatis and hiv increase the risk of acquisition of hrhpv infection [ 3742 ] . the impact of cofactors on the acquisition of infections with multiple hrhpv types has not been studied largely [ 43 , 44 ] . stis are a serious health problem in developing countries , and several studies indicate that conventional stis increase the likelihood of hiv transmission . hpv infections in both females and males are also risk factors for hiv acquisition [ 4648 ] . it is also highly likely that hpv - related neoplasia progresses faster in hiv - positive people . cervical cancer is a disease which can be prevented . similar to liver cancer that is secondary to hepatitis b infection application of cervical cytology in population - based screening programmes has significantly lowered the incidence of cervical cancer in developed countries . cervical cancer incidence and mortality decreased markedly in the nordic countries , europe , canada , and usa due to the implementation of cervical cytology in health care , most notably in population - based screening programmes [ 5154 ] . mortality from cervical cancer has also substantially declined since the 1960s in europe , but there are still large country - specific differences . cervical cancer mortality is substantially higher in eastern europe than in other parts of europe . increases in the incidence of and mortality from cervical cancer have been reported in the last 15 years resulting from hpv epidemics and a drop in the number of women participating in the screening programmes . mortality rates of cervical cancer are lower than incidence rates with a ratio of mortality to incidence of 55% . survival rates are , however , lower in the developing countries [ 5658 ] , and the differences in the ratios of mortality to incidence between developing and developed countries are significant . the differences can be explained by the stage at which cancer is detected , access to health services , and adequacy of treatment . currently there are two licensed prophylactic hpv vaccines , a bivalent vaccine ( cervarix ) against hpv types 16 and 18 and a quadrivalent ( gardasil ) vaccine against hpv types 6 , 11 , 16 , and 18 available ( fda 2006 ; emea 2007 ) . in order to be prophylactic , both vaccines need to be administered before the individual is exposed to hpv types covered by the vaccine . according to reports from the major phase iii trials , the vaccines prevent from 97 to 98% of infections caused by hpv types 16 and 18 [ 59 , 60 ] . both vaccines have shown a significant cross - protection also against hpv types 31 and 45 [ 28 , 29 ] . the bivalent vaccine has also shown cross - protection against hpv types 33 and 51 . as indicated above , over 70% of cervical cancers are attributed to hpv types 16 and 18 , and approximately 20% to hpv types 31 , 33 , 35 , 45 , 52 , and 58 [ 3 , 27 ] which fits the cross - protection efficacy and reported 87% overall vaccine efficacy against cin3 + . it is assumed that both hpv vaccines , in preventing hrhpv infections , prevent other hpv - related cancers besides cervical cancer with high to moderate efficacy . it is known that the existing vaccines are most efficient for antibody production when administered to early adolescents . both males and females had higher antibody responses at the age of 915 compared to the age of 1626 [ 62 , 63 ] . there is , however , no concrete information available on long - term ( > 10 years ) efficacy of the vaccines and necessity for a booster . one model has predicted an over 20-year protection but at the moment the predictions rely only on assumptions . it is not known if hiv infection will affect the efficacy of hpv vaccines , but smoking does not seem to affect hpv vaccine - induced antibody response . booster doses work very well and produce higher antibody levels when measured one month later . hiv positivity as such does not hamper development of hpv antibodies following natural infection , whereas smoking does . type replacement , that is , how nontargeted hrhpv types that may have competitive advantage will behave following mass vaccination , is an open question , but the likelihood of the kind of type replacement seen following bacterial vaccination is small due to the different biology of viral and bacterial infections [ 68 , 69 ] . according to who , 22 countries in low - resource regions have included an hpv vaccine in their vaccine programmes . adopted vaccination strategies include offering the vaccine only to 1215-year - old girls or only to a certain proportion of the female population of the same age . the vaccination strategies are in line with the results of numerous cost - effectiveness studies which suggest that with high vaccine coverage ( 75% ) vaccination of males would not be cost effective [ 7182 ] . vaccinating males becomes cost effective by assuming a low to moderate ( 30%50% ) coverage in females . the assumptions behind these recommendations and the superior cost - effectiveness of female vaccination strategy are that the health service system covers all the regions equitably and that adolescent girls have both access to the health services and are willing to use it . however , the probability of preventing other hpv - related cancers both in females and males has not been taken into account . emerging information on hpv - related cancers in men may change the conclusions of cost - effectiveness modelling . in australia , it is estimated that one quarter of the preventable cancers are in men , but this most likely varies between countries . there is not enough information on how vaccinating males would change the transmission of hpv , but it is possible that vaccinating only females could result in an increase in hpv transmission like in the case of rubella vaccination and rubella acquisition by young adult females in the uk . british data also suggests that due to sexual behaviour characteristics british men are at greater risk of being exposed to , contracting , and transmitting hpv infection than females . it is important to estimate if this assumption is true , and the extent of the possible difference . overall , the highly infectious nature ( high transmission probability ) of hpv supports the idea of vaccinating both sexes . it is also possible that vaccinating the same number of males and females as in the female - only vaccination strategy would decrease the prevalence of hrhpv infections slightly in a steady state of hrhpv epidemics and in dynamic state of the hrhpv epidemics ( as is the case in many countries ) , and it is likely that the impact of male vaccination would be higher . the questions concerning hpv vaccine efficacy on males and the possible effect of vaccination on hpv transmission are valid as it is known that the vaccine against herpes simplex virus type 2 is not effective on males but it could still have a profound effect on hsv-2 occurrence through herd immunity provided viral shedding is significantly reduced . prevention of infectious diseases comparable to hpv with vaccines is based on producing herd immunity through a sufficient coverage in susceptible individuals to reduce transmission . the quadrivalent hpv vaccine is proven efficacious in males , likely prevents hpv transmission , and has been shown to reduce hpv 6/11-associated disease burden . vaccinating males is currently not recommended by the who , but the impact of herd immunity on female cancers and other hpv - related cancers may need to be reconsidered [ 89 , 90 ] . high vaccine coverage is needed to produce herd immunity , and sufficient coverage has been achieved only in the uk ( 75% ) and australia ( 70% ) . in the netherlands ( 50% ) , germany ( 40% ) , and usa ( 25% ) , the coverage is neither enough to protect significant proportions of females nor to produce herd immunity . protection is effective on an individual level , but in the absence of herd immunity the unvaccinated remain unprotected . at present , the questions concerning coverage in both sexes and the magnitude of herd immunity remain open [ 65 , 87 , 90 ] . mathematical models suggest that vaccinating both males and females could produce herd immunity and an impact both on hrhpv prevalence and occurrence of cervical cancer with considerably lower coverage than vaccinating females only [ 12 , 89 , 90 ] . this could be a decisive factor in the low - income areas where there are problems with access to health services . other reports indicate better results ( reduction of hpv prevalence in unvaccinated females by 8696% versus 731% ) with a vaccine coverage of 80% in females and males versus 80% in females only , but there is no evidence - based data available yet . the need for cervical cancer prevention is the greatest in developing countries where the burden of cervical cancer and other hpv - associated cancers is the heaviest , and preventive measures have not been / cannot be implemented consistently . from the point of view of global justice , the prevention of cervical cancer should be a priority in countries where its burden is the heaviest . the global alliance for vaccines and immunisation ( gavi ) considers hpv vaccines among the vaccines that would have the biggest impact on the disease burden in developing countries . within the developing countries ( and in some developed or middle - income countries ) , the situation in rural regions with major problems of access to health services poses various questions concerning equity and justice . in the reality of overall scarce resources , rural regions tend to suffer the most . this is exemplified in how screening has failed to make an impact in developing countries and especially in rural regions with problems of access to all cervical cancer prevention health services , including screening , diagnosis , treatment , and followup [ 53 , 94 ] . the poor ( < 50% ) acceptance of cervical screening by a significant proportion of females in the younger birth cohorts has resulted in a comparable loss of impact with consequent increase in cervical cancer incidence in the developed countries . in the developing countries the accessibility of health services is far too low to guarantee desired impact overall . hpv vaccines offer qualitatively different ( primary / complete versus secondary / incomplete prevention ) possibilities for preventing cervical cancer in women and other hpv - related cancers in both women and men . due to the assortative nature of common sexually transmitted infections like hpv , the sufficient coverage to produce herd immunity effect is relatively low . in the case of hpv , with a narrow window of applicability ( before sexual debut ) , it is advisable to decide early enough if the vaccine is offered only to females or to both sexes , that is , all potential carriers of hpv infection . the question is pertinent in all regions where achieving ( access to or acceptability of an hpv vaccine ) high vaccine coverage is challenging . the challenges are numerous varying from financial restraints of access to cultural or religious acceptability of the vaccine . in addition to access to health services ( in this case vaccination or screening ) , it should be noted that the success of a vaccination or a screening programme is partly dependent on decision making by the objects of the intervention or other parties like parents , spouses , other family members , influential persons in the community , and politicians . in the case of hpv , attitudes towards vaccination are more positive in persons who have more information on the vaccine , hpv , and the causality between hpv and cancer . it is probable that those who have the least information are less likely to participate than those with solid / improved information . the average , complete nonacceptability does not exceed 1015% [ 96106 ] but can be further reduced with health education . according to numerous studies , women and men in general are unaware of the causality between hpv and cervical cancer , and it can be assumed that the connection between hpv and hpv - related cancers is even less generally known . attitudes towards hpv vaccination are , however , generally positive which is probably due to positive attitudes towards vaccinations in general . hpv is such a common infection that a risk - group intervention alone is not likely to produce good results and have an impact . a high - risk group strategy based on behavioural characteristics would also be problematic to implement for a variety of reasons , most notably the inability to identify people at risk . moreover a useful strategy here might be to implement add - ons for risk groups in addition to a general vaccination programme . in the case of hpv , there may be two different risk groups : those who are at risk of infection because of risk - taking behaviour and those who are at risk of nondetection of sequelae because of geographical differences in access , that is , rural versus urban residence . due to the ubiquitous nature of hpv infection it is reasonable that the who recommendation takes only the latter risk group into account by recommending starting a phased introduction in populations who do not have access to screening . cervical cancer is associated with poverty on both the global and regional scalen [ 110112 ] . the same association can be seen in cancer incidence and mortality in general [ 113 , 114 ] . poverty is the most common source of inequity along with gender , ethnicity , religion , geography , age , education , and social status [ 115118 ] . it has been argued that health inequity in the developing countries is likely to increase if hpv vaccination programmes are not implemented [ 92 , 119 ] . cervical cancer mortality rate , which can indicate the effectiveness of screening , diagnosis , treatment , and followup , is proportionally higher ( up to 18 times ) in rural than in urban areas [ 110112 , 120 ] . people with low social status living in the developing ( or middle - income ) countries and in the rural regions are the least well off in the case of hpv - related diseases . this again highly favours hpv vaccination as a prevention strategy in low - income areas with special emphasis on regional characteristics . this regional aspect has its implications in terms of judging different methods and optional preventive strategies that can guarantee access to and coverage of primary prevention . in the urban areas the recommended prevention strategies might be efficient to significantly reduce hpv disease burden because there are fewer problems connected to access and coverage . the challenges of implementing hpv vaccination programmes in practice are similar to some elements of implementing screening programmes , that is , financial constraints , competing health needs , and limited human resources . the infrastructure and logistical capacity needed are much more limited in rural regions and will require investments . the question is whether we can opt for a single strategy for a country or a group of countries or we should look at regional characteristics linked to access in deciding which strategy to adopt case by case . have stated that strategies aiming at herd immunity ( providing the vaccine to males and females ) might be the best way to prevent cervical cancer from the point of view of justice . strategies aiming at herd immunity in the developed countries would also protect those who do not have access to the vaccine ( or screening ) or who do not accept vaccination or screening . strategies that are doable in developed countries may not be practical in low - resource regions . thus , it is appealing to contemplate different strategies for rural and urban regions taking into account access / distance to health services and opting for regional and/or population subgroup - specific strategies in order to achieve the highest possible impact . in case mass vaccination a few relevant regional or even community - level strategies from the point of view of developing countries and/or regions are described as follows : vaccinating females at the age of 1215 : unrealistic targeting a coverage of 70%,questionable herd immunity effect , marginalized females excluded ; unrealistic targeting a coverage of 70% , questionable herd immunity effect , marginalized females excluded ; vaccinating females when school attendance is at the highest ( 1012 years of age ) : unrealistic targeting a coverage of > 80% , prioritizing vaccinating younger girls before school attendance drops questionable herd immunity effect , marginalized females excluded ; unrealistic targeting a coverage of > 80% , prioritizing vaccinating younger girls before school attendance drops need for boosters ? questionable herd immunity effect , marginalized females excluded ; vaccinating females and males at the age of 1215 : targeting a coverage of > 40% in all regions , cost effectiveness , acceptance of the vaccine for boys?marginalization tackled by the herd immunity effect ? targeting a coverage of > 40% in all regions , acceptance of the vaccine for boys ? marginalization tackled by the herd immunity effect ? vaccinating females and males when school attendance is at the highest ( 1012 years of age ) : targeting a coverage of > 50% in all regions , acceptance of the vaccine for boys?marginalization tackled by the herd - immunity effect . targeting a coverage of > 50% in all regions , acceptance of the vaccine for boys ? all strategies would be school based with community outreach activities in regions where school attendance is low and equally there would be a need for information campaigns to adolescents , service providers , decision makers , schools , and parents . information campaigns for both sexes with a notion to all hpv - related cancers might further increase the acceptance of hpv vaccine . it would also be important to address local or cultural issues which are linked to vaccines . in terms of hpv vaccine coverage in developed countries , school - based demonstration projects have shown promise in terms of coverage and compliance . in a universal school - based vaccination programm the adolescents who go to school may bring also nonattendees to the vaccination site . school attendance of girls has increased in developing countries from 78% in 1990 to 85% in 2005 [ 125 , 126 ] . the gender gap between boys and girls has disappeared in east asia , latin america , and eastern and southern africa and is diminishing both in urban and rural regions and within economic quintiles [ 125 , 127 ] . school - based programmes may not be feasible if sufficient resources are not allocated to providers . school attendance in girls during adolescence may be lower than is needed for effective coverage , and certain high - risk groups might not be reached at all [ 124 , 129 ] . the project funded by path , an american ngo , in peru , uganda , india , and vietnam reached coverage of 8095% in 914-year - old girls in selected schools demonstrating a high acceptability of the hpv vaccine . the path project is also producing vital practical information on introducing hpv vaccines in developing countries ( e.g. , in certain cultures parents do not have documentation concerning date of birth , there may be undue concerns for fertility , emphasis on cancer prevention , etc . ) . even though hpv vaccination shares many barriers with cervical screening , it seems that barriers linked to acceptation of hpv vaccine might be easier to deal with . for those females who agreed to be vaccinated , completion of the three - dose regimen was over 90% in peru . it is not clear whether vaccination rates such as these would be achievable in a nonresearch setting . optional preventive strategies , regional strategies , or mixed strategies , offering vaccination only to girls in some wealthier regions , and to both sexes in certain low - income regions could tackle the problems linked with coverage and access . in some areas , it might be more feasible to have boys vaccinated because of higher school attendance , and this could lessen problems of coverage even though fewer girls would be reached . other preventive acts such as male circumcision and use of condoms make this a problem which has a solution that includes actions by both males and females . tackling the problem as gender - free problem might promote vaccine acceptance and create political will . hence , the proposed add - ons would include vaccinating both sexes to achieve maximal coverage and acceptance . targeting the early adolescents when school attendance is at the highest but before sexual debut might be a problem . in a study conducted in south africa , some parents expressed their fear that vaccination at 11 years or older would already be too late . the questions concerning dosage are closely linked with logistics , vaccine storage , vaccine acceptability , and cost effectiveness . the present vaccines are administered in three doses , but it is possible that a two - dose regimen could be enough to provide protection . this would have a significant effect mainly on costs but equally to vaccine acceptability and accessibility . in a study on kenyan women , the acceptability of a three - dose vaccine regimen was only 31% compared to 86% for a one - dose regimen . it is known that present vaccines do not provide effective protection in a one - dose regimen but a two - dose regimen remains possible . further cost - effectiveness studies are needed taking into account other hpv - related cancers besides cervical cancer . regional characteristics and problems linked with access and coverage vaccination strategies including catch - up vaccination in older females or addressing early adolescents after the sexual debut should be linked with the most accessible screening and treatment methods such as the single visit approach ( via and cryotherapy ) to ensure that the means of prevention would protect those who may be already hpv infected and for whom the prophylactic vaccine can not be effective . information about hpv vaccination and hpv - related cancers continues to emerge , but more research is needed especially on the long - term impact of vaccination , duration of protection , male vaccination , and reduction of hpv transmission . the gavi alliance subsidises the provision of vaccines to the poorest countries and is currently reviewing hpv vaccine as a candidate for sustainable financing . even with secured financing , there is no simple answer concerning which strategy should be adopted in the developing countries . hpv vaccination is the most promising way to prevent cervical and other hpv - related cancers in developing countries . vaccination strategies have an important effect on the success of any vaccination programme . in the case of hpv , it is crucial to reach at least 70% of females or 4050% of both sexes before sexual debut . optional preventive strategies , regional strategies , or mixed strategies in rural low - income regions could solve the problems linked with hpv vaccination coverage , access , and acceptability . regional characteristics affect fundamentally the feasibility of hpv vaccination strategies as it has already been proven with screening . differences between regions in terms of access to health services increase the need to adopt region - specific hpv vaccination strategies that are not currently deemed as cost effective . emerging information on hpv - related cancers in both women and men and the feasibility of achieving high vaccine coverage in rural regions might produce different results in terms of cost effectiveness , and this might result as a change in strategic aims and recommendations . targeting rural low - resource regions with specific vaccination strategies should be a priority from the point of view of ethics and public health . the number of cervical cancer cases is estimated to increase dramatically in developing countries if no intervention is implemented , and the trend is probably even stronger in regions where access to health services is limited . whatever option is chosen , it is vital to merge any vaccination strategy with appropriate screening methods and sexual education .

cervical cancer and other human papillomavirus- ( hpv- ) related cancers are preventable , but preventive measures implemented in developing countries and especially in low - income rural regions have not been effective . cervical cancer burden derived from sexually transmitted hpv infections is the heaviest in developing countries , and a dramatic increase in the number of cervical cancer cases is predicted , if no intervention is implemented in the near future . hpv vaccines offer an efficient way to prevent related cancers . recently implemented school - based hpv vaccination demonstration programmes can help tackle the challenges linked with vaccine coverage , and access to vaccination and health services , but prevention strategies need to be modified according to regional characteristics . in urban regions who - recommended vaccination strategies might be enough to significantly reduce hpv - related disease burden , but in the rural regions additional vaccination strategies , vaccinating both sexes rather than only females when school attendance is the highest and applying a two - dose regime , need to be considered . from the point of view of both public health and ethics identification of the most effective prevention strategies is pivotal , especially when access to health services is limited . considering cost - effectiveness versus justice further research on optional vaccination strategies is warranted .

1. Background 2. Prevention of HPV-Related Cancers 3. Primary Prevention Strategies in Developed Countries 4. Prevention Strategies of HPV-Related Cancers in the Developing Countries 5. Conclusion

human papillomavirus ( hpv ) is the main cause of cervical cancer , the third most common cancer in women . thus , hpv can be characterised as a ubiquitous , sexually transmitted infection ( sti ) causing significant disease burden in both sexes , but especially in women with up to 6 - 7% lifetime risk of developing cervical cancer in latin america . of the estimated 530000 annual cases of cervical cancer , its standardised incidence ratio ( world , 100 ) is substantially higher in the developing countries ( 116 ) than in the developed countries ( 60 ) . it is important to note that cervical cancer is the most common cancer in women in most parts of africa , central america , southern asia , and melanesia . it is also the most important cause of years of life lost in latin america and the caribbean , and among cancers in the populous regions of sub - saharan africa and south - central asia . furthermore , largely due to changes in sexual risk taking behaviour and ( in some countries ) dynamic state of epidemics by high - risk ( hr ) hpv types [ 15 , 16 ] , the number of cervical cancer cases has been predicted to rapidly increase ( up to 90% ) by 2020 in developing countries if no intervention is implemented [ 17 , 18 ] . there are clear signs that the incidence of most hpv - related cancers is increasing . increase in the incidence of anal cancer is also reported in australia . in the developed countries , also the incidence of hpv - related head and neck cancer is rapidly increasing especially in males and in the younger birth cohorts [ 5 , 6 , 911 , 24 ] . in australia , netherlands , sweden , and usa , the incidence of hpv - related tonsillar cancer has rapidly increased during recent years [ 7 , 23 , 25 , 26 ] . the risk of cervical cancer increases as the hrhpv infection persists . over 70% of cervical cancers are attributed to hpv types 16 and 18 and approximately 20% to hpv types 31 , 33 , 35 , 45 , 52 , and 58 [ 3 , 27 ] . a majority of other hpv - related cancers are also attributable to hpv types 16 and 18 . for example , it is estimated that 24% of cancers in the mouth are associated with hpv and 95% of these cancers are attributable to hpv types 16 and 18 . thus , it can be assumed that targeting hpv types 16 and 18 and a remarkable proportion of phylogenetically related hrhpv types ( 31 , 33 , 45 ) by prophylactic vaccination [ 2830 ] would play a significant role in preventing all hpv - related cancers . reasons for the recent increase of oropharyngeal hpv - related cancers , especially in younger male birth cohorts in the developed countries , are not clear , but changes in sexual behaviour , efficiency of hpv transmission through oral sex , and lack of protective immunity from hrhpv infections of the genital mucosae have been suggested [ 6 , 36 ] . various studies indicate that a high number of lifetime sexual partners , tobacco smoking , parity , oral contraceptive use , and coinfections with chlamydia trachomatis and hiv increase the risk of acquisition of hrhpv infection [ 3742 ] . stis are a serious health problem in developing countries , and several studies indicate that conventional stis increase the likelihood of hiv transmission . it is also highly likely that hpv - related neoplasia progresses faster in hiv - positive people . similar to liver cancer that is secondary to hepatitis b infection application of cervical cytology in population - based screening programmes has significantly lowered the incidence of cervical cancer in developed countries . cervical cancer incidence and mortality decreased markedly in the nordic countries , europe , canada , and usa due to the implementation of cervical cytology in health care , most notably in population - based screening programmes [ 5154 ] . mortality from cervical cancer has also substantially declined since the 1960s in europe , but there are still large country - specific differences . increases in the incidence of and mortality from cervical cancer have been reported in the last 15 years resulting from hpv epidemics and a drop in the number of women participating in the screening programmes . mortality rates of cervical cancer are lower than incidence rates with a ratio of mortality to incidence of 55% . survival rates are , however , lower in the developing countries [ 5658 ] , and the differences in the ratios of mortality to incidence between developing and developed countries are significant . the differences can be explained by the stage at which cancer is detected , access to health services , and adequacy of treatment . currently there are two licensed prophylactic hpv vaccines , a bivalent vaccine ( cervarix ) against hpv types 16 and 18 and a quadrivalent ( gardasil ) vaccine against hpv types 6 , 11 , 16 , and 18 available ( fda 2006 ; emea 2007 ) . in order to be prophylactic , both vaccines need to be administered before the individual is exposed to hpv types covered by the vaccine . according to reports from the major phase iii trials , the vaccines prevent from 97 to 98% of infections caused by hpv types 16 and 18 [ 59 , 60 ] . as indicated above , over 70% of cervical cancers are attributed to hpv types 16 and 18 , and approximately 20% to hpv types 31 , 33 , 35 , 45 , 52 , and 58 [ 3 , 27 ] which fits the cross - protection efficacy and reported 87% overall vaccine efficacy against cin3 + . it is assumed that both hpv vaccines , in preventing hrhpv infections , prevent other hpv - related cancers besides cervical cancer with high to moderate efficacy . it is not known if hiv infection will affect the efficacy of hpv vaccines , but smoking does not seem to affect hpv vaccine - induced antibody response . type replacement , that is , how nontargeted hrhpv types that may have competitive advantage will behave following mass vaccination , is an open question , but the likelihood of the kind of type replacement seen following bacterial vaccination is small due to the different biology of viral and bacterial infections [ 68 , 69 ] . according to who , 22 countries in low - resource regions have included an hpv vaccine in their vaccine programmes . adopted vaccination strategies include offering the vaccine only to 1215-year - old girls or only to a certain proportion of the female population of the same age . the vaccination strategies are in line with the results of numerous cost - effectiveness studies which suggest that with high vaccine coverage ( 75% ) vaccination of males would not be cost effective [ 7182 ] . the assumptions behind these recommendations and the superior cost - effectiveness of female vaccination strategy are that the health service system covers all the regions equitably and that adolescent girls have both access to the health services and are willing to use it . however , the probability of preventing other hpv - related cancers both in females and males has not been taken into account . emerging information on hpv - related cancers in men may change the conclusions of cost - effectiveness modelling . in australia , it is estimated that one quarter of the preventable cancers are in men , but this most likely varies between countries . there is not enough information on how vaccinating males would change the transmission of hpv , but it is possible that vaccinating only females could result in an increase in hpv transmission like in the case of rubella vaccination and rubella acquisition by young adult females in the uk . it is important to estimate if this assumption is true , and the extent of the possible difference . overall , the highly infectious nature ( high transmission probability ) of hpv supports the idea of vaccinating both sexes . it is also possible that vaccinating the same number of males and females as in the female - only vaccination strategy would decrease the prevalence of hrhpv infections slightly in a steady state of hrhpv epidemics and in dynamic state of the hrhpv epidemics ( as is the case in many countries ) , and it is likely that the impact of male vaccination would be higher . the quadrivalent hpv vaccine is proven efficacious in males , likely prevents hpv transmission , and has been shown to reduce hpv 6/11-associated disease burden . vaccinating males is currently not recommended by the who , but the impact of herd immunity on female cancers and other hpv - related cancers may need to be reconsidered [ 89 , 90 ] . high vaccine coverage is needed to produce herd immunity , and sufficient coverage has been achieved only in the uk ( 75% ) and australia ( 70% ) . in the netherlands ( 50% ) , germany ( 40% ) , and usa ( 25% ) , the coverage is neither enough to protect significant proportions of females nor to produce herd immunity . protection is effective on an individual level , but in the absence of herd immunity the unvaccinated remain unprotected . mathematical models suggest that vaccinating both males and females could produce herd immunity and an impact both on hrhpv prevalence and occurrence of cervical cancer with considerably lower coverage than vaccinating females only [ 12 , 89 , 90 ] . this could be a decisive factor in the low - income areas where there are problems with access to health services . other reports indicate better results ( reduction of hpv prevalence in unvaccinated females by 8696% versus 731% ) with a vaccine coverage of 80% in females and males versus 80% in females only , but there is no evidence - based data available yet . the need for cervical cancer prevention is the greatest in developing countries where the burden of cervical cancer and other hpv - associated cancers is the heaviest , and preventive measures have not been / cannot be implemented consistently . from the point of view of global justice , the prevention of cervical cancer should be a priority in countries where its burden is the heaviest . the global alliance for vaccines and immunisation ( gavi ) considers hpv vaccines among the vaccines that would have the biggest impact on the disease burden in developing countries . within the developing countries ( and in some developed or middle - income countries ) , the situation in rural regions with major problems of access to health services poses various questions concerning equity and justice . in the reality of overall scarce resources , rural regions tend to suffer the most . this is exemplified in how screening has failed to make an impact in developing countries and especially in rural regions with problems of access to all cervical cancer prevention health services , including screening , diagnosis , treatment , and followup [ 53 , 94 ] . the poor ( < 50% ) acceptance of cervical screening by a significant proportion of females in the younger birth cohorts has resulted in a comparable loss of impact with consequent increase in cervical cancer incidence in the developed countries . in the developing countries the accessibility of health services is far too low to guarantee desired impact overall . hpv vaccines offer qualitatively different ( primary / complete versus secondary / incomplete prevention ) possibilities for preventing cervical cancer in women and other hpv - related cancers in both women and men . in the case of hpv , with a narrow window of applicability ( before sexual debut ) , it is advisable to decide early enough if the vaccine is offered only to females or to both sexes , that is , all potential carriers of hpv infection . the question is pertinent in all regions where achieving ( access to or acceptability of an hpv vaccine ) high vaccine coverage is challenging . the challenges are numerous varying from financial restraints of access to cultural or religious acceptability of the vaccine . in addition to access to health services ( in this case vaccination or screening ) , it should be noted that the success of a vaccination or a screening programme is partly dependent on decision making by the objects of the intervention or other parties like parents , spouses , other family members , influential persons in the community , and politicians . in the case of hpv , attitudes towards vaccination are more positive in persons who have more information on the vaccine , hpv , and the causality between hpv and cancer . according to numerous studies , women and men in general are unaware of the causality between hpv and cervical cancer , and it can be assumed that the connection between hpv and hpv - related cancers is even less generally known . poverty is the most common source of inequity along with gender , ethnicity , religion , geography , age , education , and social status [ 115118 ] . it has been argued that health inequity in the developing countries is likely to increase if hpv vaccination programmes are not implemented [ 92 , 119 ] . cervical cancer mortality rate , which can indicate the effectiveness of screening , diagnosis , treatment , and followup , is proportionally higher ( up to 18 times ) in rural than in urban areas [ 110112 , 120 ] . people with low social status living in the developing ( or middle - income ) countries and in the rural regions are the least well off in the case of hpv - related diseases . this again highly favours hpv vaccination as a prevention strategy in low - income areas with special emphasis on regional characteristics . in the urban areas the recommended prevention strategies might be efficient to significantly reduce hpv disease burden because there are fewer problems connected to access and coverage . the challenges of implementing hpv vaccination programmes in practice are similar to some elements of implementing screening programmes , that is , financial constraints , competing health needs , and limited human resources . have stated that strategies aiming at herd immunity ( providing the vaccine to males and females ) might be the best way to prevent cervical cancer from the point of view of justice . strategies aiming at herd immunity in the developed countries would also protect those who do not have access to the vaccine ( or screening ) or who do not accept vaccination or screening . thus , it is appealing to contemplate different strategies for rural and urban regions taking into account access / distance to health services and opting for regional and/or population subgroup - specific strategies in order to achieve the highest possible impact . in case mass vaccination a few relevant regional or even community - level strategies from the point of view of developing countries and/or regions are described as follows : vaccinating females at the age of 1215 : unrealistic targeting a coverage of 70%,questionable herd immunity effect , marginalized females excluded ; unrealistic targeting a coverage of 70% , questionable herd immunity effect , marginalized females excluded ; vaccinating females when school attendance is at the highest ( 1012 years of age ) : unrealistic targeting a coverage of > 80% , prioritizing vaccinating younger girls before school attendance drops questionable herd immunity effect , marginalized females excluded ; unrealistic targeting a coverage of > 80% , prioritizing vaccinating younger girls before school attendance drops need for boosters ? vaccinating females and males when school attendance is at the highest ( 1012 years of age ) : targeting a coverage of > 50% in all regions , acceptance of the vaccine for boys?marginalization tackled by the herd - immunity effect . all strategies would be school based with community outreach activities in regions where school attendance is low and equally there would be a need for information campaigns to adolescents , service providers , decision makers , schools , and parents . information campaigns for both sexes with a notion to all hpv - related cancers might further increase the acceptance of hpv vaccine . in terms of hpv vaccine coverage in developed countries , school - based demonstration projects have shown promise in terms of coverage and compliance . in a universal school - based vaccination programm the adolescents who go to school may bring also nonattendees to the vaccination site . school attendance of girls has increased in developing countries from 78% in 1990 to 85% in 2005 [ 125 , 126 ] . the gender gap between boys and girls has disappeared in east asia , latin america , and eastern and southern africa and is diminishing both in urban and rural regions and within economic quintiles [ 125 , 127 ] . school - based programmes may not be feasible if sufficient resources are not allocated to providers . school attendance in girls during adolescence may be lower than is needed for effective coverage , and certain high - risk groups might not be reached at all [ 124 , 129 ] . the project funded by path , an american ngo , in peru , uganda , india , and vietnam reached coverage of 8095% in 914-year - old girls in selected schools demonstrating a high acceptability of the hpv vaccine . the path project is also producing vital practical information on introducing hpv vaccines in developing countries ( e.g. even though hpv vaccination shares many barriers with cervical screening , it seems that barriers linked to acceptation of hpv vaccine might be easier to deal with . for those females who agreed to be vaccinated , completion of the three - dose regimen was over 90% in peru . optional preventive strategies , regional strategies , or mixed strategies , offering vaccination only to girls in some wealthier regions , and to both sexes in certain low - income regions could tackle the problems linked with coverage and access . in some areas , it might be more feasible to have boys vaccinated because of higher school attendance , and this could lessen problems of coverage even though fewer girls would be reached . hence , the proposed add - ons would include vaccinating both sexes to achieve maximal coverage and acceptance . targeting the early adolescents when school attendance is at the highest but before sexual debut might be a problem . the questions concerning dosage are closely linked with logistics , vaccine storage , vaccine acceptability , and cost effectiveness . the present vaccines are administered in three doses , but it is possible that a two - dose regimen could be enough to provide protection . it is known that present vaccines do not provide effective protection in a one - dose regimen but a two - dose regimen remains possible . further cost - effectiveness studies are needed taking into account other hpv - related cancers besides cervical cancer . regional characteristics and problems linked with access and coverage vaccination strategies including catch - up vaccination in older females or addressing early adolescents after the sexual debut should be linked with the most accessible screening and treatment methods such as the single visit approach ( via and cryotherapy ) to ensure that the means of prevention would protect those who may be already hpv infected and for whom the prophylactic vaccine can not be effective . information about hpv vaccination and hpv - related cancers continues to emerge , but more research is needed especially on the long - term impact of vaccination , duration of protection , male vaccination , and reduction of hpv transmission . even with secured financing , there is no simple answer concerning which strategy should be adopted in the developing countries . hpv vaccination is the most promising way to prevent cervical and other hpv - related cancers in developing countries . in the case of hpv , it is crucial to reach at least 70% of females or 4050% of both sexes before sexual debut . optional preventive strategies , regional strategies , or mixed strategies in rural low - income regions could solve the problems linked with hpv vaccination coverage , access , and acceptability . regional characteristics affect fundamentally the feasibility of hpv vaccination strategies as it has already been proven with screening . differences between regions in terms of access to health services increase the need to adopt region - specific hpv vaccination strategies that are not currently deemed as cost effective . emerging information on hpv - related cancers in both women and men and the feasibility of achieving high vaccine coverage in rural regions might produce different results in terms of cost effectiveness , and this might result as a change in strategic aims and recommendations . targeting rural low - resource regions with specific vaccination strategies should be a priority from the point of view of ethics and public health . the number of cervical cancer cases is estimated to increase dramatically in developing countries if no intervention is implemented , and the trend is probably even stronger in regions where access to health services is limited .

molecular data are generally unavailable for members of asterinaceae and the taxonomy of most genera in this family to date relies chiefly on morphological data . overcoming this limitation will depend on recollecting the taxa described in the past and where appropriate epitypifying these after extracting and sequencing genomic dna . there have been a few cases where this was performed successfully from old herbarium specimens ( telle & thines 2008 , ogorman et al . the genera of asterinaceae are presently segregated using well - defined morphological characters , such as the presence or absence of appressoria on the external mycelium , setae on ascomata and/or on the external mycelium , appressorium type ( intercalary or lateral ) , and ascospore septation ( bezerra 2004 , hosagoudar 2012 ) . within the family , only three genera have intercalary appressoria : asterolibertia , cirsosia , and bheemamyces ( arnaud 1918 , bezerra 2004 , hosagoudar 2010 , 2012 ) . however , in bheemamyces the appressoria are both lateral and intercalary ( hosagoudar et al . 2010 ) . for almost a century , asterolibertia was considered a well - supported genus , due to the presence of intercalary appressoria as originally established by arnaud ( 1918 ) , and this has been always regarded as a strong morphological character . however , hongsanan et al . ( 2014 ) , without any molecular basis and morphological justification , recombined species of asterolibertia into asterina , a genus with species showing only lateral appressoria . such recombinations must be regarded as questionable , particularly in the absence of molecular data . currently , asterolibertia includes 35 species ( tables 12 ) found only in the tropics and mainly on the host families chrysobalanaceae , malpighiaceae , and rubiaceae ( hosagoudar 2010 , farr & rossman 2015 ) . the genus cirsosia accommodates 15 species and one variety , all from the tropics ( tables 34 ) , found mainly on arecaceae , chrysobalanaceae , dipterocarpaceae , and malpighiaceae ( hosagoudar 2010 , farr & rossman 2015 ) . asterolibertia couepiae , the type species of the genus , was collected in the brazilian cerrado by ule in 1892 , while c. manaosensis , the type species of cirsosia , also from brazil , was collected also by ule in the amazonian forest ( arnaud 1918 ) . asterolibertia species are characterized by having circular thyriothecial ascomata opening by a central star - shape fissure , adhering to the host by superficial hyphae with intercalary appressoria , and showing 2-celled ascospores . cirsosia species differ from asterolibertia in the lirelliform or v y - shaped ascomata , opening by a longitudinal fissure . however , as in asterolibertia , cirsosia species have superficial hyphae with intercalary appressoria and 2-celled ascospores ( bezerra 2004 , hosagoudar 2010 , 2012 ) . the specimens studied here were collected from different areas of the brazilian cerrado ( including a fragment of cerrado vegetation inserted in an area of the brazilian atlantic forest in the state of bahia ) and yielded what is here recognised as novel taxa and host - associations for asterolibertia and cirsosia . this paper describes four new asterolibertia species , a new variety of c. splendida , and illustrates a. licaniae in association with a new host . leaves bearing black colonies were collected and dried in a plant press before being processed and deposited in the fungarium known as the ub mycological collection , a part of herbarium ub ( universidade de braslia ) . after these had solidified and fungal structures became embedded preventing the collapse of the colonies , these were peeled from the leaf surfaces . these colonies and small samples taken directly from the leaves were mounted on slides containing lacto - glycerol for light microscopic observations . imaging and measurements were done using a leica dm 2500 light microscope adapted with a dfc 490 leica digital camera , operated by a leica qwin plus digital image - processing software . for scanning electron microscopy ( sem ) , air - dried material was fixed to disks using carbon double - sided tape , and then treated with gold using a 25-ma current , at 1.102 mbar for 2 min and 30 s. photographs were obtained using a jeol model jsm-700 1 f sem . a total of 35 species of asterolibertia have previously been described on 19 different host families ( hosagoudar 2010 , farr & rossman 2015 ) . these have been described on the families : chrysobalanaceae ( 9 species , including two new species described here ) , rubiaceae ( 4 ) ; anacardiaceae , annonaceae , dipterocarpaceae , fabaceae , and malpighiaceae ( 2 on each ) ; and achariaceae , arecaceae , bignoniaceae , bromeliaceae , burseraceae , ebenaceae ( described here ) , erythroxylaceae ( described here ) , euphorbiaceae , lauraceae , melastomataceae , myrtaceae , poaceae , polygalaceae , and styracaceae ( 1 on each ; table 2 ) . asterolibertia bredemeyerae was reported from two different families ( polygalaceae and fabaceae ) , as well as a. schroeteri ( annonaceae and chrysobalanaceae ) and a. peruviana ( bignoniaceae and chrysobalanaceae ) . however , a. peruviana apparently belongs to microthyriaceae due to the well - defined circular ostiole present on the ascomata ( hansford 1955 , wu et al . 2011 ) . as asterolibertia species are usually host specific , a reevaluation of the three species that occur in two different host families is recommended . a total of 15 species and one variety of cirsosia have been described on six different host families ( hosagoudar 2010 , hofmann & piepenbring 2014 , farr & rossman 2015 ) : dipterocarpaceae ( 5 species ) ; arecaceae ( 4 ) ; chrysobalanaceae ( 3 , including the new variety described here ) ; malpighiaceae ( 2 ) ; and burseraceae and lauraceae ( 1 on each ; table 4 ) . there is no record of the same cirsosia species being found on two different host families ( hosagoudar 2010 , farr & rossman 2015 ) . the data in tables 14 that include the characteristics of the new taxa herein proposed were used to formulate the keys provided for the identification of asterolibertia and cirsosia species , thus simplifying the text . diagnosis : asterolibertia bahiensis is quite close to a. nodulifera but differs in having smaller paraphysate ascomata and larger appressoria . type : brazil : bahia : una , bolandeira farm , on an enclosure of cerrado vegetation in the brazilian atlantic forest , close to the entrance to comandatuba island , 15 21 12.7 s 39 00 7.7 w , on leaves of erythroxylum sp . ( erythroxylaceae ) , 26 aug . 1995 , m. sanchez ( ub - mycol col . description : colonies amphigenous , circular to irregular , single to confluent , black , 112 mm diam . hyphae straight to flexuous , branching irregularly , pale brown , septate , hyphal cells cylindrical , 45 m diam , smooth . appressoria numerous , entire , sessile , intercalary , elongated with a lateral protuberance , unicellular , 1417.5 910 m , brown , penetration peg central on the appressorial cell . ascomata superficial , thyriothecia , scutiform , on top of mycelial mat , circular , single to confluent , fringed at margins , randomly distributed in the colony , 175235 m diam , opening by a central star - shaped fissure , dark brown ; wall of textura radiata , cells cylindrical . asci bitunicate in structure , fissitunicate , disposed as an upright palisade layer , globose to ovoid , 8-spored , hyaline , 3552.5 3042.5 m . ascospores cylindrical , ends rounded , straight or slightly arched , 1-septate , constricted at the septum at the supramedian septum , hyaline , becoming brown at maturity , verruculose , 32.537.5 1014 m . brazil : minas gerais : buritis , pedra grande farm , 8 may 1993 , j. c. dianese 863 ( ub - mycol col . bahia : una , on an enclosure of cerrado vegetation in the brazilian atlantic forest at fazenda bolandeira , near entrance to comandatuba island , j.c . notes : the type material was collected in a rare small enclosure of cerrado vegetation in the brazilian atlantic forest . 3934 ) was found in a typical cerrado natural landscape in buritis , minas gerais . in addition , this is the first asterolibertia species found on a member of erythroxylaceae ( hosagoudar 2010 , farr & rossman 2015 ) . diagnosis : asterolibertia barrinhensis is quite close to a. campograndensis but differs in having opposite hyphal branching , loose ascomatal fringes and verruculose ascospores . type : brazil : minas gerais : divinpolis , barrinha farm , right side of highway from divinoplis to formiga , 20 13 54.9 s 45 05 33.7 w , on leaves of diospyros burchellii ( ebenaceae ) , 16 feb . description : colonies epiphyllous , circular to irregular , single to confluent , black , 18 mm diam . hyphae straight to flexuous , with opposite branches , ferruginous to brown , septate , hyphal cells cylindrical , 45 m diam , smooth . appressoria numerous , entire , intercalary , elongated with a lateral protuberance , unicellular , 915 7.510 m , ferruginous to brown , penetration peg central on the appressorial cell . ascomata superficial , thyriothecia , scutiform , on top of mycelial mat , circular , single to confluent , fringed at the margins , randomly distributed in the colony , 105167.5 m diam , opening by a central star - shaped fissure , brown ; wall of textura radiata , cells isodiametric to cylindrical . asci bitunicate in structure , fissitunicate , forming as an upright palisade layer , globose to ovoid , 8-spored , hyaline , 3042.5 m diam . ascospores cylindrical , oblong - clavate , ends broadly rounded , straight , 1-septate , slightly constricted at supramedian septum , hyaline , becoming brown at maturity , verruculose , 2027.5 912.5 m . 1994 , j. c. dianese ( ub - mycol col . 5891 , and 5901 ) ; gois : mineiros , parque nacional das emas , gua ruim , 18 8 12.04 s 52 58 44.06 w , 7 apr . notes : this new asterolibertia species is the first reported on a member of ebenaceae ( hosagoudar 2010 , farr & rossman 2015 ) . it shows characteristics in common with several species ( tables 12 ) , including the one described below , but clear differences persist as shown in the discussion that follows the description of a. campograndensis . etymology : epithet refers to the city where the fungus was collected , campo grande . diagnosis : asterolibertia campograndensis differs from a. parinarii in having larger hyphae , appressoria and ascospores , and globose to ovoid asci . type : brazil : mato grosso do sul : campo grande , left lane of br-163 highway , 200 m from the roundabout turn to so paulo , behind cerealista juliana , 20 35 8.58 s 54 34 49.51 w , on leaves of hirtella glandulosa ( chrysobalanaceae ) , 22 aug . description : colonies epiphyllous , circular to irregular , single to confluent , black , 16 mm diam . hyphae straight to flexuous , mostly showing opposite seldom irregular branches , ferruginous to brown , septate , hyphal cells cylindrical , 57 m diam , smooth . appressoria numerous , entire , intercalary , elongated with a lateral protuberance , unicellular , 914 7.512 m , ferruginous to brown , penetration peg central on the appressorial cells . ascomata superficial , thyriothecia , scutiform , on top of mycelial mat , circular , single to confluent , fringed at margins , massed in the centre of the colony , 75160 m diam , opening by a central star - shaped fissure , dark brown ; wall of textura radiata to irregulata , cells cylindrical to irregular . asci bitunicate in structure , fissitunicate , disposed as an upright palisade layer , globose to ovoid , 8-spored , hyaline , 3040 2032.5 m . ascospores oblong - clavate , rounded ends , straight , 1-septate , septum supramedian , constricted at septum , hyaline , becoming brown at maturity , smooth , 22.530 9.510.5 m . notes : seven species of asterolibertia have been reported previously in association with living leaves of chrysobalanaceous hosts . four of these were recorded from brazil : a. couepiae on couepia grandiflora , a. licaniae and a. licaniicola on licania sp . , and a. peruviana on licania macrophylla . additionally , a. nodulifera was recorded on angelesia splendens from the philippines , a. parinarii on parinari subcordata from the democratic republic of the congo , and a. schroeteri on chrysobalanus icaco from india ( arnaud 1918 , hansford 1947 , 1949 , 1955 , mller & von arx 1962 , hosagoudar 2010 , hofmann & piepenbring 2014 , farr & rossman 2015 ) . asterolibertia campograndensis differs from the species previously reported on chrysobalanaceae ( table 1 ) ( arnaud 1918 , hansford 1947 , 1949 , 1955 , mller & von arx 1962 , hosagoudar 2010 , hofmann & piepenbring 2014 , farr & rossman 2015 ) . it is closest to a. parinarii , which has smaller appressoria and ascospores , narrower hyphae , and ellipsoid to subglobose asci . asterolibertia couepiae is distinct from the new species in having black hyphae , larger thyriothecial ascomata , lacking pseudoparaphyses , 46-spored asci , and ovoid ascospores . asterolibertia nodulifera has amphigenous colonies , no pseudoparaphyses , larger ascomata and larger , echinulate ascospores . asterolibertia licaniae differs from a. campograndensis in the dark brown hyphae , barrel - shaped and larger appressoria , ascomatal dehiscence by an irregular fissure , a lack of fringes at the margins of the ascomata , the absence of pseudoparaphyses , and finally larger , ellipsoidal , dark brown ascospores with a central septum . asterolibertia licaniicola differs from a. campograndensis in the wider hyphae , barrel - shaped appressoria , larger ascomata , absence of pseudoparaphyses , and dark brown ascospores with a central septum . asterolibertia peruviana has narrower appressoria , larger ascomata that are not fringed at the margins , and smaller appressoria and ascospores . asterolibertia schroeteri differs in the larger ascomata with an irregular fissure , the absence of pseudoparaphyses , and larger asci and ascospores ( tables 12 ) ( arnaud 1918 , hansford 1947 , 1949 , 1955 , mller & von arx 1962 , hofmann & piepenbring 2014 ) . however , these species differ in important morphological details such as ascospore ornamentation , the shape of the ascomatal fringes ( loosely set in a. barrinhensis ) , and hyphal branching patterns ( opposite in a. barrinhesis , and irregular in a. campograndensis ) . asterolibertia campograndensis is the fifth species of asterolibertia reported on hosts belonging to chrysobalanaceae in brazil , and the first on hirtella . diagnosis : asterolibertia parinaricola differs from a. licaniicola in having conspicuous lateral protuberance of the appressoria , presence of pseudoparaphyses , and much larger , verruculose ascospores constricted at a supramedian septum . type : brazil : distrito federal : braslia , pad - df , on leaves of parinari obtusifolia ( chrysobalanaceae ) , 10 nov . description : colonies epiphyllous , circular or irregular , single or confluent , black , 310 mm diam . hyphae straight , with opposite branches , brown , septate , hyphal cells cylindrical , 4.55.5 m diam , smooth . appressoria numerous , entire , intercalary , elliptical or with a lateral protuberance , unicellular , 1015 79 m , brown , penetration peg central on the appressorial cells . ascomata superficial , thyriothecia , scutiform , on top of a mycelial mat , circular , single to confluent , fringed at the margins , randomly distributed in the colony , 150207 m diam , opening by a central star - shaped fissure , dark brown ; wall textura radiata , with isodiametrical cells . asci bitunicate in structure , fissitunicate , disposed as an upright palisade layer , globose to ovoid , 8-spored , hyaline , 37.547.5 2932.5 m . ascospores oblong to oblong - clavate , ends rounded , straight to slightly arched , 1-septate , constricted at the supramedian septum , hyaline , becoming pale brown to brown at maturity , verruculose , 3440 1014 m . brazil : maranho : nogueiras , 60 km north of balsas , 6 57 52.47 notes : seven species of asterolibertia have been reported previously in association with living leaves of chrysobalanaceous hosts . asterolibertia couepiae on couepia grandiflora from brazil , a. nodulifera on angelesia splendens from the philippines , a. licaniae and a. licaniicola on licania sp . from brazil , a. parinarii on parinari subcordata from the democratic republic of the congo , a. peruviana on licania macrophylla from brazil , and a. schroeteri on chrysobalanus icaco from india ( arnaud 1918 , hansford 1947 , 1949 , 1955 , mller & von arx 1962 , hosagoudar 2010 , hofmann & piepenbring 2014 , farr & rossman 2015 ) . asterolibertia parinaricola differs from the species previously reported on chrysobalanaceae ( table 1 ) ( arnaud 1918 , hansford 1947 , 1949 , 1955 , mller & von arx 1962 , hosagoudar 2010 , hofmann & piepenbring 2014 , farr & rossman 2015 ) , and is most similar to a. licaniicola . however , the latter has barrel - shaped appressoria , no pseudoparaphyses , and much smaller , smooth ascospores constricted at the central septum . asterolibertia couepiae differs from the new species in the wider hyphae , lack of pseudoparaphyses , smaller asci and ascospores , and smooth ascospores . asterolibertia nodulifera differs in the amphigenous colonies , smaller thyrothecia , lack of pseudoparaphyses , and echinulate ascospores . additionally , a. licaniae differs from the new species in the larger thyriothecia and wider hyphae , barrel - shaped appressoria , the lack of pseudoparaphyses , and smaller , ellipsoidal , smooth ascospores that are constricted at the central septum . asterolibertia parinarii has narrower hyphae , unbranched pseudoparaphyses , smaller appressoria , asci and ascospores , and also smooth ascospores . asterolibertia parinaricola and a. parinarii are described on the same host genus , but on different parinari species . besides , there is no record of the host species of a. parinarii in brazil , further supporting that they are really distinct species ( sothers et al . asterolibertia peruviana has narrower appressoria without a lateral protuberance to one side of the hypha , and smaller , smooth ascospores . finally , a. schroeteri differs from a. parinaricola in the larger thyriothecia , asci and hyphae , the lack of pseudoparaphyses , and smooth ascospores ( tables 12 ) ( arnaud 1918 , hansford 1947 , 1949 , 1955 , mller & von arx 1962 , hofmann & piepenbring 2014 ) . asterolibertia campograndensis , newly described above , differs in the unbranched pseudoparaphyses and smaller , smooth ascospores . asterolibertia parinaricola is distinct from all seven species known on chrysobalanaceae , and represents the sixth species of asterolibertia reported on this host family in brazil . this is the second species of asterolibertia described on parinari , and the first species found on p. obtusifolia . specimen examined : brazil : rondnia : ro-494 highway , 82 km from pimenta bueno towards parecis , 11 45 16.43 s 61 18 54.45 w , on leaves of hirtella gracilipes ( chrysobalanaceae ) , 13 jul . description : colonies epiphyllous , circular to irregular , single to confluent , dark brown to black , 35 mm diam . hyphae straight or flexuous , branching irregularly , pale brown to brown , septate , hyphal cells cylindrical , 58 m diam , smooth . appressoria numerous , entire , intercalary , elliptical , unicellular , 915 7.510 m , pale brown , penetration peg central on the appressorial cell . ascomata , superficial , thyriothecial , scutiform , radiate , arising on top of a mycelial mat , circular , single to confluent , fringed at the margins , randomly distributed in the colony , 180410 m diam , opening by a central star - shaped fissure , dark brown ; wall textura radiata , cells cylindrical . asci bitunicate in structure , fissitunicate , disposed as an upright palisade layer , globose , 8-spored , hyaline , 57.565 m diam . ascospores oblong , ends rounded , straight , 1-septate , constricted at the median septum , hyaline , becoming brown to ferruginous at maturity , smooth , 3035 1922.5 m . notes : the specimen described above was collected in the state of rondnia on living leaves of hirtella gracilipes , a new host for a. licaniae . this species was originally described by hansford ( 1949 ) based on material from brazil collected on leaves of licania sp . see tables 12 for further information on the characters of the species keyed out here . a. thaxteri 2 ( 1 ) ascospores smooth ........................................................................................................................................................ 3 ascospores verruculose ............................................................................................................................................... 24 3 ( 2 ) ascospores medianly constricted ................................................................................................................................... 4 ascospores constricted supramedianly ........................................................................................................................... 9 4 ( 3 ) ascomata with fringed margin ......................................................................................................................................... 5 ascomata with uniform margin ......................................................................................................................... a. licaniae 5 ( 4 ) ascomata opening by a stellar fissure ............................................................................................................................ 6 ascomata opening by an irregular fissure ........................................................................................................ a. bredemeyerae 7 ( 6 ) ascospores more than 16 m in length .......................................................................................................................... 8 ascospores 1416 6.57 m ................................................................................................................... a. hiiranensis 8 ( 7 ) ascospores 1921 910 m ................................................................................................................ a. nothopegiae ascospores 2428 1215 m .................................................................................................................. 14 10 ( 9 ) ascomata with a fringed margin ..................................................................................................................................... 11 ascomata with a uniform margin ................................................................................................................................... a. santiriae 12 ( 10 ) ascomata over 90 m diam ......................................................................................................................................... 13 ascomata to 90 m diam ................................................................................................................................ a. cryptocaryae 14 ( 9 ) ascomata opening by stellar or irregular fissure ............................................................................................................ 15 ascomata opening by ostiole [ may belong in mycrothyriaceae ] .................................................................. a. peruviana 15 ( 14 ) pseudoparaphyses present .......................................................................................................................................... 16 pseudoparaphyses absent ........................................................................................................................................... a. parinarii 17 ( 15 ) ascomata with a fringed margin .................................................................................................................................... 18 ascomata with a uniform margin ................................................................................................................ a. hydnocarpi 18 ( 17 ) no leaf discoloration under the colonies ........................................................................................................................ 19 conspicuous leaf discoloration under the colonies ....................................................................................... a. crustacea 19 ( 18 ) ascomata opening by a stellar fissure ........................................................................................................................... 20 ascomata opening by an irregular fissure .................................................................................................... a. schroeteri 20 ( 19 ) appressoria showing a lateral protuberance .................................................................................................................. 21 appressoria barrel - shaped to cylindrical ....................................................................................................................... 22 21 ( 20 ) a. myocoproides 27 ( 26 ) appressoria with a lateral protuberance ........................................................................................................ a. malpighii appressoria barrel - shape to subglobose without a lateral protuberance ....................................................... 34 29 ( 28 ) pseudoparaphyses present .......................................................................................................................................... 30 pseudoparaphyses absent ........................................................................................................................................... 31 30 ( 29 ) pseudoparaphyses branched ................................................................................................................... 32 32 ( 31 ) hyphae 35 m wide ......................................................................................................................................... a. bakeri hyphae more than 5 m wide ....................................................................................................................................... 33 33 ( 32 ) ascospores 2430 1217 m ............................................................................................................................... a. ulei ascospores 3240 1825 m ................................................................................................................... type : brazil : mato grosso do sul : campo grande , br-163 highway left lane , 200 m from the roundabout turn to so paulo , behind cerealista juliana , 20 35 8.58 s 54 34 49.51 w , on leaves of hirtella glandulosa ( chrysobalanaceae ) , 22 aug . 1996 , m. sanchez ( ub - mycol col description : sexual morph : colonies hypophyllous , circular or irregular , single or confluent , black , 19 mm diam . hyphae straight or flexuous , with opposite branches , rarely unilateral or irregular , brown , septate , hyphal cells cylindrical , 2.55 m wide , smooth . appressoria numerous , entire , intercalary , elongated with a lateral protuberance , unicellular , 1015 4.57.5 m , brown , penetration peg central or at the distal part of the appressorial cell . ascomata superficial , hysterothecia , lirelliform , v y - shaped , on top of a mycelia mat , single to confluent , fringed at margins , randomly distributed in the colony , 110290 6090 m , opening by longitudinal fissures , brown ; wall of textura radiata , cells isodiametric to cylindrical . asci bitunicate in structure , fissitunicate , disposed as an upright palisade layer , globose to subclavate , 8-spored , hyaline , 2537.5 17.527.5 m . ascospores cylindrical to oblong - clavate , ends rounded , straight , 1-septate , slightly constricted at the supramedian septum , hyaline , becoming brown to ferruginous at maturity , smooth , 17.527.5 69.5 m . asexual morph : colonies amphigenous , circular or irregular , single or confluent , black , 18 mm diam . hyphae straight or flexuous , branching irregularly , septate , hyphal cells cylindrical , 2.53 m wide , smooth . appressoria numerous , entire , intercalary , elongated with a lateral protuberance , unicellular , 1015 57.5 m , brown , penetration peg central on the appressorial cell . conidiomata superficial , pycnothyrial , scutiform , on top of a mycelium mat , circular , single to confluent , fringed at margins , randomly distributed in the colony , 80120 m diam , centrally ostiolate , light to dark brown ; wall textura radiata , cells isodiametric to cylindrical . conidia initially 1celled becoming 2celled at maturity , ellipsoidal , upper cell with rounded end and lower cell with a truncate base , ends rounded when mature , straight , medianly or supramedianly 1-septate , not constricted at the septum , hyaline , becoming brown to ferruginous at maturity , smooth , 2025 4.55 m . other specimen examined : brazil : rondnia : ro494 highway , 82 km from pimenta bueno towards parecis , on leaves of hirtella gracilipes ( chrysobalanaceae ) , 13 jul . notes : two species of cirsosia have been reported previously in association with living leaves of chrysobalanaceous hosts : c. moquileae on licania tomentosa from brazil , and c. splendida on hirtella americana , chrysobalanus icaco , and h. triandra from brazil , panama , and puerto rico , respectively ( batista & maia 1960b , hosagoudar 2010 , hofmann & piepenbring 2014 , farr & rossman 2015 ) . laevigata , the first asterinaceae reported on h. glandulosa and h. gracilipes , is almost identical to c. splendida , except for the smaller ascomata , presence of pseudoparaphyses , and smooth ascospores ( batista & maia 1960b , hofmann & piepenbring 2014 ) . such differences are here considered enough to recognize the specimen studied as a new variety of c. splendida . furthermore , both occur on the same host genus in brazil , but on different species . finally , cirsosia moquileae differs from the new variety by dehiscence through a central star - shaped fissure ( instead of the longitudinal fissure normally shown in cirsosia species ) , and narrower ascospores ( batista & maia 1960b ) . indeed , such a form of dehiscence and the shape of the ascomata in c. moquileae indicate that that species could be better accommodated in asterolibertia ( batista & maia 1960b ) . see tables 34 for further information on the characters of the species keyed out here . 3 ascospores supramedianly constricted ........................................................................................................................... 7 3 ( 2 ) colonies amphigenous ................................................................................................................................................... 4 colonies hypophyllous or epiphyllous .............................................................................................................................. 5 4 ( 3 ) ascospores 2832 1518 m ....................................................................................................................... c. vateriae ascospores 32.535 1921.5 m ................................................................................................................ c. santiriae 5 ( 3 ) colonies epiphyllous ....................................................................................................................................................... 6 colonies hypophyllous ....................................................................................................................................... c. litseae 6 ( 5 ) asci 4-spored .......................................................................................................................................... c. arecacearum asci 8-spored ............................................................................................................................................. c. globulifera 7 ( 2 ) ascomata opening by a longitudinal fissure ..................................................................................................................... 8 ascomata opening by a stellar fissure ......................................................................................................... c. moquileae 8 ( 7 ) appressoria having a lateral protuberance ...................................................................................................................... 9 appressoria globose ...................................................................................................................................... c. hughesii 9 ( 8) ascospores 17.527.5 69.5 m ........................................................................................ c. splendida var . laevigata ascospores 2633.5 1520.5 m ..................................................................................................... c. moulmeinensis 10 ( 1 ) ascospores medianly constricted .................................................................................................................................. splendida 11 ( 10 ) colonies epiphyllous ..................................................................................................................................................... 12 colonies hypophyllous ................................................................................................................................ c. irregularis 12 ( 11 ) appressoria with a lateral protuberance ........................................................................................................................ 13 appressoria barrel - shaped to globose ........................................................................................................................... 14 13 ( 12 ) appressoria 810 13.515 m ............................................................................................................... c. dipterocarpi appressoria 1114 78 m ................................................................................................................... c. transversalis 14 ( 12 ) hosts in malpighiaceae ................................................................................................................................................. 15 hosts in dipterocarpaceae ............................................................................................................................... c. hopeae 15 ( 14 ) asci 2729.5 1924 m ........................................................................................................................... c. flabellariae asci 5565 4550 m .......................................................................................................................... c. manaosensis see tables 12 for further information on the characters of the species keyed out here . 1 colonies amphigenous or epiphyllous ............................................................................................................................ 2 colonies hypophyllous .................................................................................................................................... ascospores medianly constricted ................................................................................................................................... 4 ascospores constricted supramedianly ........................................................................................................................... 9 4 ( 3 ) ascomata with fringed margin ......................................................................................................................................... a. licaniae 5 ( 4 ) ascomata opening by a stellar fissure ............................................................................................................................ 6 ascomata opening by an irregular fissure ........................................................................................................ a. bredemeyerae 7 ( 6 ) ascospores more than 16 m in length .......................................................................................................................... 8 ascospores 1416 6.57 m ................................................................................................................... a. hiiranensis 8 ( 7 ) ascospores 1921 910 m ................................................................................................................ a. nothopegiae ascospores 2428 1215 m .................................................................................................................. 14 10 ( 9 ) ascomata with a fringed margin ..................................................................................................................................... 11 ascomata with a uniform margin ................................................................................................................................... 12 11 ( 10 ) ascospores 1518 56.5 m ........................................................................................................................ a. randiae ascospores 3236 1722 m ..................................................................................................................... a. santiriae 12 ( 10 ) ascomata over 90 m diam ......................................................................................................................................... 13 ascomata to 90 m diam ................................................................................................................................ ........................................................................................................................ a. parinarii 17 ( 15 ) ascomata with a fringed margin .................................................................................................................................... 18 ascomata with a uniform margin ................................................................................................................ a. hydnocarpi 18 ( 17 ) no leaf discoloration under the colonies ........................................................................................................................ 19 conspicuous leaf discoloration under the colonies ....................................................................................... a. crustacea 19 ( 18 ) ascomata opening by a stellar fissure ........................................................................................................................... 20 ascomata opening by an irregular fissure .................................................................................................... a. schroeteri 20 ( 19 ) appressoria showing a lateral protuberance .................................................................................................................. 21 appressoria barrel - shaped to cylindrical ....................................................................................................................... ............................................................................................................ a. pogonophorae 22 ( 20 ) ascospores less than 30 m in length ........................................................................................................................... 23 ascospores 3542 1619 m .................................................................................................................. a. nodulosa 28 ( 25 ) colonies epiphyllous ..................................................................................................................................................... 29 colonies amphigenous ................................................................................................................................................. 34 29 ( 28 ) pseudoparaphyses present .......................................................................................................................................... a. anisopterae appressoria barrel - shaped to subglobose without a lateral protuberance ..................................................................... 32 32 ( 31 ) hyphae 35 m wide ......................................................................................................................................... type : brazil : mato grosso do sul : campo grande , br-163 highway left lane , 200 m from the roundabout turn to so paulo , behind cerealista juliana , 20 35 8.58 s 54 34 49.51 w , on leaves of hirtella glandulosa ( chrysobalanaceae ) , 22 aug . 1996 , m. sanchez ( ub - mycol col . description : sexual morph : colonies hypophyllous , circular or irregular , single or confluent , black , 19 mm diam . hyphae straight or flexuous , with opposite branches , rarely unilateral or irregular , brown , septate , hyphal cells cylindrical , 2.55 m wide , smooth . appressoria numerous , entire , intercalary , elongated with a lateral protuberance , unicellular , 1015 4.57.5 m , brown , penetration peg central or at the distal part of the appressorial cell . ascomata superficial , hysterothecia , lirelliform , v y - shaped , on top of a mycelia mat , single to confluent , fringed at margins , randomly distributed in the colony , 110290 6090 m , opening by longitudinal fissures , brown ; wall of textura radiata , cells isodiametric to cylindrical . asci bitunicate in structure , fissitunicate , disposed as an upright palisade layer , globose to subclavate , 8-spored , hyaline , 2537.5 17.527.5 m . ascospores cylindrical to oblong - clavate , ends rounded , straight , 1-septate , slightly constricted at the supramedian septum , hyaline , becoming brown to ferruginous at maturity , smooth , 17.527.5 69.5 m . asexual morph : colonies amphigenous , circular or irregular , single or confluent , black , 18 mm diam . hyphae straight or flexuous , branching irregularly , septate , hyphal cells cylindrical , 2.53 m wide , smooth . appressoria numerous , entire , intercalary , elongated with a lateral protuberance , unicellular , 1015 57.5 m , brown , penetration peg central on the appressorial cell . conidiomata superficial , pycnothyrial , scutiform , on top of a mycelium mat , circular , single to confluent , fringed at margins , randomly distributed in the colony , 80120 m diam , centrally ostiolate , light to dark brown ; wall textura radiata , cells isodiametric to cylindrical . conidia initially 1celled becoming 2celled at maturity , ellipsoidal , upper cell with rounded end and lower cell with a truncate base , ends rounded when mature , straight , medianly or supramedianly 1-septate , not constricted at the septum , hyaline , becoming brown to ferruginous at maturity , smooth , 2025 4.55 m . other specimen examined : brazil : rondnia : ro494 highway , 82 km from pimenta bueno towards parecis , on leaves of hirtella gracilipes ( chrysobalanaceae ) , 13 jul . notes : two species of cirsosia have been reported previously in association with living leaves of chrysobalanaceous hosts : c. moquileae on licania tomentosa from brazil , and c. splendida on hirtella americana , chrysobalanus icaco , and h. triandra from brazil , panama , and puerto rico , respectively ( batista & maia 1960b , hosagoudar 2010 , hofmann & piepenbring 2014 , farr & rossman 2015 ) laevigata , the first asterinaceae reported on h. glandulosa and h. gracilipes , is almost identical to c. splendida , except for the smaller ascomata , presence of pseudoparaphyses , and smooth ascospores ( batista & maia 1960b , hofmann & piepenbring 2014 ) . such differences are here considered enough to recognize the specimen studied as a new variety of c. splendida . furthermore , both occur on the same host genus in brazil , but on different species . finally , cirsosia moquileae differs from the new variety by dehiscence through a central star - shaped fissure ( instead of the longitudinal fissure normally shown in cirsosia species ) , and narrower ascospores ( batista & maia 1960b ) . indeed , such a form of dehiscence and the shape of the ascomata in c. moquileae indicate that that species could be better accommodated in asterolibertia ( batista & maia 1960b ) . see tables 34 for further information on the characters of the species keyed out here . 3 ascospores supramedianly constricted ........................................................................................................................... 7 3 ( 2 ) colonies amphigenous ................................................................................................................................................... 4 colonies hypophyllous or epiphyllous .............................................................................................................................. 5 4 ( 3 ) ascospores 2832 1518 m ....................................................................................................................... c. vateriae ascospores 32.535 1921.5 m ................................................................................................................ c. santiriae 5 ( 3 ) colonies epiphyllous ....................................................................................................................................................... 6 colonies hypophyllous ....................................................................................................................................... c. litseae 6 ( 5 ) asci 4-spored .......................................................................................................................................... c. arecacearum asci 8-spored ............................................................................................................................................. c. globulifera 7 ( 2 ) ascomata opening by a longitudinal fissure ..................................................................................................................... 8 ascomata opening by a stellar fissure ......................................................................................................... c. moquileae 8 ( 7 ) appressoria having a lateral protuberance ...................................................................................................................... 9 appressoria globose ...................................................................................................................................... c. hughesii 9 ( 8) ascospores 17.527.5 69.5 m ........................................................................................ c. splendida var . laevigata ascospores 2633.5 1520.5 m ..................................................................................................... c. moulmeinensis 10 ( 1 ) ascospores medianly constricted .................................................................................................................................. c. irregularis 12 ( 11 ) appressoria with a lateral protuberance ........................................................................................................................ 13 appressoria barrel - shaped to globose ........................................................................................................................... 14 13 ( 12 ) appressoria 810 13.515 m ............................................................................................................... c. dipterocarpi appressoria 1114 78 m ................................................................................................................... c. transversalis 14 ( 12 ) hosts in malpighiaceae ................................................................................................................................................. 15 hosts in dipterocarpaceae ............................................................................................................................... c. hopeae 15 ( 14 ) asci 2729.5 1924 m ........................................................................................................................... c. flabellariae asci 5565 4550 m .......................................................................................................................... c. manaosensis we dedicate this publication to the memory of mariza sanchez recently deceased , ub mycol . we thank cnpq for fellowships , and acknowledge the financial support from ppbio / cnpq / mcti- projeto fungos do cerrado .

four new asterolibertia species and a new variety of cirsosia splendida , all found on native cerrado plants , belonging to three host families are described , illustrated and named as : a. bahiensis sp . nov . on erythroxylum sp . ( erythroxylaceae ) ; a. barrinhensis sp . nov . on diospyros burchellii ( ebenaceae ) ; a. campograndensis sp . nov . on hirtella glandulosa ( chrysobalanaceae ) ; a. parinaricola sp . nov . on parinari obtusifolia ( chrysobalanaceae ) ; and cirsosia splendida var . laevigata var . nov . , showing both sexual and asexual morphs , on h. glandulosa and h. gracilipes ( chrysobalanaceae ) . finally , a. licaniae is reported on a new host , h. gracilipes . keys to all the known species of asterolibertia and cirsosia are included .

INTRODUCTION MATERIAL AND METHODS RESULTS TAXONOMY Key to the known Key to the known ACKNOWLEDGEMENTS

( 2014 ) , without any molecular basis and morphological justification , recombined species of asterolibertia into asterina , a genus with species showing only lateral appressoria . the genus cirsosia accommodates 15 species and one variety , all from the tropics ( tables 34 ) , found mainly on arecaceae , chrysobalanaceae , dipterocarpaceae , and malpighiaceae ( hosagoudar 2010 , farr & rossman 2015 ) . asterolibertia couepiae , the type species of the genus , was collected in the brazilian cerrado by ule in 1892 , while c. manaosensis , the type species of cirsosia , also from brazil , was collected also by ule in the amazonian forest ( arnaud 1918 ) . the specimens studied here were collected from different areas of the brazilian cerrado ( including a fragment of cerrado vegetation inserted in an area of the brazilian atlantic forest in the state of bahia ) and yielded what is here recognised as novel taxa and host - associations for asterolibertia and cirsosia . this paper describes four new asterolibertia species , a new variety of c. splendida , and illustrates a. licaniae in association with a new host . a total of 35 species of asterolibertia have previously been described on 19 different host families ( hosagoudar 2010 , farr & rossman 2015 ) . these have been described on the families : chrysobalanaceae ( 9 species , including two new species described here ) , rubiaceae ( 4 ) ; anacardiaceae , annonaceae , dipterocarpaceae , fabaceae , and malpighiaceae ( 2 on each ) ; and achariaceae , arecaceae , bignoniaceae , bromeliaceae , burseraceae , ebenaceae ( described here ) , erythroxylaceae ( described here ) , euphorbiaceae , lauraceae , melastomataceae , myrtaceae , poaceae , polygalaceae , and styracaceae ( 1 on each ; table 2 ) . asterolibertia bredemeyerae was reported from two different families ( polygalaceae and fabaceae ) , as well as a. schroeteri ( annonaceae and chrysobalanaceae ) and a. peruviana ( bignoniaceae and chrysobalanaceae ) . as asterolibertia species are usually host specific , a reevaluation of the three species that occur in two different host families is recommended . a total of 15 species and one variety of cirsosia have been described on six different host families ( hosagoudar 2010 , hofmann & piepenbring 2014 , farr & rossman 2015 ) : dipterocarpaceae ( 5 species ) ; arecaceae ( 4 ) ; chrysobalanaceae ( 3 , including the new variety described here ) ; malpighiaceae ( 2 ) ; and burseraceae and lauraceae ( 1 on each ; table 4 ) . the data in tables 14 that include the characteristics of the new taxa herein proposed were used to formulate the keys provided for the identification of asterolibertia and cirsosia species , thus simplifying the text . type : brazil : bahia : una , bolandeira farm , on an enclosure of cerrado vegetation in the brazilian atlantic forest , close to the entrance to comandatuba island , 15 21 12.7 s 39 00 7.7 w , on leaves of erythroxylum sp . in addition , this is the first asterolibertia species found on a member of erythroxylaceae ( hosagoudar 2010 , farr & rossman 2015 ) . type : brazil : minas gerais : divinpolis , barrinha farm , right side of highway from divinoplis to formiga , 20 13 54.9 s 45 05 33.7 w , on leaves of diospyros burchellii ( ebenaceae ) , 16 feb . notes : this new asterolibertia species is the first reported on a member of ebenaceae ( hosagoudar 2010 , farr & rossman 2015 ) . type : brazil : mato grosso do sul : campo grande , left lane of br-163 highway , 200 m from the roundabout turn to so paulo , behind cerealista juliana , 20 35 8.58 s 54 34 49.51 w , on leaves of hirtella glandulosa ( chrysobalanaceae ) , 22 aug . notes : seven species of asterolibertia have been reported previously in association with living leaves of chrysobalanaceous hosts . four of these were recorded from brazil : a. couepiae on couepia grandiflora , a. licaniae and a. licaniicola on licania sp . additionally , a. nodulifera was recorded on angelesia splendens from the philippines , a. parinarii on parinari subcordata from the democratic republic of the congo , and a. schroeteri on chrysobalanus icaco from india ( arnaud 1918 , hansford 1947 , 1949 , 1955 , mller & von arx 1962 , hosagoudar 2010 , hofmann & piepenbring 2014 , farr & rossman 2015 ) . asterolibertia licaniae differs from a. campograndensis in the dark brown hyphae , barrel - shaped and larger appressoria , ascomatal dehiscence by an irregular fissure , a lack of fringes at the margins of the ascomata , the absence of pseudoparaphyses , and finally larger , ellipsoidal , dark brown ascospores with a central septum . however , these species differ in important morphological details such as ascospore ornamentation , the shape of the ascomatal fringes ( loosely set in a. barrinhensis ) , and hyphal branching patterns ( opposite in a. barrinhesis , and irregular in a. campograndensis ) . asterolibertia campograndensis is the fifth species of asterolibertia reported on hosts belonging to chrysobalanaceae in brazil , and the first on hirtella . type : brazil : distrito federal : braslia , pad - df , on leaves of parinari obtusifolia ( chrysobalanaceae ) , 10 nov . brazil : maranho : nogueiras , 60 km north of balsas , 6 57 52.47 notes : seven species of asterolibertia have been reported previously in association with living leaves of chrysobalanaceous hosts . asterolibertia couepiae on couepia grandiflora from brazil , a. nodulifera on angelesia splendens from the philippines , a. licaniae and a. licaniicola on licania sp . from brazil , a. parinarii on parinari subcordata from the democratic republic of the congo , a. peruviana on licania macrophylla from brazil , and a. schroeteri on chrysobalanus icaco from india ( arnaud 1918 , hansford 1947 , 1949 , 1955 , mller & von arx 1962 , hosagoudar 2010 , hofmann & piepenbring 2014 , farr & rossman 2015 ) . additionally , a. licaniae differs from the new species in the larger thyriothecia and wider hyphae , barrel - shaped appressoria , the lack of pseudoparaphyses , and smaller , ellipsoidal , smooth ascospores that are constricted at the central septum . asterolibertia parinaricola and a. parinarii are described on the same host genus , but on different parinari species . finally , a. schroeteri differs from a. parinaricola in the larger thyriothecia , asci and hyphae , the lack of pseudoparaphyses , and smooth ascospores ( tables 12 ) ( arnaud 1918 , hansford 1947 , 1949 , 1955 , mller & von arx 1962 , hofmann & piepenbring 2014 ) . asterolibertia parinaricola is distinct from all seven species known on chrysobalanaceae , and represents the sixth species of asterolibertia reported on this host family in brazil . this is the second species of asterolibertia described on parinari , and the first species found on p. obtusifolia . specimen examined : brazil : rondnia : ro-494 highway , 82 km from pimenta bueno towards parecis , 11 45 16.43 s 61 18 54.45 w , on leaves of hirtella gracilipes ( chrysobalanaceae ) , 13 jul . notes : the specimen described above was collected in the state of rondnia on living leaves of hirtella gracilipes , a new host for a. licaniae . a. thaxteri 2 ( 1 ) ascospores smooth ........................................................................................................................................................ 3 ascospores verruculose ............................................................................................................................................... 24 3 ( 2 ) ascospores medianly constricted ................................................................................................................................... 4 ascospores constricted supramedianly ........................................................................................................................... 9 4 ( 3 ) ascomata with fringed margin ......................................................................................................................................... 5 ascomata with uniform margin ......................................................................................................................... a. licaniae 5 ( 4 ) ascomata opening by a stellar fissure ............................................................................................................................ 6 ascomata opening by an irregular fissure ........................................................................................................ a. bredemeyerae 7 ( 6 ) ascospores more than 16 m in length .......................................................................................................................... 8 ascospores 1416 6.57 m ................................................................................................................... a. hiiranensis 8 ( 7 ) ascospores 1921 910 m ................................................................................................................ a. nothopegiae ascospores 2428 1215 m .................................................................................................................. 14 10 ( 9 ) ascomata with a fringed margin ..................................................................................................................................... 11 ascomata with a uniform margin ................................................................................................................................... a. santiriae 12 ( 10 ) ascomata over 90 m diam ......................................................................................................................................... 13 ascomata to 90 m diam ................................................................................................................................ a. cryptocaryae 14 ( 9 ) ascomata opening by stellar or irregular fissure ............................................................................................................ 15 ascomata opening by ostiole [ may belong in mycrothyriaceae ] .................................................................. a. peruviana 15 ( 14 ) pseudoparaphyses present .......................................................................................................................................... 16 pseudoparaphyses absent ........................................................................................................................................... a. parinarii 17 ( 15 ) ascomata with a fringed margin .................................................................................................................................... 18 ascomata with a uniform margin ................................................................................................................ a. hydnocarpi 18 ( 17 ) no leaf discoloration under the colonies ........................................................................................................................ 19 conspicuous leaf discoloration under the colonies ....................................................................................... a. crustacea 19 ( 18 ) ascomata opening by a stellar fissure ........................................................................................................................... 20 ascomata opening by an irregular fissure .................................................................................................... a. schroeteri 20 ( 19 ) appressoria showing a lateral protuberance .................................................................................................................. 21 appressoria barrel - shaped to cylindrical ....................................................................................................................... 22 21 ( 20 ) a. myocoproides 27 ( 26 ) appressoria with a lateral protuberance ........................................................................................................ a. malpighii appressoria barrel - shape to subglobose without a lateral protuberance ....................................................... 34 29 ( 28 ) pseudoparaphyses present .......................................................................................................................................... 30 pseudoparaphyses absent ........................................................................................................................................... 31 30 ( 29 ) pseudoparaphyses branched ................................................................................................................... 32 32 ( 31 ) hyphae 35 m wide ......................................................................................................................................... a. bakeri hyphae more than 5 m wide ....................................................................................................................................... 33 33 ( 32 ) ascospores 2430 1217 m ............................................................................................................................... a. ulei ascospores 3240 1825 m ................................................................................................................... type : brazil : mato grosso do sul : campo grande , br-163 highway left lane , 200 m from the roundabout turn to so paulo , behind cerealista juliana , 20 35 8.58 s 54 34 49.51 w , on leaves of hirtella glandulosa ( chrysobalanaceae ) , 22 aug . other specimen examined : brazil : rondnia : ro494 highway , 82 km from pimenta bueno towards parecis , on leaves of hirtella gracilipes ( chrysobalanaceae ) , 13 jul . notes : two species of cirsosia have been reported previously in association with living leaves of chrysobalanaceous hosts : c. moquileae on licania tomentosa from brazil , and c. splendida on hirtella americana , chrysobalanus icaco , and h. triandra from brazil , panama , and puerto rico , respectively ( batista & maia 1960b , hosagoudar 2010 , hofmann & piepenbring 2014 , farr & rossman 2015 ) . laevigata , the first asterinaceae reported on h. glandulosa and h. gracilipes , is almost identical to c. splendida , except for the smaller ascomata , presence of pseudoparaphyses , and smooth ascospores ( batista & maia 1960b , hofmann & piepenbring 2014 ) . such differences are here considered enough to recognize the specimen studied as a new variety of c. splendida . finally , cirsosia moquileae differs from the new variety by dehiscence through a central star - shaped fissure ( instead of the longitudinal fissure normally shown in cirsosia species ) , and narrower ascospores ( batista & maia 1960b ) . 1 colonies amphigenous or epiphyllous ............................................................................................................................ 2 colonies hypophyllous .................................................................................................................................... ascospores medianly constricted ................................................................................................................................... 4 ascospores constricted supramedianly ........................................................................................................................... 9 4 ( 3 ) ascomata with fringed margin ......................................................................................................................................... a. licaniae 5 ( 4 ) ascomata opening by a stellar fissure ............................................................................................................................ 6 ascomata opening by an irregular fissure ........................................................................................................ a. bredemeyerae 7 ( 6 ) ascospores more than 16 m in length .......................................................................................................................... 8 ascospores 1416 6.57 m ................................................................................................................... a. hiiranensis 8 ( 7 ) ascospores 1921 910 m ................................................................................................................ a. nothopegiae ascospores 2428 1215 m .................................................................................................................. 14 10 ( 9 ) ascomata with a fringed margin ..................................................................................................................................... 11 ascomata with a uniform margin ................................................................................................................................... 12 11 ( 10 ) ascospores 1518 56.5 m ........................................................................................................................ a. randiae ascospores 3236 1722 m ..................................................................................................................... a. santiriae 12 ( 10 ) ascomata over 90 m diam ......................................................................................................................................... 13 ascomata to 90 m diam ................................................................................................................................ ........................................................................................................................ a. parinarii 17 ( 15 ) ascomata with a fringed margin .................................................................................................................................... 18 ascomata with a uniform margin ................................................................................................................ a. hydnocarpi 18 ( 17 ) no leaf discoloration under the colonies ........................................................................................................................ 19 conspicuous leaf discoloration under the colonies ....................................................................................... a. crustacea 19 ( 18 ) ascomata opening by a stellar fissure ........................................................................................................................... 20 ascomata opening by an irregular fissure .................................................................................................... a. schroeteri 20 ( 19 ) appressoria showing a lateral protuberance .................................................................................................................. 21 appressoria barrel - shaped to cylindrical ....................................................................................................................... ............................................................................................................ a. pogonophorae 22 ( 20 ) ascospores less than 30 m in length ........................................................................................................................... 23 ascospores 3542 1619 m .................................................................................................................. a. nodulosa 28 ( 25 ) colonies epiphyllous ..................................................................................................................................................... 29 colonies amphigenous ................................................................................................................................................. 34 29 ( 28 ) pseudoparaphyses present .......................................................................................................................................... a. anisopterae appressoria barrel - shaped to subglobose without a lateral protuberance ..................................................................... 32 32 ( 31 ) hyphae 35 m wide ......................................................................................................................................... type : brazil : mato grosso do sul : campo grande , br-163 highway left lane , 200 m from the roundabout turn to so paulo , behind cerealista juliana , 20 35 8.58 s 54 34 49.51 w , on leaves of hirtella glandulosa ( chrysobalanaceae ) , 22 aug . other specimen examined : brazil : rondnia : ro494 highway , 82 km from pimenta bueno towards parecis , on leaves of hirtella gracilipes ( chrysobalanaceae ) , 13 jul . notes : two species of cirsosia have been reported previously in association with living leaves of chrysobalanaceous hosts : c. moquileae on licania tomentosa from brazil , and c. splendida on hirtella americana , chrysobalanus icaco , and h. triandra from brazil , panama , and puerto rico , respectively ( batista & maia 1960b , hosagoudar 2010 , hofmann & piepenbring 2014 , farr & rossman 2015 ) laevigata , the first asterinaceae reported on h. glandulosa and h. gracilipes , is almost identical to c. splendida , except for the smaller ascomata , presence of pseudoparaphyses , and smooth ascospores ( batista & maia 1960b , hofmann & piepenbring 2014 ) . such differences are here considered enough to recognize the specimen studied as a new variety of c. splendida . finally , cirsosia moquileae differs from the new variety by dehiscence through a central star - shaped fissure ( instead of the longitudinal fissure normally shown in cirsosia species ) , and narrower ascospores ( batista & maia 1960b ) .

the fundamental principle of adhesion to tooth substrate is based upon an exchange process by which inorganic tooth material is replaced with synthetic resin26 . using adhesive systems , the exchange of substance between adhesive resin and tooth tissue is carried out in one , two , or three clinical application steps , depending on the bonding protocol used . in addition to the number of steps , adhesive systems can further be classified based on the underlying adhesion strategy as etch - and - rinse and self - etch systems . self - etch adhesive systems use non - rinse acidic monomers that simultaneously etchs and prime enamel and dentin . self - etch adhesive systems that can have one or two steps and are classified as mild ( ph>2 ) , intermediate ( ph 1 - 2 ) , and strong ( ph<1 ) according to their acidity . new adhesive systems are continuously being introduced to the dentistry , unfortunately often without sufficient clinical validation . although clinical trials have demostrated that reliable adhesive restorations can be achieved using three - step etch - and - rinse adhesive systems , more randomized clinical studies should be performed to evaluate the clinical performance of new adhesive systems . these lesions serve as ideal test cavities because they are relatively common and are located mainly in dentin in which it is more difficult to achieve bonding than enamel . they present no macro - mechanical undercuts , and are usually found in anterior teeth or premolars and offer good access . a drawback related to the use of non - carious cervical lesions might be the substantial differences in the composition of the bonding surface . non - carious cervical lesions have a high degree of sclerosis and hybrid layer formation on this hyperminearlized dentin is more difficult . alternative strategies for adhesion to sclerotic dentin have been recommended by previous researchers who evaluated available techniques . one recommended technique is the removal of the top layer of the sclerotic lesion using a bur . using phosphoric acid conditioning before self - etch primers is another possible adaptive strategy for improving retention of resins to sclerotic dentin . the objective of this study was to evaluate the clinical perfomance of cervical restorations using a nanofilled composite resin and three different adhesive systems and to determine the effect of bur removal and phosphoric acid prior to self - etch primer of dentin for improving micromechanical retention of adhesive resins after years of clinical service . the null hypothesis to be tested was that the clinical performance of cervical restorations does not vary with different adhesive systems or with different adhesive strategies like bur removal of the outer dentin layer and pre - etching . a total of 252 restorations were placed in 29 subjects ( 16 males and 13 females ; age range 30 - 70 years ) being treated for non - carious cervical lesions . patients with a medically compromised history , periodontitis , extreme caries risk and heavy bruxism were excluded from the study . extreme caries risk was evaluated according to a large number of decayed and filled tooth surfaces and missing teeth ( not those removed for orthodontic reasons ) , infrequent use of toothpaste and toothbrush , frequent consumption of fermentable carbohydrates and low socio - economic status . heavy bruxism was evaluated according to presence of multiple wear facets on the occlusal surfaces of the teeth . additionally , all restored teeth made contact with the opposing teeth in class i cusp fossa or cusp marginal ridge occlusion relationships , and the participants had normal periodontal health ( no gingival inflammation or pocket formation around the teeth ) . prior to participating in the study , all patients signed a written informed consent form . the clinical trial protocol was approved by the gazi university commission for medical ethics . one operator who was familiar with adhesive dentistry , placed all restorations to the non - carious cervical lesions . the occlusal cavosurface margins of the lesion involved enamel , and all axial surfaces and gingival cavosurface margin of the non - carious cervical lesion involved dentin . patients who had at least 6 and no more than 20 non - carious cervical lesions were had the teeth restored with treated groups randomly in order . the adhesive systems , their manufacturers and compositions are shown in figure 2 . summary of treated groups si : single bond , cl : clearfil se , xe : xeno iii , b : bur ; p : 37% phosphoric acid composition of materials used group si : lesion surfaces were etched for 15 s with 35% phosphoric acid gel ( vococid , voco , cuxhaven , germany ) , rinsed with water and gently air dried . single bond total - etch adhesive system was applied to the etched surfaces , gently air dried and light - cured for 20 s. group si - b : the outer surface of the sclerotic dentin was removed by roughening with a diamond bur ( diatech , coltene , whaledent ag , switzerland ) at a highspeed handpiece with water spray and single bond was applied in the same manner as described for group si . group cl : clearfil se primer self - etch primer was applied to the lesion surfaces for 20 s and gently air dried . clearfil se bond was applied , air thinned and light - cured for 10 s. group cl - b : the outer surface of the sclerotic dentin was removed by roughening with a water - cooled high - speed diamond bur and clearfil se bond system was applied in the same manner as described for group cl . group xe : xeno iii single step self etching dental adhesive was applied to the lesion surfaces for 10 s , air - thinned and light - cured for 10 s. group xe - b : the outer surface of the sclerotic dentin was removed by roughening with a water - cooled high - speed diamond bur and xeno iii was applied to the roughened surfaces in the same as explained for group xe . group cl - bp : after removing the outer surface of the sclerotic dentin with a water - cooled high - speed diamond bur , the remaining dentin was etched with a 37% phosphoric acid gel for 15 s , rinsed with water and gently air dried , and clearfil se bond system was applied in the same manner as described for group cl . group xe - bp : after removing the outer surface of the sclerotic dentin with a water - cooled high - speed diamond bur , the remaining dentin was etched with a 37% phosphoric acid gel for 15 s , rinsed with water and gently air dried , and xeno iii was then applied as explained for group xe . other sclerotic lesions were randomly chosen to be roughened with a diamond bur at a high speed handpiece with water spray before phosphoric acid etching ( figure 3 ) . distribution of restorations by treatment , tooth type , sclerosis and configuration si : single bond , cl : clearfil se , xe : xeno iii , b : bur , p : 37% phosphoric acid an : anterior , prm : premolar , mo : molar , ws : wedge - shaped . ss : saucer - shaped in all groups , lesions were restored with the nanofilled composite resin filtek supreme . the resin was placed in at least two 2-mm - thick increments , which were light - cured for 40 s each using a halogen lamp ( hilux , benliolu dental inc . , turkey ) at 620 mw / cm maintaining the light - guide tip at a distance of 1 mm from the composite surface . restorations were then finished with po - go disks ( de trey dentsply , konstanz , germany ) . the restorations were evaluated by a single investigator ( not the operator ) at baseline , 3 months , 1 year and 2 years after placement using the modified usphs criteria ( figure 4 ) . marginal adaptation , gingival tissue response and wear were evaluated using a mirror and a probe . gingival tissue response was evaluated according to presence of red , hypertrophic gingiva and gingival bleeding on probing around the cervical restoration . postoperative sensitivity was evaluated before and after the restorative procedures with a 3-s air blast applied directly at the restoration site from a distance of 1 inch . tooth vitality was evaluated with an electronic digital pulp tester ( parkell , parkell electronics division , farmingdale , ny , usa ) . kruskal wallis and mann - whitney u test were used to determine the statistical differences in clinical marginal adaptation and marginal staining data between the groups . four patients with 20 lesions each , one patient with 6 lesions and one patient with 8 lesions could not be evaluated at the 3-month recall due to nonattendance ( 63% recall rate ) . all of the remaining restorations were evaluated at 3 months , 1 year and 2 years . number of retained restorations and retention rates of the groups are shown in table 1 . at 2 years , retention rates of the groups si , si - b , cl , cl - b , xe , xe - b , cl - bp , xe - bp were 70.6% , 86.7% , 78.1% , 95.5% , 70% , 85.7% , 93.3% , 93.8% , respectively . at all time number , and percentages of retained restorations and comparisons between the groups at 3 months , 1 year and 2 years ( p<0.05 ) ( total no . of restorations / no . of restorations ) lower cases represent the significant differences between the groups ( p<0.05 ) the same lower cases showed nonsignificant difference between the groups . capitals stated at the bottom , represent the significant differences between the time ( 3 month , 1 year , 2 years ) ( p<0.05 ) , the same capitals showed non - significant difference between the time . number of restorations that had no marginal staining ( alpha ) and comparisons of the groups are shown in table 2 . , significant difference was found between group xe and other groups ( p<0.05 ) . in addition , only group xe showed significant difference according to time ( between 3 months and 1 year , between 3 months and 2 years ) ( p<0.05 ) . number and percentages of restorations that have no marginal staining ( alpha ) and comparisons of the groups at 3 months , 1 year and 2 years ( p<0.05 ) . of restorations ) lower cases represent the significant differences between the groups ( p<0.05 ) the same lower cases showed nonsignificant difference between the groups . capitals stated at the bottom , represent the significant differences between the time ( 3 month , 1 year , 2 years ) ( p<0.05 ) , the same capitals showed non - significant difference between the time . number of restorations that had undetectable margins ( alpha ) and comparisons of the groups are shown in table 3 . at all time intervals ( 3 months , 1 year , 2 years ) when groups si , cl and xe were compared , no significant difference was found between the groups cl and si ( p>0.05 ) , but significant differerence was found between groups si and xe ( p<0.05 ) . although no significant difference was found between the groups cl and xe at 3 months and 1 year ( p>0.05 ) , significant difference was detected at 2 years ( p<0.05 ) . at all time intervals , there was no significant difference among the groups si - b , cl - b and xe - b ( p>0.05 ) . no significant difference on marginal adaptation was observed by bur removal of dentin at 3 months and 1 year ( p>0.05 ) between groups si and si - b , groups cl and cl - b , and groups xe and xe - b . at 2 years , significant difference was found between group xe and group xe - b ( p<0.05 ) . bur removal of dentin and pre - etching with phosphoric acid in the groups with self - etch adhesive systems ( groups cl and cl - bp , and groups xe and xe - bp ) showed a significant difference at all time intervals ( p<0.05 ) . number and percentages of restorations that have undetectable margin ( alpha ) and comparison of the groups at 3 months , 1 year and 2 years ( p < 0.05).(total no . of restorations / no . of restorations ) lower cases represent the significant differences between the groups ( p<0.05 ) the same lower cases showed nonsignificant difference between the groups . capitals stated at the bottom , represent the significant differences between the time ( 3 month , 1 year , 2 years ) ( p<0.05 ) , the same capitals showed non - significant difference between the time . when the groups were evaluated according to time , marginal adaptation did not decreased over time in groups si - b , cl - bp and xe - bp ( p>0.05 ) . other groups showed significant difference between 3 months and 1 year and between 3 months and 2 years ( p<0.05 ) . while group xe - bp showed the best marginal adaptation , group xe showed the worst marginal adaptation at 2 years . none of the restorations showed postoperative sensitivity , gingival tissue response or secondary caries and all of the retained restorations were clinically and aesthetically acceptable . when failed restorations were evaluated , 54% of totally lost restorations were in premolars , 58% in wedge - shaped lesions and 42% in saucer - shaped lesions . failed restorations occurred almost equally in both arches , so no correlation was determined between arch and restoration failure . number of retained restorations and retention rates of the groups are shown in table 1 . at 2 years , retention rates of the groups si , si - b , cl , cl - b , xe , xe - b , cl - bp , xe - bp were 70.6% , 86.7% , 78.1% , 95.5% , 70% , 85.7% , 93.3% , 93.8% , respectively . at all time intervals no significant difference was found among the groups ( p>0.05 ) . number , and percentages of retained restorations and comparisons between the groups at 3 months , 1 year and 2 years ( p<0.05 ) ( total no . of restorations / no . of restorations ) lower cases represent the significant differences between the groups ( p<0.05 ) the same lower cases showed nonsignificant difference between the groups . capitals stated at the bottom , represent the significant differences between the time ( 3 month , 1 year , 2 years ) ( p<0.05 ) , the same capitals showed non - significant difference between the time . number of restorations that had no marginal staining ( alpha ) and comparisons of the groups are shown in table 2 . , significant difference was found between group xe and other groups ( p<0.05 ) . in addition , only group xe showed significant difference according to time ( between 3 months and 1 year , between 3 months and 2 years ) ( p<0.05 ) . number and percentages of restorations that have no marginal staining ( alpha ) and comparisons of the groups at 3 months , 1 year and 2 years ( p<0.05 ) . lower cases represent the significant differences between the groups ( p<0.05 ) the same lower cases showed nonsignificant difference between the groups . capitals stated at the bottom , represent the significant differences between the time ( 3 month , 1 year , 2 years ) ( p<0.05 ) , the same capitals showed non - significant difference between the time . number of restorations that had undetectable margins ( alpha ) and comparisons of the groups are shown in table 3 . at all time intervals ( 3 months , 1 year , 2 years ) when groups si , cl and xe were compared , no significant difference was found between the groups cl and si ( p>0.05 ) , but significant differerence was found between groups si and xe ( p<0.05 ) . although no significant difference was found between the groups cl and xe at 3 months and 1 year ( p>0.05 ) , significant difference was detected at 2 years ( p<0.05 ) . at all time intervals , there was no significant difference among the groups si - b , cl - b and xe - b ( p>0.05 ) . no significant difference on marginal adaptation was observed by bur removal of dentin at 3 months and 1 year ( p>0.05 ) between groups si and si - b , groups cl and cl - b , and groups xe and xe - b . at 2 years , significant difference was found between group xe and group xe - b ( p<0.05 ) . bur removal of dentin and pre - etching with phosphoric acid in the groups with self - etch adhesive systems ( groups cl and cl - bp , and groups xe and xe - bp ) showed a significant difference at all time intervals ( p<0.05 ) . number and percentages of restorations that have undetectable margin ( alpha ) and comparison of the groups at 3 months , 1 year and 2 years ( p < 0.05).(total no . of restorations / no . of restorations ) lower cases represent the significant differences between the groups ( p<0.05 ) the same lower cases showed nonsignificant difference between the groups . capitals stated at the bottom , represent the significant differences between the time ( 3 month , 1 year , 2 years ) ( p<0.05 ) , the same capitals showed non - significant difference between the time . when the groups were evaluated according to time , marginal adaptation did not decreased over time in groups si - b , cl - bp and xe - bp ( p>0.05 ) . other groups showed significant difference between 3 months and 1 year and between 3 months and 2 years ( p<0.05 ) . while group xe - bp showed the best marginal adaptation , group xe showed the worst marginal adaptation at 2 years . none of the restorations showed postoperative sensitivity , gingival tissue response or secondary caries and all of the retained restorations were clinically and aesthetically acceptable . when failed restorations were evaluated , 54% of totally lost restorations were in premolars , 58% in wedge - shaped lesions and 42% in saucer - shaped lesions . failed restorations occurred almost equally in both arches , so no correlation was determined between arch and restoration failure . clinical trials are the ultimate test for assessment of bonding effectiveness of adhesive materials . peumans , et al.15 ( 2005 ) reviewed clinical studies published between 1998 and 2004 and concluded that three - step etch - and - rinse and two - step selfetch adhesive systems showed a clinically reliable and sufficiently predictable clinical performance . the clinical performance of two - step etch - andrinse adhesive systems was less favorable , while one - step self - etch adhesive systems had an unacceptable clinical performance . in the present study , three adhesive systems were compared and two - step self - etch adhesive ( clearfil se ) showed more reliable marginal adaptation than the one - step self - etch adhesive ( xeno iii ) in the groups without bur removal of dentin at 2 years . there was no difference between the two - step self - etch adhesive ( clearfil se ) and the two - step etch - rinse adhesive ( single bond ) with respect to marginal adaptation . clearfil se is a two - step self - etch adhesive whose self - etching primer contains 10-methacryloxydecyl dihydrogen phosphate ( 10-mdp ) as a functional monomer , which is dissolved in water resulting in a ph of approximately 3 . in previous studies , 10-mdp has been shown to chemically react with hydroxyapatite . the resulting two - fold micromechanical and chemical bonding mechanism might have led to the better marginal adaptation of clearfil se over xeno iii at 2 years . in fact , at 2 years , xeno iii showed the worst marginal adaptation of all three systems . this may be due to the weak bond between dentin and xeno iii adhesive resin , which may be more affected than the other adhesive systems used in this study by occlusal stresses and intraoral temperature changes in the oral cavity . water trees in the hybrid layer may be another explanation of the lower marginal adaptation of xeno iii to dentin surfaces . water trees represent an area where a certain volume of water is retained causing incomplete polymerization of the adhesive . it has been speculated that this volume of water may cause degradation of the bonded surface due to hydrolysis . tay , et al.(2003 ) suggested that water treeing in the hybrid layer may explain the initial problems associated with the bonding of simplified adhesive systems and the underlying causes of their relative lack of durability . tay , et al.(2001 ) noted that onestep self - etch adhesive systems are permeable membranes that permit diffusion of water , and this residual water within the adhesive layer may lead to areas of incomplete polymerization of the adhesive . in previous nanoleakage studies , it was reported that the lowest occurrence of nanoleakage within the hybrid layer occurred with clearfil se . in the present study , however , the retention results were different from those of marginal adaptation , as no significant difference was found among the groups . ( 2005 ) evaluated the clinical performance of the mild twostep self - etch adhesive clearfil se in non - carious cervical lesions . they reported 100% retention rate after 3 years and 98% retention rate after 5 years . burrow and tyas(2007 ) compared clearfil se , single bond and a resin - modified glass ionomer cement in cervical non - carious cervical lesions , and found that rmgic performed the best clinical success ( 97% ) , followed by clearfil se ( 90% ) and single bond ( 77% ) . in the present study , clearfil se showed 77.12% retention rate with no bur removal of dentin , and 95.45% retention rate with bur removal of dentin after two years . the difference between previous studies and this current study may be due to factors associated with individual patients ( such as occlusal loading , dentin sclerosis ) . ( 2007 ) restored class v cavities with xeno iii and composite resin , and evaluated the outcomes at recall intervals up to 18 months . similar to these results , trkn23 ( 2003 ) reported a 96% retention rate for xeno iii and no significant difference was found between xeno iii and a two - step self - etch adhesive ( clearfil protect bond ) . in the present study , retention rates for xeno iii ( 70% ) were not significantly different from those for the other groups . the removal of the outer surface of the sclerotic dentin by roughening with a diamond bur has been recommended in the literature in order to create a better hybrid layer . van dijken(2004 ) determined that roughening of sclerotic dentin surfaces with a diamond bur did not increase the retention of restorations . in the present study , although the group of xe - b , tended to show better marginal adaptation , marginal staining and retention than the group of xe , it is found that no significant differences were determined between them . a significantly better marginal adaptation was determined as a result of the study after 2 years . on the other hand , no significant differences were observed between groups si and si - b , and groups cl and cl - b , in which the adhesive systems were applied with or without bur removal of dentin . the different results between these adhesive systems may depend on their different bonding ability to sclerotic dentin . sclerotic dentin has diffusion barriers ( obliteration of dentin tubules with sclerotic casts and presence of acid - resistant hypermineralized layer ) , which can inhibit the acid demineralization and compromise bonding . unlike single bond and clearfil se , xeno iii was completely ineffective in overcoming the diffusion barriers in sclerotic dentin and could not bond sufficiently to the intact substrate . however , roughening the sclerotic dentin with a diamond bur increased the bonding ability of xeno iii . different strategies were used in previous studies to increase the bond strength of adhesive systems to sclerotic dentin , such as bur removal of the most superficial sclerotic dentin layer or pre - etching with phosphoric acid . however , none of these earlier studies investigated these two mechanisms together . in the present study , bur removal of dentin and additional phosphoric acid etching before self - etch primer application increased marginal adaptation of both self - etch adhesive systems , but did not change their retention rates . although van landuyt , et al . ( 2006 ) reported that prior acidetching significantly decreased the bond strength of self - etch adhesive systems to sound dentin , in the current study sclerotic dentin surfaces were used as a bonding substrate . the different dentin substrates ( sclerotic versus sound dentin ) may be the explanation for the different results obtained in the studies . several clinical co - variables that are unique to the oral environment have been described to affect the clinical performance of adhesive systems . with regard to location , researchers determined that the retention of cervical restorations was significantly higher in the maxillary arch than in the mandibular arch . differently from previous studies , in the current study , failed restorations occurred almost equally in both arches . however , most of the failed restorations occurred in premolars ( 54% ) . using finite element method ( fem ) analysis , researchers compared tooth groups and reported that the magnitude of cervical stress was highest for premolars , followed by incisors and lowest for canines . powell , et al.(1995 ) reported that the shapes of the lesions do not affect the retention of restorations . on the other hand , eliguzeloglu , ( 2011 ) compared the effect of cavity shape on the stress distribution of cervical lesions which were restored with a composite using fem analysis , and it is determined that the stress distribution of saucer - shaped non - carious cervical lesions have more advantages than wedge - shaped lesions . in the present study , 58% of the lost restorations had been placed in wedge - shaped lesions , and 42% of them in saucer - shaped lesions . these results are in good aggrement with the authors ' previous fem study . to be considered clinically effective , adhesive systems should keep the restoration in place and completely seal the restoration margins against the ingress of oral fluids and microorganisms . incomplete marginal seal will result in postplacement sensitivity , marginal staining and , eventually , recurrent caries , which are still the most common symptoms associated with clinical failure of adhesive restorations . marginal staining is probably caused by microleakage or an accumulation of stains at a marginal defect , such as the chip fracture of a slight excess of material covering unground and/or non - treated tooth surface . in the present study , the sealing capacity of adhesive systems was assessed with the marginal staining index . in the present study , marginal staining increased over time in all groups , but only in group xe significant difference was found between the time intervals . in conclusion , the null hpothesis could not be accepted because ; the two - step self - etch adhesive system clearfil se showed better marginal adaptation than the one - step self - etch adhesive system xeno iii after 2 years ; clearfil se showed similar marginal adaptation , retention and marginal staining to those of the etch - and - rinse adhesive system single bond after 2 years ; the removal of the outer surface layer of sclerotic dentin did not increase the retention and marginal staining of the adhesive systems evaluated in this study ; the removal of the outer surface layer of dentin and additional phosphoric acid etching , increased the marginal adaptation of xeno iii and clearfil se after 2 years .

objectives adhesive systems are continuously being introduced to dentistry , unfortunately often without sufficient clinical validation . the aim of this study was to evaluate the clinical performance of cervical restorations done with three different adhesive systems . material and methods 158 non - carious cervical lesions of 23 patients were restored with a nanofilled composite resin ( filtek supreme , 3m / espe ) combined with single bond ( 3m / espe , group si ) , clearfil se ( kuraray medical inc . , group cl ) and xeno iii ( de trey dentsply , group xe ) . in groups si - b , cl - b and xe - b , the outer surface of the sclerotic dentin was removed by roughening with a diamond bur before application of the respective adhesive systems . in groups cl - bp and xe - bp , after removal of the outer surface of the sclerotic dentin with the bur , the remaining dentin was etched with 37% phosphoric acid and the self - etch adhesive systems clearfil se and xeno iii were applied , respectively . lesions were evaluated at baseline , and restorations after 3 months , 1 year and 2 years using modified usphs criteria . results after 2 years , no significant difference was found between the retention rates of the groups ( p > 0.05 ) . although groups cl and si showed significantly better marginal adaptation than group xe ( p<0.05 ) at 2 years , no significant difference was found between the marginal adaptation of the groups si - b , cl - b and xe - b ( p>0.05 ) . after 2 years no significant difference was observed among the marginal staining results of all groups ( p>0.05 ) . conclusion although all adhesive systems showed similar retention rates , clearfil se and single bond showed better marginal adaptation than xeno iii after 2 years of follow - up .

INTRODUCTION MATERIAL AND METHODS RESULTS Retention Marginal staining Marginal adaptation Remaining clinical variables Failure analysis DISCUSSION CONCLUSIONS

self - etch adhesive systems use non - rinse acidic monomers that simultaneously etchs and prime enamel and dentin . self - etch adhesive systems that can have one or two steps and are classified as mild ( ph>2 ) , intermediate ( ph 1 - 2 ) , and strong ( ph<1 ) according to their acidity . new adhesive systems are continuously being introduced to the dentistry , unfortunately often without sufficient clinical validation . although clinical trials have demostrated that reliable adhesive restorations can be achieved using three - step etch - and - rinse adhesive systems , more randomized clinical studies should be performed to evaluate the clinical performance of new adhesive systems . a drawback related to the use of non - carious cervical lesions might be the substantial differences in the composition of the bonding surface . the objective of this study was to evaluate the clinical perfomance of cervical restorations using a nanofilled composite resin and three different adhesive systems and to determine the effect of bur removal and phosphoric acid prior to self - etch primer of dentin for improving micromechanical retention of adhesive resins after years of clinical service . the null hypothesis to be tested was that the clinical performance of cervical restorations does not vary with different adhesive systems or with different adhesive strategies like bur removal of the outer dentin layer and pre - etching . one operator who was familiar with adhesive dentistry , placed all restorations to the non - carious cervical lesions . the occlusal cavosurface margins of the lesion involved enamel , and all axial surfaces and gingival cavosurface margin of the non - carious cervical lesion involved dentin . patients who had at least 6 and no more than 20 non - carious cervical lesions were had the teeth restored with treated groups randomly in order . summary of treated groups si : single bond , cl : clearfil se , xe : xeno iii , b : bur ; p : 37% phosphoric acid composition of materials used group si : lesion surfaces were etched for 15 s with 35% phosphoric acid gel ( vococid , voco , cuxhaven , germany ) , rinsed with water and gently air dried . single bond total - etch adhesive system was applied to the etched surfaces , gently air dried and light - cured for 20 s. group si - b : the outer surface of the sclerotic dentin was removed by roughening with a diamond bur ( diatech , coltene , whaledent ag , switzerland ) at a highspeed handpiece with water spray and single bond was applied in the same manner as described for group si . clearfil se bond was applied , air thinned and light - cured for 10 s. group cl - b : the outer surface of the sclerotic dentin was removed by roughening with a water - cooled high - speed diamond bur and clearfil se bond system was applied in the same manner as described for group cl . group xe : xeno iii single step self etching dental adhesive was applied to the lesion surfaces for 10 s , air - thinned and light - cured for 10 s. group xe - b : the outer surface of the sclerotic dentin was removed by roughening with a water - cooled high - speed diamond bur and xeno iii was applied to the roughened surfaces in the same as explained for group xe . group cl - bp : after removing the outer surface of the sclerotic dentin with a water - cooled high - speed diamond bur , the remaining dentin was etched with a 37% phosphoric acid gel for 15 s , rinsed with water and gently air dried , and clearfil se bond system was applied in the same manner as described for group cl . group xe - bp : after removing the outer surface of the sclerotic dentin with a water - cooled high - speed diamond bur , the remaining dentin was etched with a 37% phosphoric acid gel for 15 s , rinsed with water and gently air dried , and xeno iii was then applied as explained for group xe . other sclerotic lesions were randomly chosen to be roughened with a diamond bur at a high speed handpiece with water spray before phosphoric acid etching ( figure 3 ) . distribution of restorations by treatment , tooth type , sclerosis and configuration si : single bond , cl : clearfil se , xe : xeno iii , b : bur , p : 37% phosphoric acid an : anterior , prm : premolar , mo : molar , ws : wedge - shaped . ss : saucer - shaped in all groups , lesions were restored with the nanofilled composite resin filtek supreme . the restorations were evaluated by a single investigator ( not the operator ) at baseline , 3 months , 1 year and 2 years after placement using the modified usphs criteria ( figure 4 ) . all of the remaining restorations were evaluated at 3 months , 1 year and 2 years . at 2 years , retention rates of the groups si , si - b , cl , cl - b , xe , xe - b , cl - bp , xe - bp were 70.6% , 86.7% , 78.1% , 95.5% , 70% , 85.7% , 93.3% , 93.8% , respectively . at all time number , and percentages of retained restorations and comparisons between the groups at 3 months , 1 year and 2 years ( p<0.05 ) ( total no . of restorations ) lower cases represent the significant differences between the groups ( p<0.05 ) the same lower cases showed nonsignificant difference between the groups . capitals stated at the bottom , represent the significant differences between the time ( 3 month , 1 year , 2 years ) ( p<0.05 ) , the same capitals showed non - significant difference between the time . , significant difference was found between group xe and other groups ( p<0.05 ) . in addition , only group xe showed significant difference according to time ( between 3 months and 1 year , between 3 months and 2 years ) ( p<0.05 ) . number and percentages of restorations that have no marginal staining ( alpha ) and comparisons of the groups at 3 months , 1 year and 2 years ( p<0.05 ) . of restorations ) lower cases represent the significant differences between the groups ( p<0.05 ) the same lower cases showed nonsignificant difference between the groups . capitals stated at the bottom , represent the significant differences between the time ( 3 month , 1 year , 2 years ) ( p<0.05 ) , the same capitals showed non - significant difference between the time . at all time intervals ( 3 months , 1 year , 2 years ) when groups si , cl and xe were compared , no significant difference was found between the groups cl and si ( p>0.05 ) , but significant differerence was found between groups si and xe ( p<0.05 ) . although no significant difference was found between the groups cl and xe at 3 months and 1 year ( p>0.05 ) , significant difference was detected at 2 years ( p<0.05 ) . at all time intervals , there was no significant difference among the groups si - b , cl - b and xe - b ( p>0.05 ) . no significant difference on marginal adaptation was observed by bur removal of dentin at 3 months and 1 year ( p>0.05 ) between groups si and si - b , groups cl and cl - b , and groups xe and xe - b . at 2 years , significant difference was found between group xe and group xe - b ( p<0.05 ) . bur removal of dentin and pre - etching with phosphoric acid in the groups with self - etch adhesive systems ( groups cl and cl - bp , and groups xe and xe - bp ) showed a significant difference at all time intervals ( p<0.05 ) . number and percentages of restorations that have undetectable margin ( alpha ) and comparison of the groups at 3 months , 1 year and 2 years ( p < 0.05). of restorations ) lower cases represent the significant differences between the groups ( p<0.05 ) the same lower cases showed nonsignificant difference between the groups . capitals stated at the bottom , represent the significant differences between the time ( 3 month , 1 year , 2 years ) ( p<0.05 ) , the same capitals showed non - significant difference between the time . when the groups were evaluated according to time , marginal adaptation did not decreased over time in groups si - b , cl - bp and xe - bp ( p>0.05 ) . other groups showed significant difference between 3 months and 1 year and between 3 months and 2 years ( p<0.05 ) . while group xe - bp showed the best marginal adaptation , group xe showed the worst marginal adaptation at 2 years . at 2 years , retention rates of the groups si , si - b , cl , cl - b , xe , xe - b , cl - bp , xe - bp were 70.6% , 86.7% , 78.1% , 95.5% , 70% , 85.7% , 93.3% , 93.8% , respectively . at all time intervals no significant difference was found among the groups ( p>0.05 ) . number , and percentages of retained restorations and comparisons between the groups at 3 months , 1 year and 2 years ( p<0.05 ) ( total no . of restorations ) lower cases represent the significant differences between the groups ( p<0.05 ) the same lower cases showed nonsignificant difference between the groups . capitals stated at the bottom , represent the significant differences between the time ( 3 month , 1 year , 2 years ) ( p<0.05 ) , the same capitals showed non - significant difference between the time . , significant difference was found between group xe and other groups ( p<0.05 ) . in addition , only group xe showed significant difference according to time ( between 3 months and 1 year , between 3 months and 2 years ) ( p<0.05 ) . number and percentages of restorations that have no marginal staining ( alpha ) and comparisons of the groups at 3 months , 1 year and 2 years ( p<0.05 ) . lower cases represent the significant differences between the groups ( p<0.05 ) the same lower cases showed nonsignificant difference between the groups . capitals stated at the bottom , represent the significant differences between the time ( 3 month , 1 year , 2 years ) ( p<0.05 ) , the same capitals showed non - significant difference between the time . at all time intervals ( 3 months , 1 year , 2 years ) when groups si , cl and xe were compared , no significant difference was found between the groups cl and si ( p>0.05 ) , but significant differerence was found between groups si and xe ( p<0.05 ) . although no significant difference was found between the groups cl and xe at 3 months and 1 year ( p>0.05 ) , significant difference was detected at 2 years ( p<0.05 ) . at all time intervals , there was no significant difference among the groups si - b , cl - b and xe - b ( p>0.05 ) . no significant difference on marginal adaptation was observed by bur removal of dentin at 3 months and 1 year ( p>0.05 ) between groups si and si - b , groups cl and cl - b , and groups xe and xe - b . at 2 years , significant difference was found between group xe and group xe - b ( p<0.05 ) . bur removal of dentin and pre - etching with phosphoric acid in the groups with self - etch adhesive systems ( groups cl and cl - bp , and groups xe and xe - bp ) showed a significant difference at all time intervals ( p<0.05 ) . number and percentages of restorations that have undetectable margin ( alpha ) and comparison of the groups at 3 months , 1 year and 2 years ( p < 0.05). of restorations ) lower cases represent the significant differences between the groups ( p<0.05 ) the same lower cases showed nonsignificant difference between the groups . capitals stated at the bottom , represent the significant differences between the time ( 3 month , 1 year , 2 years ) ( p<0.05 ) , the same capitals showed non - significant difference between the time . when the groups were evaluated according to time , marginal adaptation did not decreased over time in groups si - b , cl - bp and xe - bp ( p>0.05 ) . other groups showed significant difference between 3 months and 1 year and between 3 months and 2 years ( p<0.05 ) . while group xe - bp showed the best marginal adaptation , group xe showed the worst marginal adaptation at 2 years . the clinical performance of two - step etch - andrinse adhesive systems was less favorable , while one - step self - etch adhesive systems had an unacceptable clinical performance . in the present study , three adhesive systems were compared and two - step self - etch adhesive ( clearfil se ) showed more reliable marginal adaptation than the one - step self - etch adhesive ( xeno iii ) in the groups without bur removal of dentin at 2 years . there was no difference between the two - step self - etch adhesive ( clearfil se ) and the two - step etch - rinse adhesive ( single bond ) with respect to marginal adaptation . the resulting two - fold micromechanical and chemical bonding mechanism might have led to the better marginal adaptation of clearfil se over xeno iii at 2 years . in fact , at 2 years , xeno iii showed the worst marginal adaptation of all three systems . (2001 ) noted that onestep self - etch adhesive systems are permeable membranes that permit diffusion of water , and this residual water within the adhesive layer may lead to areas of incomplete polymerization of the adhesive . in the present study , however , the retention results were different from those of marginal adaptation , as no significant difference was found among the groups . ( 2005 ) evaluated the clinical performance of the mild twostep self - etch adhesive clearfil se in non - carious cervical lesions . burrow and tyas(2007 ) compared clearfil se , single bond and a resin - modified glass ionomer cement in cervical non - carious cervical lesions , and found that rmgic performed the best clinical success ( 97% ) , followed by clearfil se ( 90% ) and single bond ( 77% ) . similar to these results , trkn23 ( 2003 ) reported a 96% retention rate for xeno iii and no significant difference was found between xeno iii and a two - step self - etch adhesive ( clearfil protect bond ) . the removal of the outer surface of the sclerotic dentin by roughening with a diamond bur has been recommended in the literature in order to create a better hybrid layer . van dijken(2004 ) determined that roughening of sclerotic dentin surfaces with a diamond bur did not increase the retention of restorations . in the present study , although the group of xe - b , tended to show better marginal adaptation , marginal staining and retention than the group of xe , it is found that no significant differences were determined between them . a significantly better marginal adaptation was determined as a result of the study after 2 years . on the other hand , no significant differences were observed between groups si and si - b , and groups cl and cl - b , in which the adhesive systems were applied with or without bur removal of dentin . however , roughening the sclerotic dentin with a diamond bur increased the bonding ability of xeno iii . different strategies were used in previous studies to increase the bond strength of adhesive systems to sclerotic dentin , such as bur removal of the most superficial sclerotic dentin layer or pre - etching with phosphoric acid . in the present study , bur removal of dentin and additional phosphoric acid etching before self - etch primer application increased marginal adaptation of both self - etch adhesive systems , but did not change their retention rates . ( 2006 ) reported that prior acidetching significantly decreased the bond strength of self - etch adhesive systems to sound dentin , in the current study sclerotic dentin surfaces were used as a bonding substrate . on the other hand , eliguzeloglu , ( 2011 ) compared the effect of cavity shape on the stress distribution of cervical lesions which were restored with a composite using fem analysis , and it is determined that the stress distribution of saucer - shaped non - carious cervical lesions have more advantages than wedge - shaped lesions . in the present study , the sealing capacity of adhesive systems was assessed with the marginal staining index . in the present study , marginal staining increased over time in all groups , but only in group xe significant difference was found between the time intervals . in conclusion , the null hpothesis could not be accepted because ; the two - step self - etch adhesive system clearfil se showed better marginal adaptation than the one - step self - etch adhesive system xeno iii after 2 years ; clearfil se showed similar marginal adaptation , retention and marginal staining to those of the etch - and - rinse adhesive system single bond after 2 years ; the removal of the outer surface layer of sclerotic dentin did not increase the retention and marginal staining of the adhesive systems evaluated in this study ; the removal of the outer surface layer of dentin and additional phosphoric acid etching , increased the marginal adaptation of xeno iii and clearfil se after 2 years .

early childhood caries ( ecc ) is a common childhood disease in the united states ( us ) and worldwide . until recently , the standard of care for ecc has been primarily surgical and restorative treatment with relatively little emphasis on the prevention and management of disease . in the us , young children who are not cooperative are commonly sedated or treated under general anesthesia ( ga ) . however , despite receiving costly treatment under ga , such as in the operating room ( or ) [ 35 ] , children all too often develop new and recurrent caries [ 610 ] . it is now accepted that surgical repair alone does not address the underlying etiology of the disease . unless the caries balance is altered , new and recurrent caries are likely to occur . on the other hand , a successful rebalance of risk and protective factors may slow down or completely halt the disease process , resulting in caries arrest , if not also preventing the onset of new disease . contemporary approaches to caries prevention and management modeled after medical management of chronic conditions such as diabetes , obesity and asthma , have been described in the scientific literature and are herein known as chronic disease management ( dm ) [ 11 , 1315 ] . dm differs from the traditional approach of oral health care providers relying on a surgical treatment model in response to the disease , while telling the patients what to do . instead , it assumes that patients have a central role in determining the care of their chronic condition . a close collaboration between the healthcare provider and patient is required , ideally in a culturally and linguistically appropriate manner . in practice , healthcare providers coach patients and parents about the factors that lead to and protect against dental disease and assist them in selecting self - management goals to improve their own and their children 's risk for disease . treatment decisions are based on the latest evidence - based guidelines that are customized to patients ' individual needs . risk - based dm of ecc requires significant patient and family engagement and empowerment from the provider and care team in effective day - to - day behavior modifications ( e.g. , tooth brushing , topical fluorides , and dietary control ) that address disease etiology . family - centered behavior plans lead to real behavior change and maintenance of oral health behaviors in the child 's home . at the same time , the dental practice has a reciprocal role in tracking and managing the care of patients . phase 1 in 2008 , a risk - based dm approach to address preschool children with ecc was implemented and tested as a quality improvement ( qi ) demonstration project at boston children 's hospital in boston , massachusetts ( bch ) , and st . joseph health services of rhode island in providence , ri ( sjh ) . the clinical protocol and project results have been previously published . thirty months of results found that children in the ecc group experienced lower rates of new cavitated lesions , pain , and referrals for restorative treatment under general anesthesia in the or as compared to baseline historical controls ( table 1 ) . at bch , the ecc group experienced a 62% lower rate of new cavitation compared to the historical control group . structured interviews with phase 1 parents revealed that most believed the dm approach to be helpful for their children ; almost all parents appreciated given reasons as to why their children may have developed ecc . the collaborative approach allowed clinicians to engage parents or caregivers to better understand that they have a voice in the care their child receives . in 2008 , a risk - based dm approach to address preschool children with ecc was implemented and tested as a quality improvement ( qi ) demonstration project at boston children 's hospital in boston , massachusetts ( bch ) , and st . joseph health services of rhode island in providence , ri ( sjh ) . the clinical protocol and project results have been previously published . thirty months of results found that children in the ecc group experienced lower rates of new cavitated lesions , pain , and referrals for restorative treatment under general anesthesia in the or as compared to baseline historical controls ( table 1 ) . at bch , the ecc group experienced a 62% lower rate of new cavitation compared to the historical control group . structured interviews with phase 1 parents revealed that most believed the dm approach to be helpful for their children ; almost all parents appreciated given reasons as to why their children may have developed ecc . the collaborative approach allowed clinicians to engage parents or caregivers to better understand that they have a voice in the care their child receives . phase 2building upon the promising results of phase 1 , the project was expanded in 2011 to include five additional teams in the us . phase 2 further tested the feasibility and effectiveness of the dm approach to reduce ecc in more diverse settings . the five additional teams were in the following locations across the us : holyoke health center ( holyoke , ma ) ; native american health center ( san francisco , ca ) ; nationwide children 's hospital ( columbus , oh ) , neighborcare health ( seattle , wa ) ; and university pediatric dentistry ( buffalo , ny ) . the purpose of this report is to describe the phase 2 project and experiences , present the results after 18 months , and discuss the implications of what was learned . building upon the promising results of phase 1 , the project was expanded in 2011 to include five additional teams in the us . phase 2 further tested the feasibility and effectiveness of the dm approach to reduce ecc in more diverse settings . the five additional teams were in the following locations across the us : holyoke health center ( holyoke , ma ) ; native american health center ( san francisco , ca ) ; nationwide children 's hospital ( columbus , oh ) , neighborcare health ( seattle , wa ) ; and university pediatric dentistry ( buffalo , ny ) . the purpose of this report is to describe the phase 2 project and experiences , present the results after 18 months , and discuss the implications of what was learned . phase 2 was implemented as an 18-month qi learning collaborative . using established qi methods , a nationwide collection of staff , experts , and faculty provided training and technical assistance to the seven participating teams , which included the two teams that were part of phase 1 . teams were required to attend three on - site learning sessions where each of the seven teams received didactic education and training on qi concepts and activities . the learning session curriculum focused on the use of logic models , measurement plans , plan - do - study - act ( pdsa ) cycles , dm of ecc such as caries risk assessment ( cra ) , self - management goals ( smgs ) , effective patient - provider communication , and fluorides and other remineralizing modalities . the learning sessions provided invaluable opportunities for synergy as teams exchanged approaches to dm during consultations with experts , faculty , and staff , who provided coaching and support . qi has been defined as the combined and unceasing efforts of everyone to make changes that will lead to better patient outcomes ( health ) , system performance ( care ) , and professional development ( learning ) . qi is intended to support the redesign of care processes based on a system of learning , incremental change , and the incorporation of empirically supported best practices from evaluating performance and outcome measures . unlike a protracted randomized trial , qi uses systematic , data - guided activities designed to bring about immediate improvements in health care delivery in particular settings and can be considered as the scientific method used for action - oriented learning . the model for improvement developed by associates in process improvement was used as the essential framework to guide changes made by each team 's care delivery system in order to use a dm approach to address ecc . the collaborative developed a driver diagram outlining three main outcomes of interests : ( 1 ) new cavitation ; ( 2 ) pain related to untreated caries ; and ( 3 ) referral to the or , along with primary and secondary drivers affecting those outcomes ( figure 1 ) . pdsa cycles are small - scale tests of change in real work settings by planning a test , trying it , observing the results , and acting on what is learned . pdsa cycles promote creativity , offer quick results , and empirically support approaches to dm that are specific to the clinical teams . for example , pdsas served as learning opportunities for phase 2 teams to use evidence to determine how to perform activities such as cra , smgs , and patient scheduling to support the additional dm visits required . the phase 2 clinical dm protocol ( shown in figure 2 ) cra allows for a customized prevention and maintenance plan to be developed that is appropriate for the child and the family . cra involves asking parents a few questions to assess each child 's risk for caries at the initial visit and every visit thereafter . this form was adapted from the american academy of pediatric dentistry ( aapd cra ) form and the pediatric caries management by risk assessment ( cambra ) form . teams were able to customize forms for use with their specific patient populations and organizational context provided that they included at least the basic questions seen in figure 3 . children who had at least one tooth with clinical manifestation of caries tooth decay ( including demineralization)or who had a history of carious lesions was considered an ecc patient . at the initial and recall visits , a clinical examination and charting were performed to allow for the tracking of caries presence and activity by tooth and surface , since decay may progress and become inactive at different sites of the dentition at the same time . parents of ecc patients were engaged and coached about the factors that lead to caries and tooth decay by dentists , hygienist , dental assistants , and/or support staff . parents learned about the caries process as they were informed of the ways that tooth decay can be prevented and stopped . in addition , parents learned that without a change in diet and home care , new cavities and broken fillings will likely result . providers and care team members worked with parents to select smgs to improve their child 's disease risk . such goals include basic caries control strategies such as more frequent tooth brushing , using topical fluorides at home , and modifying one 's diet to include fewer and less frequent intake of sugary products . the frequency of return dm visits for patients and parents in - office and at the clinic site was based on their caries risk . whenever possible , the dm activities were coordinated with restorative treatment . table 2 shows the dm protocol with return visit intervals based on the most recent caries risk status in conjunction with restorative care as needed and as desired by the parent and dental provider . the in - office dm protocol was based on the assumption that children who initially presented as high caries risk may improve their risk over time . children who were assessed to be high caries risk were advised to return in 13 months for a dm visit . medium or moderate risk children returned in 36 months , while low risk children returned in 612 months . in some cases , accurate clinical assessment was hampered by the presence of heavy plaque and/or a lack of patient cooperation . as a result , a one - month follow - up visit for a child assessed to be high caries risk allowed for a more accurate assessment of demineralized , cavitated , and remineralized tooth surfaces . during each recall or subsequent dm visit , a cra was performed . providers asked parents to report on their experiences with the smgs in order to assess the level of compliance and the utility of the agreed - upon smgs . a clinical examination was also performed , reassessing for the presence of new demineralization and cavitation along with caries remineralization . parents were given the full range of options for restorative treatment , which included pharmacologic management ( i.e. , use of nitrous oxide , sedation , or ga / or ) as needed by the patient and as desired by the parent . restorative options included conventional treatment and minimally invasive restorative treatment ( i.e. , interim therapeutic restorations ( itr ) ) . if the destruction of the tooth structure by the caries process was minimal , caries arrest was possible with remineralization of the tooth structure . the restorative treatment was then deferred in patients if the caries process was stabilized , especially in a child unable to cooperate for restorative treatment . however , close follow - up and preventive care based on caries risk were essential to safeguard from relapse . if the decay had progressed into dentin and caries arrest was not achieved , itr was offered as an alternative treatment with early cavitated lesions . a secondary gain from more frequent visits for preventive care was usually a reduction in a child 's fears and a gain in trust between the dental provider and the child over time , allowing for restorative treatment to be completed with greater ease at a later time . in order for teams and their sites to support risk - based disease prevention and management of ecc , dentists , staff , patients , and families who were accustomed to conventional surgical and restorative care were educated about and guided to accept a contemporary approach that emphasizes risk assessment , individualized disease prevention , and management and maintenance of health . for example , scheduling systems , typically set up to accommodate recall preventive visits every six months as allowed by insurance , had to be adjusted to allow for more frequent preventive return visits for high caries risk patients . before and during the implementation of the project , senior leaders and clinical champions of each team provided training to their dental providers and staff about the dm protocol . they shared what they learned from the learning sessions and monthly calls on dm and qi methods with those who were involved in the day - to - day work of implementing the ecc dm protocol . teams were expected to hold regular meetings to address questions about the protocol and care management of patients , review project progress , plan pdsas , and institute change . most teams chose to begin the protocol initially with a few providers , followed by spreading to additional providers over time . in phase 2 , teams collected process and outcome measurement data for the purpose of evaluating improvement trends in care processes and patient outcomes over time . each month , teams randomly selected 20 patient records ( charts ) of their ecc patients to record the results of some measures . meanwhile , on a quarterly basis , they selected 30 charts of ecc patients to record results of other measures . the deidentified data were collected and managed using redcap ( research electronic data capture ) electronic data capture tools hosted at bch . redcap is a secure , web - based application designed to support data capture for research studies providing ( 1 ) an intuitive interface for validated data entry ; ( 2 ) audit trails for tracking data manipulation and export procedures ; ( 3 ) automated export procedures for seamless data downloads to common statistical packages ; and ( 4 ) procedures for importing data from external sources . bch staff retrieved and processed the data , screened for errors , and managed the deidentified data . each month , run charts were produced by bch and sent to the collaborative staff . in turn , the collaborative sent run charts to each team for use in monitoring progress toward improvement . during monthly collaborative calls , trends in the run charts were reviewed . in addition , any questions and concerns from the teams were addressed by the collaborative staff , faculty , and improvement advisor . although this collaborative was designed as a qi initiative that aimed to identify positive trends in process and outcome measures which would signify improvements in care and outcomes , a trends analysis would not necessarily infer causality . therefore , there was a need to compare the project outcome data to baseline data derived from historical controls ( i.e. , patients treated by the teams prior to the start of phase 2 ) . in the last several months of the collaborative , after obtaining irb approval , teams collected data , on the three outcomes of interest ( percentage of patients with new cavitation , pain , and unplanned referrals to the or ) by randomly selecting 50 charts of their ecc patients and 50 charts of baseline historical control patients . at each site , a computer generated randomized scheme identified the potential ecc and control patients . qualifying ecc patients were those who had been ( a ) in phase 2 for a minimum of 6 months and ( b ) had at least one formally scheduled preventive visit ( recall visit ) whereby caries charting was performed and documented . all sites reviewed their phase 2 records ( i.e. excel spreadsheets or billing generated report ) and randomly selected 50 ecc patients ( one site had only 46 patients ) . qualifying historical control patients were children who were younger than 60 months of age and had ( 1 ) a history of decay ; ( 2 ) at least one recall visit six months after the initial visit ; and ( 3 ) a last visit that was at least six months prior to the start of the phase 2 . for each team , a computer generated randomized scheme identified potential patients based on age and whose billing records were reviewed to determine whether they had a recall visit at least 6 months prior to the start of phase 2 . these patient records were selected for further review to determine those patients who met the qualifying criteria . the following information was documented for both ecc and historical control patients by visit date : ( 1 ) type of visit ( preventive , restorative , sedation , or , missed , or canceled ) , ( 2 ) new cavitation identified , ( 3 ) pain identified , and ( 4 ) referral to or . the first visit was determined for each patient as that which decay was initially charted or documented in the patient 's clinical notes . pain and or referral at first visit , including pain unrelated to untreated decay at any visit were excluded . from this information , the percentage of patients with new cavitation , pain identified , and referral to or were determined . the ecc and historical control data were collected by most teams onto paper collection forms . figure 4 shows some of the trend data for the seven phase 2 teams . over time , the teams demonstrated a highly consistent level of performance with their providers performing cras and smgs . most teams saw a reduction of ecc children deemed as high caries risk and an increase in ecc children with improved caries risk from the first visit . table 3 shows a comparison of the rates of new cavitation , pain , and referral to or between ecc patients and the baseline patients for phase 2 . these results reflect a random sample of 316 historical control children and 344 ecc children drawn from a total phase 2 population of 3,030 . in the aggregate , children in the ecc dm group experienced lower rates of new cavitated lesions , pain , and referrals for restorative treatment under general anesthesia in the operating room ( or ) as compared to baseline historical controls , although there was variability from site to site . in phase 2 of the ecc learning collaborative , by using qi methods to change their systems of care , teams were able to efficiently implement the dm protocol into their clinical practice . in the aggregate for phase 2 , fewer ecc children experienced new cavitation , pain , and referrals to the or compared to baseline historical controls . there was discrepancy from team to team in the degree of improvement in the process measures and outcomes achieved . the variation in outcomes among teams , as expected , may be attributed to differences in each team 's use of the dm protocol or to distinct cultural and socioeconomic differences in patients and families among the sites . similarly , phase 1 also found imbalance in terms of outcomes achieved at the two sites . the phase 1 team that demonstrated a relatively less dramatic improvement in new cavitation rates had predominantly english speaking providers serving predominantly latino populations who spoke spanish as their native language . unfortunately , demographic data were not collected in phase 2 . in terms of limitations , 50 randomly selected charts for each of the ecc and historical control groups at each site may be insufficiently representative of the groups for each team . at the same time , an 18-month follow - up period may be an inadequate length of time to evaluate the clinical outcomes of some children who were enrolled as ecc patients over time . in addition , although dental providers at all sites received training on the dm protocol to chart decay ( by using a modified icdas system ) , they did not receive calibration to chart new cavitated or precavitated lesions . however , by protocol , ecc patients were seen more frequently for dm visits , during which time they were examined for new cavitation and thus may have received increased opportunities for new cavitated lesions to be identified . during their participation in phase 2 , teams shared their experiences including their knowledge and skills gained , lessons learned , and tools developed with other collaborative participants . examples of new skills include training of support staff and employing them to assist with cra and patient education , collaborating with pediatric medical providers to enhance the referral of young children for early preventive dental visits , scheduling differently to accommodate the more frequent return needs of the ecc children for dm visits , and managing no - show appointments by using a registry to track patient visits . two teams were located in community health centers that initially saw a limited number of young children . these teams developed pdsas to specifically focus on increasing referrals from pediatricians within their centers , and they were successful in their efforts . one site had a baby 's clinic already in place prior to joining phase 2 and a hygienist to see children specifically younger than age three years for infant oral health visits ; this hygienist was already using a cra tool . this site incorporated the dm protocol , smgs , and more frequent visits first into their baby 's clinic and later spread the dm protocol to other providers in their main dental clinic over time . tools developed , enhanced , and willingly shared among the collaborative teams included forms for conducting and documenting carious lesions , cra , smgs , and tracking of the ecc patients . at the conclusion of phase 2 , team leaders convened for a final summary conference . all teams agreed that the dm approach to address ecc was a logical change in practice , albeit not easy to implement . challenges included having to accommodate the additional dm visits and the time required for each visit . in some cases initially , most providers and teams struggled with having to fit in the dm visits , especially if their schedules were booked out in advance . since most dental insurance plans do not cover more than two diagnostic / preventive visits each year , the additional dm visits and lost time for reimbursable restorative care posed as additional obstacles . changes that worked were implemented across the practice and continually improved upon through further testing . providers received training and coaching to be able to perform cra , explain the causes of the caries process , and work with the caregivers to select smgs more effectively and with greater efficiency . providers and families began to accept a paradigm shift that addressed disease etiology in lieu of relying solely on a surgical model of treatment . the use of qi methods was useful in facilitating the adoption of dm to address ecc , first by getting buy - in from the early adopters at each site , followed by later adopters . some teams had spread the protocol to more providers within their primary site , while others had spread it to all their providers . one team successfully spread the protocol beyond their primary site to other dental sites that were a part of their community center network . most sites embedded their dm protocols into their systems of care such that dismantling those systems would require effort . what impact did the collaborative have on you ? responses included it made me a better provider , a better teacher ; and i no longer view children 05 the same way ( i do not pick up the hand - piece first ) . we demonstrated the feasibility of an innovative approach to address ecc utilizing dm protocols that can be successfully implemented into dental practice using qi methods in a learning collaborative model . although not easy to implement , after 18 months , all teams reported that the dm approach resulted in overall improved care delivery and patient outcomes ( new cavitation , pain , and referrals to the or ) . teams recognized that while a dm model can be implemented into practice , policy and payment reforms are needed to facilitate a wider - scaled adoption of the dm protocols . elements to be addressed include compensation for providers ' time and efforts to perform cra , smgs , patient education and engagement , and the dm visits . future demonstration should quantify opportunities for cost savings to be realized by avoiding more costly restorative treatment . future policy changes are necessary to support a paradigm shift from surgical treatment of caries toward an individualized risk - based disease prevention and management model as the new standard of care . at the same time , the use of qi methods may help accelerate the adoption and spread of dm protocols into any dental practice .

until recently , the standard of care for early childhood caries ( ecc ) has been primarily surgical and restorative treatment with little emphasis on preventing and managing the disease itself . it is now recognized that surgical treatment alone does not address the underlying etiology of the disease . despite costly surgeries and reparative treatment , the onset and progression of caries are likely to continue . a successful rebalance of risk and protective factors may prevent , slow down , or even arrest dental caries and its progression . an 18-month risk - based chronic disease management ( dm ) approach to address ecc in preschool children was implemented as a quality improvement ( qi ) collaborative by seven teams of oral health care providers across the united states . in the aggregate , fewer dm children experienced new cavitation , pain , and referrals to the operating room ( or ) for restorative treatment compared to baseline historical controls . the teams found that qi methods facilitated adoption of the dm approach and resulted in improved care to patients and better outcomes overall . despite these successes , the wide scale adoption and spread of the dm approach may be limited unless health policy and payment reforms are enacted to compensate providers for implementing dm protocols in their practice .

1. Background 2. Methods 3. Results 4. Discussion 5. Conclusion

early childhood caries ( ecc ) is a common childhood disease in the united states ( us ) and worldwide . until recently , the standard of care for ecc has been primarily surgical and restorative treatment with relatively little emphasis on the prevention and management of disease . in the us , young children who are not cooperative are commonly sedated or treated under general anesthesia ( ga ) . however , despite receiving costly treatment under ga , such as in the operating room ( or ) [ 35 ] , children all too often develop new and recurrent caries [ 610 ] . it is now accepted that surgical repair alone does not address the underlying etiology of the disease . unless the caries balance is altered , new and recurrent caries are likely to occur . on the other hand , a successful rebalance of risk and protective factors may slow down or completely halt the disease process , resulting in caries arrest , if not also preventing the onset of new disease . contemporary approaches to caries prevention and management modeled after medical management of chronic conditions such as diabetes , obesity and asthma , have been described in the scientific literature and are herein known as chronic disease management ( dm ) [ 11 , 1315 ] . dm differs from the traditional approach of oral health care providers relying on a surgical treatment model in response to the disease , while telling the patients what to do . a close collaboration between the healthcare provider and patient is required , ideally in a culturally and linguistically appropriate manner . in practice , healthcare providers coach patients and parents about the factors that lead to and protect against dental disease and assist them in selecting self - management goals to improve their own and their children 's risk for disease . treatment decisions are based on the latest evidence - based guidelines that are customized to patients ' individual needs . risk - based dm of ecc requires significant patient and family engagement and empowerment from the provider and care team in effective day - to - day behavior modifications ( e.g. , tooth brushing , topical fluorides , and dietary control ) that address disease etiology . family - centered behavior plans lead to real behavior change and maintenance of oral health behaviors in the child 's home . at the same time , the dental practice has a reciprocal role in tracking and managing the care of patients . phase 1 in 2008 , a risk - based dm approach to address preschool children with ecc was implemented and tested as a quality improvement ( qi ) demonstration project at boston children 's hospital in boston , massachusetts ( bch ) , and st . joseph health services of rhode island in providence , ri ( sjh ) . thirty months of results found that children in the ecc group experienced lower rates of new cavitated lesions , pain , and referrals for restorative treatment under general anesthesia in the or as compared to baseline historical controls ( table 1 ) . at bch , the ecc group experienced a 62% lower rate of new cavitation compared to the historical control group . structured interviews with phase 1 parents revealed that most believed the dm approach to be helpful for their children ; almost all parents appreciated given reasons as to why their children may have developed ecc . the collaborative approach allowed clinicians to engage parents or caregivers to better understand that they have a voice in the care their child receives . in 2008 , a risk - based dm approach to address preschool children with ecc was implemented and tested as a quality improvement ( qi ) demonstration project at boston children 's hospital in boston , massachusetts ( bch ) , and st . thirty months of results found that children in the ecc group experienced lower rates of new cavitated lesions , pain , and referrals for restorative treatment under general anesthesia in the or as compared to baseline historical controls ( table 1 ) . at bch , the ecc group experienced a 62% lower rate of new cavitation compared to the historical control group . structured interviews with phase 1 parents revealed that most believed the dm approach to be helpful for their children ; almost all parents appreciated given reasons as to why their children may have developed ecc . the collaborative approach allowed clinicians to engage parents or caregivers to better understand that they have a voice in the care their child receives . phase 2building upon the promising results of phase 1 , the project was expanded in 2011 to include five additional teams in the us . phase 2 further tested the feasibility and effectiveness of the dm approach to reduce ecc in more diverse settings . the five additional teams were in the following locations across the us : holyoke health center ( holyoke , ma ) ; native american health center ( san francisco , ca ) ; nationwide children 's hospital ( columbus , oh ) , neighborcare health ( seattle , wa ) ; and university pediatric dentistry ( buffalo , ny ) . the purpose of this report is to describe the phase 2 project and experiences , present the results after 18 months , and discuss the implications of what was learned . building upon the promising results of phase 1 , the project was expanded in 2011 to include five additional teams in the us . phase 2 further tested the feasibility and effectiveness of the dm approach to reduce ecc in more diverse settings . the five additional teams were in the following locations across the us : holyoke health center ( holyoke , ma ) ; native american health center ( san francisco , ca ) ; nationwide children 's hospital ( columbus , oh ) , neighborcare health ( seattle , wa ) ; and university pediatric dentistry ( buffalo , ny ) . the purpose of this report is to describe the phase 2 project and experiences , present the results after 18 months , and discuss the implications of what was learned . phase 2 was implemented as an 18-month qi learning collaborative . using established qi methods , a nationwide collection of staff , experts , and faculty provided training and technical assistance to the seven participating teams , which included the two teams that were part of phase 1 . teams were required to attend three on - site learning sessions where each of the seven teams received didactic education and training on qi concepts and activities . the learning session curriculum focused on the use of logic models , measurement plans , plan - do - study - act ( pdsa ) cycles , dm of ecc such as caries risk assessment ( cra ) , self - management goals ( smgs ) , effective patient - provider communication , and fluorides and other remineralizing modalities . the learning sessions provided invaluable opportunities for synergy as teams exchanged approaches to dm during consultations with experts , faculty , and staff , who provided coaching and support . qi has been defined as the combined and unceasing efforts of everyone to make changes that will lead to better patient outcomes ( health ) , system performance ( care ) , and professional development ( learning ) . qi is intended to support the redesign of care processes based on a system of learning , incremental change , and the incorporation of empirically supported best practices from evaluating performance and outcome measures . unlike a protracted randomized trial , qi uses systematic , data - guided activities designed to bring about immediate improvements in health care delivery in particular settings and can be considered as the scientific method used for action - oriented learning . the model for improvement developed by associates in process improvement was used as the essential framework to guide changes made by each team 's care delivery system in order to use a dm approach to address ecc . the collaborative developed a driver diagram outlining three main outcomes of interests : ( 1 ) new cavitation ; ( 2 ) pain related to untreated caries ; and ( 3 ) referral to the or , along with primary and secondary drivers affecting those outcomes ( figure 1 ) . pdsa cycles are small - scale tests of change in real work settings by planning a test , trying it , observing the results , and acting on what is learned . pdsa cycles promote creativity , offer quick results , and empirically support approaches to dm that are specific to the clinical teams . for example , pdsas served as learning opportunities for phase 2 teams to use evidence to determine how to perform activities such as cra , smgs , and patient scheduling to support the additional dm visits required . this form was adapted from the american academy of pediatric dentistry ( aapd cra ) form and the pediatric caries management by risk assessment ( cambra ) form . children who had at least one tooth with clinical manifestation of caries tooth decay ( including demineralization)or who had a history of carious lesions was considered an ecc patient . at the initial and recall visits , a clinical examination and charting were performed to allow for the tracking of caries presence and activity by tooth and surface , since decay may progress and become inactive at different sites of the dentition at the same time . parents of ecc patients were engaged and coached about the factors that lead to caries and tooth decay by dentists , hygienist , dental assistants , and/or support staff . parents learned about the caries process as they were informed of the ways that tooth decay can be prevented and stopped . such goals include basic caries control strategies such as more frequent tooth brushing , using topical fluorides at home , and modifying one 's diet to include fewer and less frequent intake of sugary products . the frequency of return dm visits for patients and parents in - office and at the clinic site was based on their caries risk . whenever possible , the dm activities were coordinated with restorative treatment . table 2 shows the dm protocol with return visit intervals based on the most recent caries risk status in conjunction with restorative care as needed and as desired by the parent and dental provider . as a result , a one - month follow - up visit for a child assessed to be high caries risk allowed for a more accurate assessment of demineralized , cavitated , and remineralized tooth surfaces . providers asked parents to report on their experiences with the smgs in order to assess the level of compliance and the utility of the agreed - upon smgs . parents were given the full range of options for restorative treatment , which included pharmacologic management ( i.e. , use of nitrous oxide , sedation , or ga / or ) as needed by the patient and as desired by the parent . restorative options included conventional treatment and minimally invasive restorative treatment ( i.e. , interim therapeutic restorations ( itr ) ) . if the destruction of the tooth structure by the caries process was minimal , caries arrest was possible with remineralization of the tooth structure . the restorative treatment was then deferred in patients if the caries process was stabilized , especially in a child unable to cooperate for restorative treatment . if the decay had progressed into dentin and caries arrest was not achieved , itr was offered as an alternative treatment with early cavitated lesions . a secondary gain from more frequent visits for preventive care was usually a reduction in a child 's fears and a gain in trust between the dental provider and the child over time , allowing for restorative treatment to be completed with greater ease at a later time . in order for teams and their sites to support risk - based disease prevention and management of ecc , dentists , staff , patients , and families who were accustomed to conventional surgical and restorative care were educated about and guided to accept a contemporary approach that emphasizes risk assessment , individualized disease prevention , and management and maintenance of health . before and during the implementation of the project , senior leaders and clinical champions of each team provided training to their dental providers and staff about the dm protocol . they shared what they learned from the learning sessions and monthly calls on dm and qi methods with those who were involved in the day - to - day work of implementing the ecc dm protocol . teams were expected to hold regular meetings to address questions about the protocol and care management of patients , review project progress , plan pdsas , and institute change . redcap is a secure , web - based application designed to support data capture for research studies providing ( 1 ) an intuitive interface for validated data entry ; ( 2 ) audit trails for tracking data manipulation and export procedures ; ( 3 ) automated export procedures for seamless data downloads to common statistical packages ; and ( 4 ) procedures for importing data from external sources . bch staff retrieved and processed the data , screened for errors , and managed the deidentified data . each month , run charts were produced by bch and sent to the collaborative staff . in turn , the collaborative sent run charts to each team for use in monitoring progress toward improvement . during monthly collaborative calls , trends in the run charts were reviewed . in addition , any questions and concerns from the teams were addressed by the collaborative staff , faculty , and improvement advisor . although this collaborative was designed as a qi initiative that aimed to identify positive trends in process and outcome measures which would signify improvements in care and outcomes , a trends analysis would not necessarily infer causality . therefore , there was a need to compare the project outcome data to baseline data derived from historical controls ( i.e. , patients treated by the teams prior to the start of phase 2 ) . in the last several months of the collaborative , after obtaining irb approval , teams collected data , on the three outcomes of interest ( percentage of patients with new cavitation , pain , and unplanned referrals to the or ) by randomly selecting 50 charts of their ecc patients and 50 charts of baseline historical control patients . qualifying historical control patients were children who were younger than 60 months of age and had ( 1 ) a history of decay ; ( 2 ) at least one recall visit six months after the initial visit ; and ( 3 ) a last visit that was at least six months prior to the start of the phase 2 . for each team , a computer generated randomized scheme identified potential patients based on age and whose billing records were reviewed to determine whether they had a recall visit at least 6 months prior to the start of phase 2 . the following information was documented for both ecc and historical control patients by visit date : ( 1 ) type of visit ( preventive , restorative , sedation , or , missed , or canceled ) , ( 2 ) new cavitation identified , ( 3 ) pain identified , and ( 4 ) referral to or . the first visit was determined for each patient as that which decay was initially charted or documented in the patient 's clinical notes . from this information , the percentage of patients with new cavitation , pain identified , and referral to or were determined . figure 4 shows some of the trend data for the seven phase 2 teams . over time , the teams demonstrated a highly consistent level of performance with their providers performing cras and smgs . most teams saw a reduction of ecc children deemed as high caries risk and an increase in ecc children with improved caries risk from the first visit . table 3 shows a comparison of the rates of new cavitation , pain , and referral to or between ecc patients and the baseline patients for phase 2 . in the aggregate , children in the ecc dm group experienced lower rates of new cavitated lesions , pain , and referrals for restorative treatment under general anesthesia in the operating room ( or ) as compared to baseline historical controls , although there was variability from site to site . in phase 2 of the ecc learning collaborative , by using qi methods to change their systems of care , teams were able to efficiently implement the dm protocol into their clinical practice . in the aggregate for phase 2 , fewer ecc children experienced new cavitation , pain , and referrals to the or compared to baseline historical controls . there was discrepancy from team to team in the degree of improvement in the process measures and outcomes achieved . the variation in outcomes among teams , as expected , may be attributed to differences in each team 's use of the dm protocol or to distinct cultural and socioeconomic differences in patients and families among the sites . the phase 1 team that demonstrated a relatively less dramatic improvement in new cavitation rates had predominantly english speaking providers serving predominantly latino populations who spoke spanish as their native language . in terms of limitations , 50 randomly selected charts for each of the ecc and historical control groups at each site may be insufficiently representative of the groups for each team . at the same time , an 18-month follow - up period may be an inadequate length of time to evaluate the clinical outcomes of some children who were enrolled as ecc patients over time . in addition , although dental providers at all sites received training on the dm protocol to chart decay ( by using a modified icdas system ) , they did not receive calibration to chart new cavitated or precavitated lesions . however , by protocol , ecc patients were seen more frequently for dm visits , during which time they were examined for new cavitation and thus may have received increased opportunities for new cavitated lesions to be identified . during their participation in phase 2 , teams shared their experiences including their knowledge and skills gained , lessons learned , and tools developed with other collaborative participants . examples of new skills include training of support staff and employing them to assist with cra and patient education , collaborating with pediatric medical providers to enhance the referral of young children for early preventive dental visits , scheduling differently to accommodate the more frequent return needs of the ecc children for dm visits , and managing no - show appointments by using a registry to track patient visits . these teams developed pdsas to specifically focus on increasing referrals from pediatricians within their centers , and they were successful in their efforts . one site had a baby 's clinic already in place prior to joining phase 2 and a hygienist to see children specifically younger than age three years for infant oral health visits ; this hygienist was already using a cra tool . this site incorporated the dm protocol , smgs , and more frequent visits first into their baby 's clinic and later spread the dm protocol to other providers in their main dental clinic over time . tools developed , enhanced , and willingly shared among the collaborative teams included forms for conducting and documenting carious lesions , cra , smgs , and tracking of the ecc patients . all teams agreed that the dm approach to address ecc was a logical change in practice , albeit not easy to implement . in some cases initially , most providers and teams struggled with having to fit in the dm visits , especially if their schedules were booked out in advance . since most dental insurance plans do not cover more than two diagnostic / preventive visits each year , the additional dm visits and lost time for reimbursable restorative care posed as additional obstacles . changes that worked were implemented across the practice and continually improved upon through further testing . providers received training and coaching to be able to perform cra , explain the causes of the caries process , and work with the caregivers to select smgs more effectively and with greater efficiency . the use of qi methods was useful in facilitating the adoption of dm to address ecc , first by getting buy - in from the early adopters at each site , followed by later adopters . most sites embedded their dm protocols into their systems of care such that dismantling those systems would require effort . what impact did the collaborative have on you ? we demonstrated the feasibility of an innovative approach to address ecc utilizing dm protocols that can be successfully implemented into dental practice using qi methods in a learning collaborative model . although not easy to implement , after 18 months , all teams reported that the dm approach resulted in overall improved care delivery and patient outcomes ( new cavitation , pain , and referrals to the or ) . teams recognized that while a dm model can be implemented into practice , policy and payment reforms are needed to facilitate a wider - scaled adoption of the dm protocols . elements to be addressed include compensation for providers ' time and efforts to perform cra , smgs , patient education and engagement , and the dm visits . future demonstration should quantify opportunities for cost savings to be realized by avoiding more costly restorative treatment . future policy changes are necessary to support a paradigm shift from surgical treatment of caries toward an individualized risk - based disease prevention and management model as the new standard of care . at the same time , the use of qi methods may help accelerate the adoption and spread of dm protocols into any dental practice .

two of these disorders , autism spectrum disorders ( asds ) and schizophrenia ( sczd ) , combine to affect nearly 2% of the global population . asds are complex neurodevelopmental disorders characterized by severe impairments in social interactions , communication and behavior ( kanner , 1943 ; american psychiatric association , 2000 ) . sczd is characterized by three severe classes of symptoms : positive ( hallucinations and delusions ) , negative ( inability to speak , express emotion or find pleasure ) and cognitive ( deficits in attention , memory and planning ) ( american psychiatric association , 2000 ; carpenter and buchanan , 1994 ) ; it often leads to premature death from poverty , homelessness , substance abuse and poor health maintenance ( brown et al . , 2000 ) . of these two psychiatric disorders , twin studies estimate the heritability of asds to be greatest , as high as 90% ( ritvo et al . , 1985 ) , whereas estimates of the heritability of sczd range from 7085% ( tsuang et al . , 2001 ; sullivan et al . , 2003 ) . genome - wide association scans for asds and sczd suggest that common genetic loci influence susceptibility to many psychiatric disorders ( lewis et al . , 2003 ; fanous et al . , 2007 ) . it is now believed that many rare but penetrant mutations contribute to asds and sczd , and that hundreds of common variants with mild effects are shared by sczd and bipolar disorder ( odonovan et al . it is generally very difficult to obtain live neurons from patients affected with asds or sczd to study . in addition , because most psychiatric disorders are polygenic , few relevant animal models have been developed . although transgenic mice demonstrate some abnormalities in behavior in specific experimental conditions ( clapcote et al . , 2007 ; , 2007 ; barros et al . , 2009 ) , it is difficult to determine how well these mouse models recapitulate the human condition because it is difficult to assess complex phenotypes such as hallucinations and delusions in mice . to date , human studies of these psychiatric disorders have relied primarily on brain imaging of patients , postmortem studies of brains and genetic studies of patient lymphocytes . many notable insights have been made through these methods , but the molecular and cellular defects that contribute to disease initiation and progression in neurons have been difficult to elucidate . although they are poorly understood at the molecular and cellular levels , psychiatric disorders are now believed to be developmental disorders . therefore , human - cell - based models representing genetic backgrounds known to develop these neurological diseases might be the best method to identify the neuronal defects associated with them . in this commentary , we review recent studies that use sophisticated human - cell - based models to study the pathways underlying psychiatric disorders . we limit our discussion to approaches that focus on neuronal cell types , although non - neuronal cell - based approaches have also provided useful information ( box 1 ) . in 2006 , takahashi and yamanaka identified four factors ( oct3/4 , klf4 , sox2 and myc ) whose transient expression was sufficient to directly reprogram mouse adult somatic cells into an induced pluripotent stem cell ( ipsc ) state ( takahashi and yamanaka , 2006 ) . one year later , groups led by shinya yamanaka and james thomson ( takahashi et al . , 2007 ; yu et al . , 2007 ) demonstrated for the first time that it was also possible to reprogram human fibroblasts . ipscs can be generated from either mouse or human skin cells and are nearly identical to embryonic stem ( es ) cells with respect to gene expression , dna methylation and chromatin modifications . both mouse ipscs ( mipscs ) and human ipscs ( hipscs ) are believed to be capable of differentiating into every cell type found in the adult ( maherali et al . because hipscs can be derived from adult humans , after the development of disease , hipscs represent a potentially limitless source of human cells with which to study disease , even without knowing which genes are interacting to produce the disease state in an individual patient . there is a long history of non - neuronal cell - based studies comparing patients and controls , particularly in the study of sczd . the best established of these are studies of fibroblasts , lymphocytes and olfactory neural stem cells . non - neuronal cells typically express lower levels of neuronal genes than do mature neurons , so it remains uncertain to what extent findings from these studies generate insights into the mechanism of disease . fibroblasts : skin fibroblasts have been used as cellular models of sczd ( mahadik and mukherjee , 1996 ) . although some comparisons between control and sczd fibroblasts do not show robust patient - versus - control differences ( matigian et al . , 2008 ) lymphocytes : a small number of microarray analysis studies using immortalized lymphocytes derived from patients with sczd have been reported ; however , whether immortalized lymphocytes are a relevant alternative to neuronal tissue remains controversial ( matigian et al . , 2010 ) . lymphocyte studies have identified aberrant expression of disc1 , serotonin transporter ( 5-htt ) , dysbindin-1 and neuregulin-1 ( nrg-1 ) ( hranilovic et al . , 2004 ; maeda et al . , 2006 ; yamamori et al . , 2011 ) in samples from patients with sczd . olfactory neural stem cells : matigian et al . compared patient - derived olfactory neural stem cells from nine patients with sczd and 14 controls , and identified sczd - associated differences in gene expression and protein levels in neurodevelopmental pathways , including cell migration and axon guidance ( matigian et al . , 2010 ) . it is important to note that genetic and epigenetic mutations can and do occur during the reprogramming process . sometimes hipsc lines contain abnormal karyotypes after reprogramming ( mayshar et al . , 2010 ) , and large copy number variations ( cnvs ) have also been associated with both the reprogramming and differentiation of hipscs ( laurent et al . , 2011 ) . however , significantly more cnvs are present in early - passage hipscs than in higher - passage hipscs , fibroblasts or human es cells ; apparently , most novel cnvs generated during early stages of the reprogramming process are subsequently lost from the hipsc population ( hussein et al . , 2011 ) . additionally , among the 22 hipsc lines studied , an average of five protein - coding point mutations were identified ( gore et al . , 2011 ) . furthermore , hipscs share megabase - scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming ( lister et al . , 2011 ) . importantly , whereas hipscs do acquire genetic modifications in addition to epigenetic modifications , and although extensive genetic screening should become a standard procedure to ensure hipsc safety before clinical use , it is extremely unlikely that de novo mutations affect disease - specific hipsc lines in a meaningfully different way than control hipsc lines . although spontaneous mutations can occur during the reprogramming process , random mutations should not interfere with the ability to draw meaningful conclusions from hipsc - based studies of psychiatric disorders when studies include comparisons of neurons generated from multiple hipsc lines for each control and diseased patient . work with es cells first demonstrated methods to efficiently differentiate pluripotent stem cells into neurons ( tropepe et al . , 2001 ) . during embryonic development , embryonic neuroepithelia are believed to be first specified to a forebrain fate and are then subsequently transformed with caudalizing signals to midbrain / hindbrain and spinal cord fates in a gradient manner ( stern , 2001 ) . similarly , through the addition of various morphogens in vitro , es cells and ipscs can be directed to differentiate into regional identities including forebrain ( watanabe et al . , 2005 ) , midbrain / hindbrain ( kawasaki et al . , 2000 ; perrier et al . , 2004 ) and spinal cord ( wichterle et al . , 2002 ; li et al . , 2005 owing to an excellent characterization of the transcription factors used to specify fate in embryonic mice , the best - characterized hipsc differentiation protocol is generally accepted to be one for motor neurons 2008 ) , although a highly efficient method for generating midbrain dopaminergic neurons was recently published ( chambers et al . , 2009 ) . much of the developmental potential that is present in vivo seems to be maintained in vitro . the choice of specific subtypes of neurons to be compared using hipsc - derived neurons warrants careful consideration because the brain regions and cell types affected differ between diseases . magnetic resonance imaging ( mri ) studies have observed structural differences in the brains of many patients affected by psychiatric disorders compared with those of the normal population . for example , patients with sczd often have decreased brain volume , particularly in the hippocampus , thalamus and cortex ( vita et al . , 2006 ; ellison - wright et al . , 2008 ) , whereas patients with asds show transient increases in brain volume from ages 2 to 4 years , notably in the cortex , cerebellum and amygdala , coinciding with the onset of symptoms ( courchesne et al . , 2007 ) . notably , although these mri studies provide information regarding the brain regions that seem to be affected by these disorders , they do not reveal the cell types implicated in disease , but some insights have been provided through pharmacological studies . with respect to sczd , for example , dopamine receptor antagonists , and perhaps also glutamate receptor ( glur2/3 ) agonists , alleviate psychosis ( patil et al . , 2007 ) . conversely , when administered to healthy human subjects , dopamine agonists ( such as amphetamine ) and glutamate antagonists ( such as ketamine ) induce hallucinations and cognitive deficits similar to those associated with sczd ( krystal et al . , 1994 ) . pharmacological studies of asds have been less successful in identifying abnormalities in the activity of a specific neurotransmitter . although the specific cell subtypes that should ideally be examined to provide insight into these diseases remain uncertain , it is generally believed that cortical neurons are a relevant cell type for the study of many psychiatric disorders . rigorous validation of the neurons that are ultimately generated from hipscs is crucial for establishing the maturity and identity of the neuronal cultures that are generated in vitro . electrophysiological characterization should verify that hipsc neurons have membrane potentials , undergo induced action potentials and show evidence of spontaneous synaptic activity . for example , mature dopaminergic neurons express tyrosine hydroxylase ( th ) and aromatic l - amino acid decarboxylase ( aadc ) ( ang , 2006 ) , whereas mature cortical neurons express ctip2 , a transcriptional repressor that is required for the maturation of cortical axons , and otx1 , a transcription factor that is highly expressed in layer 5 cortical neurons ( frantz et al . , 1994 ; weimann et al . it is important to note that researchers can not generate artificial brain structures in vitro , such as specific brain regions implicated in neurological disease for example , the prefrontal cortex for studies of asd or the hippocampus for studies of sczd . although the relative frequency of a specific neuronal cell type might be favored , differentiated populations generally contain several types of neurons , as well as astrocytes , oligodendrocytes , neural precursors and even non - neural cells . this strategy permits study of the innate neuron - specific deficits in psychiatric disease in a manner that is not confounded by environmental factors that typically plague studies of sczd , such as treatment history , drug and alcohol abuse or poverty . these reports have all been published within the last year ; each supports our contention that hipsc - derived neurons from patients with psychiatric disorders show significant aberrations compared with those of healthy controls . hipscs differentiate robustly to forebrain neurons and , by co - culturing hipsc forebrain neurons with hek293 t cells , kim et al . showed that hek293 t cells expressing wild - type nlgn3 and nlgn4 , but not those containing asd - associated mutations in these genes , can induce presynaptic differentiation in hipsc - derived neurons ( kim et al . , 2011 ) . although this work used only hipscs derived from the cells of healthy controls , these findings did establish that these cells are a viable model for the study of synaptic differentiation and that they function under normal and disorder - associated conditions . using rett syndrome ( rtt ) as an asd genetic model , we generated hipscs from patients with rtt and found early alterations in developing hipsc - derived neurons ( marchetto et al . . compared with controls , neurons derived from rtt hipscs had fewer synapses , reduced spine density , smaller soma size , altered ca signaling and electrophysiological defects ( fig . we also used rtt - hipsc - derived neurons to test the capacity of drugs to rescue synaptic defects ( marchetto et al . , 2010 ) . subsequently , a second group repeated the finding of reduced neuronal soma size in rtt - hipsc - derived neurons ( cheung et al . , 2011 ) together , these studies established that assays of neuronal size , synaptic structure and synaptic function can detect differences between control and disordered hipsc - derived neurons in vitro , demonstrating that neurodevelopmental diseases can be modeled in this manner . notably , rtt is usually associated with the complete loss of function of a single gene ( mecp2 ) and shows rapid disease progression within the first years of life ; the first studies of complex genetic forms of asd have not yet been reported . b ) rtt - hipsc - derived neurons have reduced density of excitatory synapses along dendrites compared with neurons derived from healthy controls . the staining shows that hipsc - derived neurons from rtt patients have fewer vglut1-positive glutamatergic synaptic puncta interspersed along map2-positive dendrites . ( c ) tracking fluorescence intensity changes representing intracellular ca fluctuations provides evidence of reduced synaptic activity in rtt - hipsc - derived neurons relative to controls . chiang et al . recently reported the generation of integration - free hipscs from sczd patients with a disc1 mutation ( chiang et al . , 2011 ) , but have not yet reported a characterization of neurons differentiated from these hipscs . these first reports of hipscs generated from patients with psychiatric disease studied monogenetic disorders . given that mouse models of rtt ( mecp2 null ) and mutated disc1 ( dndisc ) exist , these early studies allow a direct comparison between in vitro hipsc studies and in vivo animal experiments . it will be interesting to learn whether disc1-hipsc - derived neurons recapitulate the cellular phenotypes observed in dndisc mice as faithfully as rtt - hipsc - derived neurons recapitulated findings in mecp2-null mice . with these important proof - of - principle validations of hipsc models completed , future hipsc - based studies must begin to take full advantage of the ability of hipscs to model complex genetic cases of psychiatric disease , in which interactions between as - yet - unidentified genes result in the disease state . we recently reported the use of hipscs to model a complex genetic psychiatric disorder in which hipsc - derived neurons from four patients with sczd were compared with those from controls . sczd - hipsc - derived neurons had reduced neuronal connectivity , reduced outgrowths from soma , reduced psd95 dendritic protein levels and altered gene expression profiles relative to controls ( fig . 2 ) ; defects in neuronal connectivity and gene expression were ameliorated following treatment with the antipsychotic drug loxapine ( brennand et al . , 2011 ) . nearly 25% of genes that showed altered expression compared with controls had been previously implicated in sczd , although the expression profiling data also suggested that a number of pathways not previously implicated in sczd might contribute to the disorder , such as notch signaling , cell adhesion and slit - robo - mediated axon guidance . we predict that , as future studies increase the number of sczd - patient - derived neurons , core pathways of genes that contribute to this disorder will be identified . hipsc - based studies show great potential use to model complex genetic disorders for which the genes responsible for producing the disease state differ between individuals . we hope that hipscs will permit direct evaluations of several hypotheses that are not easy to address in psychiatric disease research , such as : does severity of clinical outcome predict magnitude of cellular phenotype ? is clinical pharmacological response predictable by hipsc neuronal drug response ? to discover the answers to these questions , substantially increased numbers of patient - specific hipsc - derived neurons must be generated . furthermore , hipsc - derived neurons must be derived from better - characterized patient cohorts . by moving forward with cohorts of patients for whom clinical outcome , pharmacological response , mri imaging and genotype data are available , hipsc - based models of psychiatric disorders will enable direct correlations of clinical , cellular and molecular phenotypes in psychiatric disease . sczd - hipsc - derived neurons show decreased transmission of a genetically engineered rabies virus designed specifically to indicate monosynaptic neuronal connectivity ( rabies - envag - rfp ) . adapted from brennand et al . lv - synp - htg , lentivirus expressing a fusion protein comprising histone 2b and green fluorescent protein , tva and elements of the synapsin ( syn ) promoter ; used to label neurons for microscopic analysis . the limitations of hipsc - based approaches for studying psychiatric disease are mainly ( 1 ) neuron - to - neuron variability , ( 2 ) hipsc - to - hipsc variability and ( 3 ) patient - to - patient variability . neuron - to - neuron variability encompasses differences between individual hipsc - derived neurons from a single patient ; it often reflects heterogeneity of neuronal subtypes within a neural population . uniquely among the hipsc reports discussed , kim et al . attempted to direct the regional identity of their hipsc neurons , and determined that treatment with a sonic hedgehog ( shh ) agonist increased expression of the forebrain marker bf1 ( also known as foxg1 ) , repressed the expression of the dorsal markers pax6 , emx2 and gli3 , and increased the expression of the ventral markers shh and nkx2.1 at the neural progenitor cell ( npc ) stage , suggesting that some of their hipsc neurons acquired an anterior ventral forebrain fate ( kim et al . , 2011 ) . universally , hipsc - based studies of psychiatric disorders to date have all been performed on mixed populations of neuronal subtypes , which are generally described as predominantly glutamatergic with a significant fraction of gabanergic neurons and only rare dopaminergic neurons , as assessed by expression of vglut , gad67 and th , respectively . the use of heterogeneous neuronal populations increases neuron - to - neuron variability in experimental assays , a limitation that could be overcome by studying fluorescence - activated cell sorting ( facs)-purified populations of neurons of a defined identity . a second limitation of hipsc studies is variability in hipsc lines from a single patient . this variation might reflect differences in the integration of viruses used for gene delivery in reprogramming , variation in the extent of epigenetic reprogramming , spontaneous mutations resulting from reprogramming and expansion , differences in hipsc culture technique , and even differences in the cell type of origin . just as non - trivial differences in developmental potential exist among human es cell lines and within subclones of individual es cell lines cultured in different ways , substantial variability exists between the neural potency of individual hipsc lines ( hu et al . , 2010 ) . for example , when the differentiation potential of 17 human es cell lines was compared ( osafune et al . , 2008 ) , some lines exhibited a marked propensity to differentiate into specific lineages , in some cases showing greater than 100-fold differences in lineage - specific gene expression . a third major issue in hipsc - based studies is the number of patients that are compared in each report and whether they are representative of the patient populations , particularly for complex diseases such as sczd and asds . moving forward , it is crucially important to recruit patients with well - defined clinical features as well as carefully matched healthy controls . although we believe that effect sizes will be large enough to measure differences between controls and patients , it remains to be seen whether it will be feasible to carry out synaptic assays of hipsc - derived neurons on a large scale . because the number of neuronal differentiations , hipsc lines and patients studied varies between published reports , included differentiations from one hipsc line from each of two individuals in their models of synaptic disorders ( kim et al . , 2011 ) , whereas we generated rtt hipscs from four patients , and our synaptic assays compared one or two hipsc lines from four control and three rtt patient lines ( marchetto et al . , 2010 ) . cheung et al . generated three hipsc lines from each of two female rtt patients , but analyzed hipsc - derived neurons from only one rtt patient ( cheung et al . random x - inactivation led to two mecp2-mutant lines and one isogenic control hipsc line to which they were compared . in studies of sczd , generated at least two hipsc lines from each of one control and two sczd patients carrying disc1 mutations , but did not perform any neuronal differentiations or synaptic assays ( chiang et al . although we generated one to three hipsc lines from each of five controls and four sczd patients , we generally used two or three neural progenitor cell lines derived from one hipsc line per individual in most neuronal assays ( brennand et al . , 2011 ) . to date , no studies of psychiatric disease have been able to compare synaptic maturation and/or function of neuronal differentiations from three hipsc lines for each individual . although these nested comparisons are extremely laborious , they will ultimately demonstrate the predominant source of variability in hipsc experiments . summary of hipsc - based studies of asd and sczd to produce meaningful data , each hipsc experiment should ideally compare multiple neuronal differentiations from multiple independent hipsc lines from multiple patients . however , owing to issues of cost and time , particularly when characterizing synaptic maturation and function , it is not yet feasible to complete such large experiments . hipsc - based studies will not replace mri , postmortem and genetic studies of psychiatric disorders . rather , we suggest that they are a new tool that will provide complementary approaches and insights for the study of a wide range of complex genetic disorders . an alternative approach for generating patient - specific neurons to study complex psychiatric disorders has recently been reported . starting from a pool of 19 candidate genes , researchers identified a combination of three factors ascl1 ( also known as mash1 ) , brn2 and myt1l that convert adult mouse fibroblasts into functional induced neurons ( ineurons ) in vitro ( vierbuchen et al . , 2010 ) . the process is incredibly rapid , generating neurons that are capable of spontaneous action potentials and with functional synapses within 14 days . the conversion is also relatively efficient , occurring at an estimated rate of 1.8% to 7.7% . direct reprogramming of human cells to ineurons has also recently been demonstrated using several different combinations of neurod ( pang et al . , 2011 ; yoo et al . , 2011 ) and/or the micrornas mir-9 ( yoo et al . , 2011 ) and/or mir-124 ( ambasudhan et al . , 2011 ; yoo et al . , 2011 ) . additionally , expression of ascl1 and two transcription factors that are crucial for dopaminergic differentiation , nurr1 and lmx1a , is sufficient to directly reprogram functional dopaminergic neurons from mouse and human fibroblasts ( caiazzo et al . , 2011 ) . the rapid experimental timeframe of ineuron generation and the potential to reprogram to specific neuronal subtypes make this an appealing experimental strategy for in vitro models of neurological disease . however , a key limitation of ineurons should be noted : unlike ipscs , ineurons do not have an inherent capacity for self - renewal . therefore , large numbers of patient - derived fibroblasts ( which have a finite capacity for replication ) will probably be necessary to enable experimental analyses of ineurons . two important additional issues must be considered when contemplating modeling psychiatric disorders in this manner . for example , if asds ultimately result from abnormal synaptic maturation , it is possible that direct reprogramming would bypass the developmental window in which the asd cellular phenotype can be observed in vitro . second , if aberrant ascl1 , brn2 or myt1l activity were to contribute to psychiatric disorders , the persistent overexpression required for reprogramming might be sufficient to mask cellular phenotypes in vitro . given that mutations disrupting myt1l expression and binding have been linked to sczd ( vrijenhoek et al . , 2008 ; riley et al . , 2010 ) , that brn2 is known to regulate expression of kcnn3 , a conductance calcium - activated potassium channel strongly implicated in sczd ( sun et al . , 2001 ) , and that ascl1 has been linked to parkinson s disease ( ide et al . , 2005 ) , it is not unreasonable to predict that overexpression of one or more of these key neuronal genes might affect the initiation or progression of a neurological disease . a reasonable course of action to address this issue would be to determine whether the cellular and molecular phenotypes observed in rtt- and sczd - hipsc - derived neurons are recapitulated following the reprogramming of fibroblasts from the same patients directly to ineurons . with this validation in place , we predict that many studies of psychiatric disorders using ineurons will begin . it is now possible to generate hipsc - derived neurons from the fibroblasts of patients with psychiatric disorders . defects in neuronal connectivity , synapse maturation and synaptic function have recently been reported in hipsc - derived neurons from patients with rtt and sczd . in addition , it might soon be possible to reprogram patient fibroblasts directly to ineurons as an alternative in vitro model of psychiatric disorders . by combining these new cell - based human models with better - characterized cohorts of psychiatric patients , scientists will be able to more easily study the relationship between clinical , cellular and molecular phenotypes . we predict that future in vitro studies will help to elucidate not only the precise cell types affected in these disorders but also the cellular and molecular defects that contribute to the diseases .

psychiatric disorders , including autism spectrum disorders and schizophrenia , are extremely heritable complex genetic neurodevelopmental disorders . it is now possible to directly reprogram fibroblasts from psychiatric patients into human induced pluripotent stem cells ( hipscs ) and subsequently differentiate these disorder - specific hipscs into neurons . this means that researchers can generate nearly limitless quantities of live human neurons with genetic backgrounds that are known to result in psychiatric disorders , without knowing which genes are interacting to produce the disease state in each patient . with these new human - cell - based models , scientists can investigate the precise cell types that are affected in these disorders and elucidate the cellular and molecular defects that contribute to disease initiation and progression . here , we present a short review of experiments using hipscs and other sophisticated in vitro approaches to study the pathways underlying psychiatric disorders .

Introduction Reprogramming patient fibroblasts to induced pluripotent stem cells Differentiating iPSCs to neurons Phenotypes of psychiatric disorders can be identified in hiPSC-derived neurons Limitations of hiPSC-based modeling of psychiatric disease Direct reprogramming of fibroblasts to neurons Conclusion

two of these disorders , autism spectrum disorders ( asds ) and schizophrenia ( sczd ) , combine to affect nearly 2% of the global population . asds are complex neurodevelopmental disorders characterized by severe impairments in social interactions , communication and behavior ( kanner , 1943 ; american psychiatric association , 2000 ) . sczd is characterized by three severe classes of symptoms : positive ( hallucinations and delusions ) , negative ( inability to speak , express emotion or find pleasure ) and cognitive ( deficits in attention , memory and planning ) ( american psychiatric association , 2000 ; carpenter and buchanan , 1994 ) ; it often leads to premature death from poverty , homelessness , substance abuse and poor health maintenance ( brown et al . , 2000 ) . of these two psychiatric disorders , twin studies estimate the heritability of asds to be greatest , as high as 90% ( ritvo et al . , 2001 ; sullivan et al . , 2003 ) . genome - wide association scans for asds and sczd suggest that common genetic loci influence susceptibility to many psychiatric disorders ( lewis et al . it is now believed that many rare but penetrant mutations contribute to asds and sczd , and that hundreds of common variants with mild effects are shared by sczd and bipolar disorder ( odonovan et al . it is generally very difficult to obtain live neurons from patients affected with asds or sczd to study . in addition , because most psychiatric disorders are polygenic , few relevant animal models have been developed . although transgenic mice demonstrate some abnormalities in behavior in specific experimental conditions ( clapcote et al . , 2009 ) , it is difficult to determine how well these mouse models recapitulate the human condition because it is difficult to assess complex phenotypes such as hallucinations and delusions in mice . to date , human studies of these psychiatric disorders have relied primarily on brain imaging of patients , postmortem studies of brains and genetic studies of patient lymphocytes . many notable insights have been made through these methods , but the molecular and cellular defects that contribute to disease initiation and progression in neurons have been difficult to elucidate . although they are poorly understood at the molecular and cellular levels , psychiatric disorders are now believed to be developmental disorders . therefore , human - cell - based models representing genetic backgrounds known to develop these neurological diseases might be the best method to identify the neuronal defects associated with them . in this commentary , we review recent studies that use sophisticated human - cell - based models to study the pathways underlying psychiatric disorders . we limit our discussion to approaches that focus on neuronal cell types , although non - neuronal cell - based approaches have also provided useful information ( box 1 ) . in 2006 , takahashi and yamanaka identified four factors ( oct3/4 , klf4 , sox2 and myc ) whose transient expression was sufficient to directly reprogram mouse adult somatic cells into an induced pluripotent stem cell ( ipsc ) state ( takahashi and yamanaka , 2006 ) . one year later , groups led by shinya yamanaka and james thomson ( takahashi et al . , 2007 ; yu et al . , 2007 ) demonstrated for the first time that it was also possible to reprogram human fibroblasts . ipscs can be generated from either mouse or human skin cells and are nearly identical to embryonic stem ( es ) cells with respect to gene expression , dna methylation and chromatin modifications . both mouse ipscs ( mipscs ) and human ipscs ( hipscs ) are believed to be capable of differentiating into every cell type found in the adult ( maherali et al . because hipscs can be derived from adult humans , after the development of disease , hipscs represent a potentially limitless source of human cells with which to study disease , even without knowing which genes are interacting to produce the disease state in an individual patient . there is a long history of non - neuronal cell - based studies comparing patients and controls , particularly in the study of sczd . the best established of these are studies of fibroblasts , lymphocytes and olfactory neural stem cells . fibroblasts : skin fibroblasts have been used as cellular models of sczd ( mahadik and mukherjee , 1996 ) . , 2006 ; yamamori et al . olfactory neural stem cells : matigian et al . compared patient - derived olfactory neural stem cells from nine patients with sczd and 14 controls , and identified sczd - associated differences in gene expression and protein levels in neurodevelopmental pathways , including cell migration and axon guidance ( matigian et al . it is important to note that genetic and epigenetic mutations can and do occur during the reprogramming process . , 2010 ) , and large copy number variations ( cnvs ) have also been associated with both the reprogramming and differentiation of hipscs ( laurent et al . additionally , among the 22 hipsc lines studied , an average of five protein - coding point mutations were identified ( gore et al . importantly , whereas hipscs do acquire genetic modifications in addition to epigenetic modifications , and although extensive genetic screening should become a standard procedure to ensure hipsc safety before clinical use , it is extremely unlikely that de novo mutations affect disease - specific hipsc lines in a meaningfully different way than control hipsc lines . although spontaneous mutations can occur during the reprogramming process , random mutations should not interfere with the ability to draw meaningful conclusions from hipsc - based studies of psychiatric disorders when studies include comparisons of neurons generated from multiple hipsc lines for each control and diseased patient . work with es cells first demonstrated methods to efficiently differentiate pluripotent stem cells into neurons ( tropepe et al . similarly , through the addition of various morphogens in vitro , es cells and ipscs can be directed to differentiate into regional identities including forebrain ( watanabe et al . , 2005 ) , midbrain / hindbrain ( kawasaki et al . , 2004 ) and spinal cord ( wichterle et al . , 2002 ; li et al . , 2005 owing to an excellent characterization of the transcription factors used to specify fate in embryonic mice , the best - characterized hipsc differentiation protocol is generally accepted to be one for motor neurons 2008 ) , although a highly efficient method for generating midbrain dopaminergic neurons was recently published ( chambers et al . much of the developmental potential that is present in vivo seems to be maintained in vitro . the choice of specific subtypes of neurons to be compared using hipsc - derived neurons warrants careful consideration because the brain regions and cell types affected differ between diseases . magnetic resonance imaging ( mri ) studies have observed structural differences in the brains of many patients affected by psychiatric disorders compared with those of the normal population . for example , patients with sczd often have decreased brain volume , particularly in the hippocampus , thalamus and cortex ( vita et al . notably , although these mri studies provide information regarding the brain regions that seem to be affected by these disorders , they do not reveal the cell types implicated in disease , but some insights have been provided through pharmacological studies . with respect to sczd , for example , dopamine receptor antagonists , and perhaps also glutamate receptor ( glur2/3 ) agonists , alleviate psychosis ( patil et al . conversely , when administered to healthy human subjects , dopamine agonists ( such as amphetamine ) and glutamate antagonists ( such as ketamine ) induce hallucinations and cognitive deficits similar to those associated with sczd ( krystal et al . , 1994 ) . although the specific cell subtypes that should ideally be examined to provide insight into these diseases remain uncertain , it is generally believed that cortical neurons are a relevant cell type for the study of many psychiatric disorders . rigorous validation of the neurons that are ultimately generated from hipscs is crucial for establishing the maturity and identity of the neuronal cultures that are generated in vitro . electrophysiological characterization should verify that hipsc neurons have membrane potentials , undergo induced action potentials and show evidence of spontaneous synaptic activity . for example , mature dopaminergic neurons express tyrosine hydroxylase ( th ) and aromatic l - amino acid decarboxylase ( aadc ) ( ang , 2006 ) , whereas mature cortical neurons express ctip2 , a transcriptional repressor that is required for the maturation of cortical axons , and otx1 , a transcription factor that is highly expressed in layer 5 cortical neurons ( frantz et al . it is important to note that researchers can not generate artificial brain structures in vitro , such as specific brain regions implicated in neurological disease for example , the prefrontal cortex for studies of asd or the hippocampus for studies of sczd . this strategy permits study of the innate neuron - specific deficits in psychiatric disease in a manner that is not confounded by environmental factors that typically plague studies of sczd , such as treatment history , drug and alcohol abuse or poverty . these reports have all been published within the last year ; each supports our contention that hipsc - derived neurons from patients with psychiatric disorders show significant aberrations compared with those of healthy controls . hipscs differentiate robustly to forebrain neurons and , by co - culturing hipsc forebrain neurons with hek293 t cells , kim et al . showed that hek293 t cells expressing wild - type nlgn3 and nlgn4 , but not those containing asd - associated mutations in these genes , can induce presynaptic differentiation in hipsc - derived neurons ( kim et al . , 2011 ) . although this work used only hipscs derived from the cells of healthy controls , these findings did establish that these cells are a viable model for the study of synaptic differentiation and that they function under normal and disorder - associated conditions . using rett syndrome ( rtt ) as an asd genetic model , we generated hipscs from patients with rtt and found early alterations in developing hipsc - derived neurons ( marchetto et al . . , 2010 ) . subsequently , a second group repeated the finding of reduced neuronal soma size in rtt - hipsc - derived neurons ( cheung et al . , 2011 ) together , these studies established that assays of neuronal size , synaptic structure and synaptic function can detect differences between control and disordered hipsc - derived neurons in vitro , demonstrating that neurodevelopmental diseases can be modeled in this manner . notably , rtt is usually associated with the complete loss of function of a single gene ( mecp2 ) and shows rapid disease progression within the first years of life ; the first studies of complex genetic forms of asd have not yet been reported . given that mouse models of rtt ( mecp2 null ) and mutated disc1 ( dndisc ) exist , these early studies allow a direct comparison between in vitro hipsc studies and in vivo animal experiments . it will be interesting to learn whether disc1-hipsc - derived neurons recapitulate the cellular phenotypes observed in dndisc mice as faithfully as rtt - hipsc - derived neurons recapitulated findings in mecp2-null mice . with these important proof - of - principle validations of hipsc models completed , future hipsc - based studies must begin to take full advantage of the ability of hipscs to model complex genetic cases of psychiatric disease , in which interactions between as - yet - unidentified genes result in the disease state . we recently reported the use of hipscs to model a complex genetic psychiatric disorder in which hipsc - derived neurons from four patients with sczd were compared with those from controls . , 2011 ) . nearly 25% of genes that showed altered expression compared with controls had been previously implicated in sczd , although the expression profiling data also suggested that a number of pathways not previously implicated in sczd might contribute to the disorder , such as notch signaling , cell adhesion and slit - robo - mediated axon guidance . we predict that , as future studies increase the number of sczd - patient - derived neurons , core pathways of genes that contribute to this disorder will be identified . hipsc - based studies show great potential use to model complex genetic disorders for which the genes responsible for producing the disease state differ between individuals . we hope that hipscs will permit direct evaluations of several hypotheses that are not easy to address in psychiatric disease research , such as : does severity of clinical outcome predict magnitude of cellular phenotype ? to discover the answers to these questions , substantially increased numbers of patient - specific hipsc - derived neurons must be generated . by moving forward with cohorts of patients for whom clinical outcome , pharmacological response , mri imaging and genotype data are available , hipsc - based models of psychiatric disorders will enable direct correlations of clinical , cellular and molecular phenotypes in psychiatric disease . lv - synp - htg , lentivirus expressing a fusion protein comprising histone 2b and green fluorescent protein , tva and elements of the synapsin ( syn ) promoter ; used to label neurons for microscopic analysis . the limitations of hipsc - based approaches for studying psychiatric disease are mainly ( 1 ) neuron - to - neuron variability , ( 2 ) hipsc - to - hipsc variability and ( 3 ) patient - to - patient variability . universally , hipsc - based studies of psychiatric disorders to date have all been performed on mixed populations of neuronal subtypes , which are generally described as predominantly glutamatergic with a significant fraction of gabanergic neurons and only rare dopaminergic neurons , as assessed by expression of vglut , gad67 and th , respectively . the use of heterogeneous neuronal populations increases neuron - to - neuron variability in experimental assays , a limitation that could be overcome by studying fluorescence - activated cell sorting ( facs)-purified populations of neurons of a defined identity . this variation might reflect differences in the integration of viruses used for gene delivery in reprogramming , variation in the extent of epigenetic reprogramming , spontaneous mutations resulting from reprogramming and expansion , differences in hipsc culture technique , and even differences in the cell type of origin . just as non - trivial differences in developmental potential exist among human es cell lines and within subclones of individual es cell lines cultured in different ways , substantial variability exists between the neural potency of individual hipsc lines ( hu et al . , 2010 ) . , 2008 ) , some lines exhibited a marked propensity to differentiate into specific lineages , in some cases showing greater than 100-fold differences in lineage - specific gene expression . a third major issue in hipsc - based studies is the number of patients that are compared in each report and whether they are representative of the patient populations , particularly for complex diseases such as sczd and asds . moving forward , it is crucially important to recruit patients with well - defined clinical features as well as carefully matched healthy controls . cheung et al . generated three hipsc lines from each of two female rtt patients , but analyzed hipsc - derived neurons from only one rtt patient ( cheung et al . random x - inactivation led to two mecp2-mutant lines and one isogenic control hipsc line to which they were compared . in studies of sczd , generated at least two hipsc lines from each of one control and two sczd patients carrying disc1 mutations , but did not perform any neuronal differentiations or synaptic assays ( chiang et al . although we generated one to three hipsc lines from each of five controls and four sczd patients , we generally used two or three neural progenitor cell lines derived from one hipsc line per individual in most neuronal assays ( brennand et al . to date , no studies of psychiatric disease have been able to compare synaptic maturation and/or function of neuronal differentiations from three hipsc lines for each individual . although these nested comparisons are extremely laborious , they will ultimately demonstrate the predominant source of variability in hipsc experiments . summary of hipsc - based studies of asd and sczd to produce meaningful data , each hipsc experiment should ideally compare multiple neuronal differentiations from multiple independent hipsc lines from multiple patients . however , owing to issues of cost and time , particularly when characterizing synaptic maturation and function , it is not yet feasible to complete such large experiments . hipsc - based studies will not replace mri , postmortem and genetic studies of psychiatric disorders . rather , we suggest that they are a new tool that will provide complementary approaches and insights for the study of a wide range of complex genetic disorders . an alternative approach for generating patient - specific neurons to study complex psychiatric disorders has recently been reported . starting from a pool of 19 candidate genes , researchers identified a combination of three factors ascl1 ( also known as mash1 ) , brn2 and myt1l that convert adult mouse fibroblasts into functional induced neurons ( ineurons ) in vitro ( vierbuchen et al . the process is incredibly rapid , generating neurons that are capable of spontaneous action potentials and with functional synapses within 14 days . direct reprogramming of human cells to ineurons has also recently been demonstrated using several different combinations of neurod ( pang et al . , 2011 ) and/or the micrornas mir-9 ( yoo et al . additionally , expression of ascl1 and two transcription factors that are crucial for dopaminergic differentiation , nurr1 and lmx1a , is sufficient to directly reprogram functional dopaminergic neurons from mouse and human fibroblasts ( caiazzo et al . the rapid experimental timeframe of ineuron generation and the potential to reprogram to specific neuronal subtypes make this an appealing experimental strategy for in vitro models of neurological disease . two important additional issues must be considered when contemplating modeling psychiatric disorders in this manner . for example , if asds ultimately result from abnormal synaptic maturation , it is possible that direct reprogramming would bypass the developmental window in which the asd cellular phenotype can be observed in vitro . second , if aberrant ascl1 , brn2 or myt1l activity were to contribute to psychiatric disorders , the persistent overexpression required for reprogramming might be sufficient to mask cellular phenotypes in vitro . , 2010 ) , that brn2 is known to regulate expression of kcnn3 , a conductance calcium - activated potassium channel strongly implicated in sczd ( sun et al . , 2001 ) , and that ascl1 has been linked to parkinson s disease ( ide et al . , 2005 ) , it is not unreasonable to predict that overexpression of one or more of these key neuronal genes might affect the initiation or progression of a neurological disease . a reasonable course of action to address this issue would be to determine whether the cellular and molecular phenotypes observed in rtt- and sczd - hipsc - derived neurons are recapitulated following the reprogramming of fibroblasts from the same patients directly to ineurons . with this validation in place , we predict that many studies of psychiatric disorders using ineurons will begin . it is now possible to generate hipsc - derived neurons from the fibroblasts of patients with psychiatric disorders . defects in neuronal connectivity , synapse maturation and synaptic function have recently been reported in hipsc - derived neurons from patients with rtt and sczd . in addition , it might soon be possible to reprogram patient fibroblasts directly to ineurons as an alternative in vitro model of psychiatric disorders . by combining these new cell - based human models with better - characterized cohorts of psychiatric patients , scientists will be able to more easily study the relationship between clinical , cellular and molecular phenotypes . we predict that future in vitro studies will help to elucidate not only the precise cell types affected in these disorders but also the cellular and molecular defects that contribute to the diseases .

aging is associated with a progressive immune deficiency rendering aged subjects susceptible to infections and displays impaired response to vaccination [ 13 ] . monocytes play an important role in defense against microbial pathogens and inflammation [ 4 , 5 ] . two distinct subpopulations of monocytes ( cd14cd16 and cd14cd16 ) were described by passlick et al . . these two subsets of monocytes are distinct with regard to certain cell surface molecules , migration properties , and their functions [ 712 ] . cd14cd16 subpopulation of monocytes expresses cd62l , cd64 , and ccr2 with low levels of cxcr1 . in contrast , cd14cd16 monocytes lack cd62l , cd64 , or ccr2 but express high levels of cx3cr1 . cd14 + cd16 + monocytes are considered pro - inflammatory monocytes since they produce increased levels of tnf- and low levels of anti - inflammatory il-10 in response to toll - like receptors ( tlr2 and tlr-4 agonists [ 1214 ] . renshaw et al . reported decreased expression of all tlrs in both splenic and activated peritoneal macrophages from aged mice as compared to young mice . furthermore , they demonstrated that macrophages from aged mice secreted significantly less il-6 and tnf- upon stimulation with tlr ligands . in the present study , we report for the first time numerical and functional analyses of four subpopulations of peripheral blood monocytes ( cd14cd16 , cd14cd16 , cd14cd16 , and cd14cd16 ) in young and aged humans , and demonstrate a functional defect in tlr1/2-induced il-6 and tnf- production in aged subjects , which appears to be associated with decreased expression of tlr1 and is likely mediated via impaired erk peripheral blood from 17 young ( age 2132 years ) and 17 aged ( age 6689 years ) subjects was used for the present study . these subjects are of middle - class social status , and aged subjects are living independent , healthy lives , and they were asked to stop taking all anti - oxidants ( which many aged subjects take on a regular basis ) for at least 1 week prior to obtaining blood samples to avoid their effects on the present investigation . the study is approved by institutional review board ( human ) of the university of california , irvine . expression of tlr1 and tlr2 on monocyte subsets was determined by flow cytometry using fitc- conjugated anti - cd14 , alexa fluor 647-labeled anti - cd16 antibodies ( bd biosciences , san diego , ca , usa ) and pe - labeled anti - tlr2 antibodies ( e - biosciences , san diego , ca , usa ) . briefly , 100 l of whole blood was incubated with 10 l of each of the anti - cd14 , anti - cd16 , and anti - tlr1 or anti - tlr2 , or isotype control antibodies for 15 min at room temperature in the dark . cells were then incubated in 2 ml of facs lysing solution ( becton - dickinson , san jose , ca , usa ) for 15 min to lyse red blood cells and washed twice with phosphate - buffered saline ( 1 pbs ) , and the cells were resuspended in 0.5 ml of 1% paraformaldehyde and analyzed by facscalibur . whole peripheral blood was activated with 5 g / ml of tlr1/tlr2 ligand pam3cys ( invivogen , san diego , ca , usa ) in the presence of brefeldin a for 4 h. after cd14/cd16 labeling , cells were washed and permeabilized using facs permeabilizing solution ( becton - dickinson ) , and subsequently incubated with the appropriate pe - conjugated anti - il-6 , anti - tnf or isotype control antibody ( becton - dickinson ) . for each sample , data from 5,000 cells were collected and analyzed using a facscalibur flow cytometer and cellquest software ( becton - dickinson ) . forward and side scatters were used to gate monocytes and to exclude cellular debris . during analysis , an electronic gate was placed on cd14cd14-positive cells , and the associated expression of cd16 and tlr1 or tlr2 or cytokines was determined by multicolor analysis . the proportion of various subsets of monocytes ( cd14cd16 , cd14cd16 , cd14cd16 , and cd14cd16 ) expressing tlr1 and tlr2 , or containing intracellular il-6 and tnf- , were analyzed for numbers and mean fluorescence channel number ( mfc # , indicator of the density of tlr receptors on cell surface ) to study tlr1/2-mediated signaling in monocyte subsets , whole peripheral blood was activated with 5 g / ml of pam3ys for 10 min . activation of erk was determined by intracellular phospho protein technique and flow cytometry using the protocol provided by the manufacturer of phophoflow kit ( bd biosciences ) . briefly , cells were fixed immediately following activation with pam3cys using bd phosflow lyse / fix buffer , permealized by adding 1ml of phosflow perm / wash buffer to 10 cells , and incubated for 10 min at room temperature with the appropriate pe - conjugated phopho erk1/2-specific anti bodies and fitc - conjugated anti - cd14 and alexa 687-labeled anti - cd16 monoclonal antibodies . fold change was calculated by dividing the median fluorescence intensity ( mfi ) of the stimulated sample ( mfi stim ) by that of the unstimulated sample ( mfi unstim ) . peripheral blood monocytes from young and aged subjects were purified by using magnetic nanobeads ( stem cell technologie , vancouver , british columbia , canada ) . monocytes were activated with 5 g / ml of pam3cys for 10 min , and lysed with lysing buffer ( tris hcl ph 7.4 20 mm , nacl 150 mm , triton x-100 1.2% , egta 1 mm , pmsf 1 mm , aprotinin 0.15 u / ml , leupeptin 10 g / ml , nan03 ) . protein concentration was determined by using a protein assay kit ( bio - rad ) . an equal amount of protein was subjected to sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transblotted onto polyvinylidene difluoride membrane ( millipore ) . after blocking , phosphorylation of erk was determined by incubating the blot with phospho specific erk antibodies ( cell signaling , danvers , ma , usa ) followed by incubation with an appropriate hrp - labeled secondary antibody . the specific protein was detected by enhanced chemiluminescence ( ecl ) ( ge - amersham , piscataway , nj , usa ) . the blots were analyzed using un - scan - it software to estimate the density of the bands in pixels . expression of tlr1 and tlr2 on monocyte subsets was determined by flow cytometry using fitc- conjugated anti - cd14 , alexa fluor 647-labeled anti - cd16 antibodies ( bd biosciences , san diego , ca , usa ) and pe - labeled anti - tlr2 antibodies ( e - biosciences , san diego , ca , usa ) . briefly , 100 l of whole blood was incubated with 10 l of each of the anti - cd14 , anti - cd16 , and anti - tlr1 or anti - tlr2 , or isotype control antibodies for 15 min at room temperature in the dark . cells were then incubated in 2 ml of facs lysing solution ( becton - dickinson , san jose , ca , usa ) for 15 min to lyse red blood cells and washed twice with phosphate - buffered saline ( 1 pbs ) , and the cells were resuspended in 0.5 ml of 1% paraformaldehyde and analyzed by facscalibur . whole peripheral blood was activated with 5 g / ml of tlr1/tlr2 ligand pam3cys ( invivogen , san diego , ca , usa ) in the presence of brefeldin a for 4 h. after cd14/cd16 labeling , cells were washed and permeabilized using facs permeabilizing solution ( becton - dickinson ) , and subsequently incubated with the appropriate pe - conjugated anti - il-6 , anti - tnf or isotype control antibody ( becton - dickinson ) . for each sample , data from 5,000 cells were collected and analyzed using a facscalibur flow cytometer and cellquest software ( becton - dickinson ) . forward and side scatters were used to gate monocytes and to exclude cellular debris . during analysis , an electronic gate was placed on cd14cd14-positive cells , and the associated expression of cd16 and tlr1 or tlr2 or cytokines was determined by multicolor analysis . the proportion of various subsets of monocytes ( cd14cd16 , cd14cd16 , cd14cd16 , and cd14cd16 ) expressing tlr1 and tlr2 , or containing intracellular il-6 and tnf- , were analyzed for numbers and mean fluorescence channel number ( mfc # , indicator of the density of tlr receptors on cell surface ) to study tlr1/2-mediated signaling in monocyte subsets , whole peripheral blood was activated with 5 g / ml of pam3ys for 10 min . activation of erk was determined by intracellular phospho protein technique and flow cytometry using the protocol provided by the manufacturer of phophoflow kit ( bd biosciences ) . briefly , cells were fixed immediately following activation with pam3cys using bd phosflow lyse / fix buffer , permealized by adding 1ml of phosflow perm / wash buffer to 10 cells , and incubated for 10 min at room temperature with the appropriate pe - conjugated phopho erk1/2-specific anti bodies and fitc - conjugated anti - cd14 and alexa 687-labeled anti - cd16 monoclonal antibodies . fold change was calculated by dividing the median fluorescence intensity ( mfi ) of the stimulated sample ( mfi stim ) by that of the unstimulated sample ( mfi unstim ) . peripheral blood monocytes from young and aged subjects were purified by using magnetic nanobeads ( stem cell technologie , vancouver , british columbia , canada ) . monocytes were activated with 5 g / ml of pam3cys for 10 min , and lysed with lysing buffer ( tris hcl ph 7.4 20 mm , nacl 150 mm , triton x-100 1.2% , egta 1 mm , pmsf 1 mm , aprotinin 0.15 u / ml , leupeptin 10 g / ml , nan03 ) . protein concentration was determined by using a protein assay kit ( bio - rad ) . an equal amount of protein was subjected to sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transblotted onto polyvinylidene difluoride membrane ( millipore ) . after blocking , phosphorylation of erk was determined by incubating the blot with phospho specific erk antibodies ( cell signaling , danvers , ma , usa ) followed by incubation with an appropriate hrp - labeled secondary antibody . the specific protein was detected by enhanced chemiluminescence ( ecl ) ( ge - amersham , piscataway , nj , usa ) . the blots were analyzed using un - scan - it software to estimate the density of the bands in pixels . four subsets of monocytes in healthy young and aged subjects monocyte subsets were analyzed in whole blood samples with fitc - anti - cd14 and alexa687-anti - cd16 monoclonal antibodies and isotype controls using facscalibur . figure 1a shows a representative cytoflourograph for the analysis of four subsets of monocytes . the order of frequency was cd14cd16 ( major subset ) > cd14cd16 > cd14cd16 > cd14cd16 . figures 1b and c show the cumulative data for the proportions and absolute numbers , respectively , for all four subsets of monocytes from 17 young subjects and 17 aged subjects . a significant increase in the proportions ( < 0.03 ) and number ( p < 0.03 ) of cd14cd16 , and cd14cd16 monocytes , and a significant decrease in the proportions ( p < 0.001 ) and numbers ( p < 0.04 ) of cd14cd16 was observed in aged subjects . no significant difference was observed between young and aged subjects in the proportions or numbers of cd14cd16 subset . a representative cytofluorograph of four subsets of monocytes based upon expression of cd14 and cd16 antigens from a young subjects . b cumulative data for the proportions and c numbers of four subsets of monocytes from 17 young and 17 aged subjects . data are expressed as mean sd . * p < 0.05 , * * p < 0.01 monocyte subsets in young and aged humans . a representative cytofluorograph of four subsets of monocytes based upon expression of cd14 and cd16 antigens from a young subjects . b cumulative data for the proportions and c numbers of four subsets of monocytes from 17 young and 17 aged subjects . * p < 0.05 , * * p < 0.01 tlr 1/2 agonist - induced cytokine production is reduced in aging aging is associated with increased levels of circulating il-6 , and tnf- , and since we observed increased numbers of monocyte subsets , we reasoned that monocyte subsets might be the major source of increased il-6 and tnf- in aging . therefore , we examined il-6 and tnf- synthesis in all four subsets of monocytes following stimulation by pam3cys . whole blood white blood cells were stimulated with pam3cys in the presence of brefeldin a to inhibit secretion of cytokines and intracellular tnf- , and il-6 synthesizing cells were measured with multiple monoclonal antibodies defining monocyte subsets and antibodies against il-6 and tnf- by multicolor flow cytometry using facscalibur . figure 1a shows a representative cytofluorgraphs of il-6 and tnf- producing subsets of monocytes in a healthy young subject . 2b ) in both subsets of cd14 monocytes ( cd14cd16 and cd14cd16 ) , as compared to two subsets of cd14 monocytes ( cd14cd16 and cd14cd16 ) in young subjects . this would be in agreement with reported increased tnf- production by cd14cd16 monocytes ; however , we did not observe any difference in il-6 among four subsets of monocytes in young subjects ( fig . furthermore , all four subpopulations of monocytes displayed significantly reduced ( p < 0.01p < 0.05 ) il-6 production in aged ( fig . 2c ) as compared to young subjects . however , the extent of decreased il-6 production by cd14 cd16 and cd14cd16 monocyte subset was significantly greater ( p < 0.01 ) than decreased il-6 production by two subsets of cd16 monocytes ( p < 0.05 ) . tnf- production was significantly decreased ( p < 0.05 ) in all but cd14 cd16 monocyte subsets in aged subjects as compared to controls ( fig . a representative cytoflourograph of il-6 and tnf- production by four subsets of monocytes from young subjects . b cumulative data for il-6 and c for tnf- from young and aged subjects cytokine production by four subsets of monocytes in young and aged subjects . a representative cytoflourograph of il-6 and tnf- production by four subsets of monocytes from young subjects . b cumulative data for il-6 and c for tnf- from young and aged subjects tlr 1 expression is reduced in aged humans since pam3cys stimulates both tlr 1 and tlr 2 , we examined whether decreased cytokine production in monocyte subsets is due to altered expression of tlr1 and/or tlr-2 . no significant difference was observed in the proportions of four subsets of monocytes expressing tlr 2 , or the density of tlr2 between young and aged subjects ( data not shown ) . figure 3a shows a representative cytoflourograph of tlr1 expression in four subsets of monocytes in a young subject . a lower proportion of cd14 cd16 and cd14 cd16 monocyte subsets expressed tlr1 as compared to cd14cd16 and cd14cd16 in young subjects . figure 3b shows cumulative data for the proportions of cell expressing tlr1 , and fig . 3c shows cumulative data of the density of tlr1 ( as determined by mean fluorescence channel numbers ( mfc # ) ) in young and aged subjects . a significantly reduced expression of tlr 1 was observed in cd14 cd16 ( p < 0.03 ) and cd14 cd16 ( p < similarly , a significantly reduced density of tlr1 was observed in cd14 cd16 and cd14 cd16 monocytes ( p < 0.05 ) . therefore , it is likely that decreased cytokine production in aging is due to an impaired expression of tlr 1 . each subset of monocytes was gated , and proportions of each subset with tlr1 expression were expressed as percent of a particular subset of monocyte . a representative cytofluorograph from a young subject , b cumulative data for the proportions of tlr-1 + subsets , and c the fluoresecence intensity of tlr1 ( mean fluorescence channel number ( mfc # ) ) of tlr-1 in each subset of monocytes from young and aged subjects expression of tlr1 on four subsets of monocytes in young and aged subjects . each subset of monocytes was gated , and proportions of each subset with tlr1 expression were expressed as percent of a particular subset of monocyte . a representative cytofluorograph from a young subject , b cumulative data for the proportions of tlr-1 + subsets , and c the fluoresecence intensity of tlr1 ( mean fluorescence channel number ( mfc # ) ) of tlr-1 in each subset of monocytes from young and aged subjects phosphorylation of erk1/2 is reduced in aged monocytes protein was purified from isolated monocytes from young and aged subjects with or without stimulation with pam3cys and analyzed by western blotting using native and phosp erk1/2 monoclonal antibodies , and quantified by densitometer we then examined phosphorylation of erk1/2 in all four subsets of monocytes from young and aged subjects with native and phopho erk1/2 antibody and multicolor flow cytometry using facscalibur . figure 4b shows a significantly decreased ( p < 0.05p < 0.005 ) phosphorylation of erk1/2 in cd14cd16 , cd14cd16 , and cd14cd16 subsets of monocytes from aged subjects as compared to young subjects . furthermore , significantly greater reduction ( p < 0.005 ) of erk1/2 phosphorylation was observed in cd14cd16 as compared to cd14cd16 ( p < 0.02 ) and cd14cd16 ( p < 0.05 ) . 4phosphorylation of erk1/2 in monocyte subsets from young and aged subjects . a representative western blot of erk1/2 phosphorylation and densitometric analysis following pam3csk4 stimulation of total monocytes from young and aged subjects . b cumulative data of phosphor erk1/2 in four subsets of monocytes by flow cytometry phosphorylation of erk1/2 in monocyte subsets from young and aged subjects . a representative western blot of erk1/2 phosphorylation and densitometric analysis following pam3csk4 stimulation of total monocytes from young and aged subjects . in this study , we have demonstrated alterations in the proportions and numbers , tlr1 expression , cytokine production , and phosphorylation of erk1/2 in subsets of monocytes . these impairments in number and functions are more consistent in cd16 + monocytes and did not correlate with cd14 or cd14 , the original subdivision of monocytes into two subsets . therefore , this study highlights the need of further subdivision of monocytes in four subsets . aging is associated with the progressive decline in immune functions and increased susceptibility to microbial infections [ 13 ] . although alterations in adaptive responses are well established , innate immune responses in human aging are not as well understood . tlrs are pathogen recognition receptors that recognize shared structural components from bacteria , fungi , and viruses [ 1619 ] . eleven tlrs in humans and 13 tlrs in mice have been described , and ligands for nine human tlrs are known . monocytes / macrophages play an important role in defense against microbes and express several tlrs . initially , two subpopulations of monocytes were described in human monocytes - based expression of cd14 and cd16 molecules , their migratory properties , and cytokine production [ 612 ] . kim et al . described three subpopulations of monocytes in simian immunodeficiency virus infection . in humans , cd14cd16 monocytes , by virtue of producing higher levels of proinflammatory cytokines ( il-6 and tnf- ) as compared to cd14cd16 monocytes , were termed proinflammatory monocytes . in this study , we analyzed four subpopulations of human monocytes , and we observed that two subpopulations of cd14 monocytes ( cd14cd16 and cd14cd16 ) contained increased levels of tnf- ; however , we did not observe any significant difference for il-6 among any of the four subsets of monocytes . a discrepancy for il-6 between our results and those of others may be due to difference in the assays used . in our study , we analyzed intracellular cytokines ( synthesized ) in four subsets , whereas others have reported secreted cytokines in the culture supernatants of two purified subsets of monocytes [ 10 , 12 ] . . observed no difference in tnf- mrna between cd14cd16 and cd14cd16 monocytes ( only two monocyte subpopulations were analyzed ) ; however , using intracellular cytokine staining , they observed higher levels of tnf- in cd14cd16 subsets . in the present study , we also analyzed four subsets of human monocytes and their functions in aged humans . proportions and numbers of cd14cd16 and cd14cd16 monocytes were increased in aged subjects , whereas proportions and numbers of cd14cd16 monocytes were decreased in aged as compared to young subjects . there was no correlation between the changes in proportions and numbers of monocyte subsets and tlr1 expression , cytokine production , or erk1/2 phophorylation ( data not shown ) . aging is associated with increased levels of proinflammatory cytokines il-6 and tnf- [ 21 , 22 ] . since we observed an increased number of monocyte subsets in aging , we expected that monocytes may be a source of increased il-6 and tnf- in aging . however , our data show that pam3cys ( a ligand for tlr1 and tlr2)-induced il-6 and tnf--producing cells were significantly reduced in aged monocyte subsets as compared to that of young subjects . this is in agreement with published data for total or two subsets of monocytes in aged subjects in response to tlr2 and tlr4 activation [ 23 , 24 ] . furthermore , subpopulations of cd14 monocytes ( cd14cd16 ) had significantly fewer changes for il-6 or no change in tnf- production in aged as compared to other monocyte subsets . several investigators have reported decreased il-6 and tnf- production by splenic and activated peritoneal macrophages from aged mice activated by the ligands of tlrs as compared to young mice . however , no significant change was observed in the expression of tlr2 ad tlr4 in aged mice as compared to young mice . van duin and colleagues also reported reduced il-6 and tnf- production by total monocyte population from aged humans following tlr1/2 stimulation . our study may have an advantage and perhaps reflect more in vivo events , since experiments were performed in whole blood and gated subpopulations of monocytes rather than isolated monocytes , as in the study of van duin et al . , which may result in a loss of a preferential subset of monocytes . in the present study , we have measured the number of various monocyte subsets producing cytokines in whole blood samples rather than cytokines produced in culture supernatants . taken together , it appears that cells other than monocyte subsets are the source of increased levels of il-6 and tnf- in aged humans . recently , we have reported that dendritic cells from aged subjects secrete increased amounts of proinflammatory cytokines , and may be the cell type contributing to the increased circulating levels of proinflammatory cytokines in aged subjects . a decreased cytokine production by monocyte subsets may result from decreased expression of tlr1 and/or tlr2 . in aging mice , renshaw et al . reported decreased expression of all tlrs mrna as compared to younger mice . however , we have observed that the proportion of cells expressing tlr2 , as well as the density of tlr2 in all four subsets of monocytes from aged subjects , was comparable to that of young subjects ( data not shown ) . in contrast , a significantly decreased expression of tlr1 was observed on cd14cd16 and cd14cd16 monocyte subsets from aged subjects . interestingly , expression of tlr1 and tlr2 on various subsets of monocytes from young subjects was different . tlr2 expression was significantly higher in cd14cd16 and cd14cd16 as compared to cd14cd16 and cd14cd16 monocytes ( data not shown ) . in contrast , tlr1 expression was predominantly on cd14cd16 and cd14cd16 as compared to cd14cd16 and cd14cd16 monocytes . van duin et al . also reported normal tlr2 expression and decreased tlr1 expression in total monocyte population in aged humans . tlr agonists induce proinflammatory cytokine secretion via activation of erk map kinase signaling pathway [ 2729 ] . kopp et al . demonstrated that the activation of adipocytes with pam3cys resulted in the secretion of il-6 , which was dependent upon phosphorylation of erk . reported that activation of dcs via tlr4 and tlr5 resulted in the phosphorylation of p38 and c - jun n - terminal kinase 1/2 ( jnk1/2 ) , whereas activation of dcs via pam3cys ( tlr2 ligand ) resulted in a sustained phosphorylation of erk1/2 . dillon and colleagues reported that the activation of dcs by zymosan , a stimulus for tlr2 , resulted in the phosphorylation of erk1/2 . in both aged mice and aged humans , macrophages / monocytes - decreased basal levels of p38 mapk have also been reported [ 2325 ] . however , no study on erk1/2 activation in aged monocytes has been reported . in this study , we observed that pam3cys - induced phosphorylation of erk1/2 was decreased in all subsets of monocytes in aging , suggesting that decreased il-6 and tnf- synthesis in aged humans appears to be in part due to impaired tlr1/2 signaling mediated via erk1/2 pathway . since tlr1/2 is a heterodimer , which is stimulated by pan3cys , it remains to be determined whether the signaling defect is via tlr1 or both tlr1 and tlr2 . in the present study and those reported by van duin et al . , tlr-2 expression is comparable between young and aged subjects ; therefore , it is likely that the defect in monocyte subsets in aged humans lies in tlr1-mediated signaling . tlr1/2 senses pathogen recognition molecules shared by a range of pathogens including mycobacterium tuberculosis , borrelia burgdorferi . this may also explain increased susceptibility to a number of microbes during aging , as well as poor response to vaccination . this would be consistent with impaired response to ospa vaccine ( ospa is a ligand for tlr1/2 ) against lyme disease in the elderly , which was associated with tlr1 defect but preserved tlr2 response . in summary , human aging is associated with alterations in the numbers of certain subpopulations of monocytes , and impaired secretion of il-6 and tnf- , which appear to be due to an impaired expression of tlr1 and signaling via erk1/2 pathway . furthermore , in aged subjects , greater and more significant changes were observed in cd16 + ( independent of level of cd14 expression , cd14 or cd14 ) monocytes as compared to cd16 monocyte subsets ( regardless of levels of cd14 expression ) . therefore , it is important to analyze monocytes into four subsets . these defects in monocytes may play a role in increased susceptibility to microbial pathogens and poor response to vaccines in aged humans .

aging is associated with increased susceptibility to microbial infections , and monocytes play an important role in microbial defense . in this study , we have identified and compared four subpopulations of monocytes ( cd14++(high)cd16 , cd14+(low)cd16 , cd14++(high)cd16 + , and cd14+(low)cd16 + ) in the peripheral blood of young and aged subjects with regard to their numbers , cytokine production , tlr expression , and phosphorylation of erk1/2 in response to pam3cys a tlr-1/2 ligand . proportions and numbers of cd14++(high)cd16 + and cd14+(low)cd16 + monocytes were significantly increased , whereas proportions of cd14+(low)cd16 monocytes were decreased in aged subjects as compared to young subjects . in aged subjects , il-6 production by all four subsets of monocytes was significantly decreased , whereas tnf- production was decreased in monocyte subsets , except the cd14+(low)cd16 subset . a significantly reduced expression of tlr1 was observed in cd14++(high)cd16 + and cd14+(low)cd16 + monocyte subsets in aged subjects . furthermore , following pam3cys stimulation , erk1/2 phosphorylation was significantly lower in cd14+(low)cd16 + , cd14++(high)cd16 + , and cd14+(low)cd16 subsets of monocytes from aged subjects . this is the first study of four subpopulations of monocytes in aging , which demonstrates that their functions are differentially impaired with regard to the production of cytokines , expression of tlr , and signaling via the erk mapk pathway . finally , changes in the number of monocyte subsets , and impairment of tlr1 expression , tnf- production , and ek1/2 phosphorylation was more consistent in cd16 + monocyte subsets regardless of expression of cd14high or cd14+low , therefore highlighting the significance of further subdivision of monocytes into four subpopulations .

Introduction Materials and Methods Determination of Monocyte Subsets and the Expression of TLR 1 and TLR2 Measurements of Intracellular IL-6 and TNF- Activation of ERK Kinases Results Discussion

aging is associated with a progressive immune deficiency rendering aged subjects susceptible to infections and displays impaired response to vaccination [ 13 ] . monocytes play an important role in defense against microbial pathogens and inflammation [ 4 , 5 ] . two distinct subpopulations of monocytes ( cd14cd16 and cd14cd16 ) were described by passlick et al . these two subsets of monocytes are distinct with regard to certain cell surface molecules , migration properties , and their functions [ 712 ] . cd14 + cd16 + monocytes are considered pro - inflammatory monocytes since they produce increased levels of tnf- and low levels of anti - inflammatory il-10 in response to toll - like receptors ( tlr2 and tlr-4 agonists [ 1214 ] . reported decreased expression of all tlrs in both splenic and activated peritoneal macrophages from aged mice as compared to young mice . in the present study , we report for the first time numerical and functional analyses of four subpopulations of peripheral blood monocytes ( cd14cd16 , cd14cd16 , cd14cd16 , and cd14cd16 ) in young and aged humans , and demonstrate a functional defect in tlr1/2-induced il-6 and tnf- production in aged subjects , which appears to be associated with decreased expression of tlr1 and is likely mediated via impaired erk peripheral blood from 17 young ( age 2132 years ) and 17 aged ( age 6689 years ) subjects was used for the present study . whole peripheral blood was activated with 5 g / ml of tlr1/tlr2 ligand pam3cys ( invivogen , san diego , ca , usa ) in the presence of brefeldin a for 4 h. after cd14/cd16 labeling , cells were washed and permeabilized using facs permeabilizing solution ( becton - dickinson ) , and subsequently incubated with the appropriate pe - conjugated anti - il-6 , anti - tnf or isotype control antibody ( becton - dickinson ) . the proportion of various subsets of monocytes ( cd14cd16 , cd14cd16 , cd14cd16 , and cd14cd16 ) expressing tlr1 and tlr2 , or containing intracellular il-6 and tnf- , were analyzed for numbers and mean fluorescence channel number ( mfc # , indicator of the density of tlr receptors on cell surface ) to study tlr1/2-mediated signaling in monocyte subsets , whole peripheral blood was activated with 5 g / ml of pam3ys for 10 min . peripheral blood monocytes from young and aged subjects were purified by using magnetic nanobeads ( stem cell technologie , vancouver , british columbia , canada ) . whole peripheral blood was activated with 5 g / ml of tlr1/tlr2 ligand pam3cys ( invivogen , san diego , ca , usa ) in the presence of brefeldin a for 4 h. after cd14/cd16 labeling , cells were washed and permeabilized using facs permeabilizing solution ( becton - dickinson ) , and subsequently incubated with the appropriate pe - conjugated anti - il-6 , anti - tnf or isotype control antibody ( becton - dickinson ) . the proportion of various subsets of monocytes ( cd14cd16 , cd14cd16 , cd14cd16 , and cd14cd16 ) expressing tlr1 and tlr2 , or containing intracellular il-6 and tnf- , were analyzed for numbers and mean fluorescence channel number ( mfc # , indicator of the density of tlr receptors on cell surface ) to study tlr1/2-mediated signaling in monocyte subsets , whole peripheral blood was activated with 5 g / ml of pam3ys for 10 min . peripheral blood monocytes from young and aged subjects were purified by using magnetic nanobeads ( stem cell technologie , vancouver , british columbia , canada ) . four subsets of monocytes in healthy young and aged subjects monocyte subsets were analyzed in whole blood samples with fitc - anti - cd14 and alexa687-anti - cd16 monoclonal antibodies and isotype controls using facscalibur . figure 1a shows a representative cytoflourograph for the analysis of four subsets of monocytes . figures 1b and c show the cumulative data for the proportions and absolute numbers , respectively , for all four subsets of monocytes from 17 young subjects and 17 aged subjects . a significant increase in the proportions ( < 0.03 ) and number ( p < 0.03 ) of cd14cd16 , and cd14cd16 monocytes , and a significant decrease in the proportions ( p < 0.001 ) and numbers ( p < 0.04 ) of cd14cd16 was observed in aged subjects . no significant difference was observed between young and aged subjects in the proportions or numbers of cd14cd16 subset . a representative cytofluorograph of four subsets of monocytes based upon expression of cd14 and cd16 antigens from a young subjects . b cumulative data for the proportions and c numbers of four subsets of monocytes from 17 young and 17 aged subjects . * p < 0.05 , * * p < 0.01 monocyte subsets in young and aged humans . a representative cytofluorograph of four subsets of monocytes based upon expression of cd14 and cd16 antigens from a young subjects . b cumulative data for the proportions and c numbers of four subsets of monocytes from 17 young and 17 aged subjects . * p < 0.05 , * * p < 0.01 tlr 1/2 agonist - induced cytokine production is reduced in aging aging is associated with increased levels of circulating il-6 , and tnf- , and since we observed increased numbers of monocyte subsets , we reasoned that monocyte subsets might be the major source of increased il-6 and tnf- in aging . therefore , we examined il-6 and tnf- synthesis in all four subsets of monocytes following stimulation by pam3cys . whole blood white blood cells were stimulated with pam3cys in the presence of brefeldin a to inhibit secretion of cytokines and intracellular tnf- , and il-6 synthesizing cells were measured with multiple monoclonal antibodies defining monocyte subsets and antibodies against il-6 and tnf- by multicolor flow cytometry using facscalibur . figure 1a shows a representative cytofluorgraphs of il-6 and tnf- producing subsets of monocytes in a healthy young subject . 2b ) in both subsets of cd14 monocytes ( cd14cd16 and cd14cd16 ) , as compared to two subsets of cd14 monocytes ( cd14cd16 and cd14cd16 ) in young subjects . this would be in agreement with reported increased tnf- production by cd14cd16 monocytes ; however , we did not observe any difference in il-6 among four subsets of monocytes in young subjects ( fig . furthermore , all four subpopulations of monocytes displayed significantly reduced ( p < 0.01p < 0.05 ) il-6 production in aged ( fig . 2c ) as compared to young subjects . however , the extent of decreased il-6 production by cd14 cd16 and cd14cd16 monocyte subset was significantly greater ( p < 0.01 ) than decreased il-6 production by two subsets of cd16 monocytes ( p < 0.05 ) . tnf- production was significantly decreased ( p < 0.05 ) in all but cd14 cd16 monocyte subsets in aged subjects as compared to controls ( fig . a representative cytoflourograph of il-6 and tnf- production by four subsets of monocytes from young subjects . b cumulative data for il-6 and c for tnf- from young and aged subjects cytokine production by four subsets of monocytes in young and aged subjects . a representative cytoflourograph of il-6 and tnf- production by four subsets of monocytes from young subjects . b cumulative data for il-6 and c for tnf- from young and aged subjects tlr 1 expression is reduced in aged humans since pam3cys stimulates both tlr 1 and tlr 2 , we examined whether decreased cytokine production in monocyte subsets is due to altered expression of tlr1 and/or tlr-2 . no significant difference was observed in the proportions of four subsets of monocytes expressing tlr 2 , or the density of tlr2 between young and aged subjects ( data not shown ) . figure 3a shows a representative cytoflourograph of tlr1 expression in four subsets of monocytes in a young subject . a lower proportion of cd14 cd16 and cd14 cd16 monocyte subsets expressed tlr1 as compared to cd14cd16 and cd14cd16 in young subjects . 3c shows cumulative data of the density of tlr1 ( as determined by mean fluorescence channel numbers ( mfc # ) ) in young and aged subjects . a significantly reduced expression of tlr 1 was observed in cd14 cd16 ( p < 0.03 ) and cd14 cd16 ( p < similarly , a significantly reduced density of tlr1 was observed in cd14 cd16 and cd14 cd16 monocytes ( p < 0.05 ) . therefore , it is likely that decreased cytokine production in aging is due to an impaired expression of tlr 1 . each subset of monocytes was gated , and proportions of each subset with tlr1 expression were expressed as percent of a particular subset of monocyte . a representative cytofluorograph from a young subject , b cumulative data for the proportions of tlr-1 + subsets , and c the fluoresecence intensity of tlr1 ( mean fluorescence channel number ( mfc # ) ) of tlr-1 in each subset of monocytes from young and aged subjects expression of tlr1 on four subsets of monocytes in young and aged subjects . each subset of monocytes was gated , and proportions of each subset with tlr1 expression were expressed as percent of a particular subset of monocyte . a representative cytofluorograph from a young subject , b cumulative data for the proportions of tlr-1 + subsets , and c the fluoresecence intensity of tlr1 ( mean fluorescence channel number ( mfc # ) ) of tlr-1 in each subset of monocytes from young and aged subjects phosphorylation of erk1/2 is reduced in aged monocytes protein was purified from isolated monocytes from young and aged subjects with or without stimulation with pam3cys and analyzed by western blotting using native and phosp erk1/2 monoclonal antibodies , and quantified by densitometer we then examined phosphorylation of erk1/2 in all four subsets of monocytes from young and aged subjects with native and phopho erk1/2 antibody and multicolor flow cytometry using facscalibur . figure 4b shows a significantly decreased ( p < 0.05p < 0.005 ) phosphorylation of erk1/2 in cd14cd16 , cd14cd16 , and cd14cd16 subsets of monocytes from aged subjects as compared to young subjects . furthermore , significantly greater reduction ( p < 0.005 ) of erk1/2 phosphorylation was observed in cd14cd16 as compared to cd14cd16 ( p < 0.02 ) and cd14cd16 ( p < 0.05 ) . 4phosphorylation of erk1/2 in monocyte subsets from young and aged subjects . a representative western blot of erk1/2 phosphorylation and densitometric analysis following pam3csk4 stimulation of total monocytes from young and aged subjects . b cumulative data of phosphor erk1/2 in four subsets of monocytes by flow cytometry phosphorylation of erk1/2 in monocyte subsets from young and aged subjects . a representative western blot of erk1/2 phosphorylation and densitometric analysis following pam3csk4 stimulation of total monocytes from young and aged subjects . in this study , we have demonstrated alterations in the proportions and numbers , tlr1 expression , cytokine production , and phosphorylation of erk1/2 in subsets of monocytes . these impairments in number and functions are more consistent in cd16 + monocytes and did not correlate with cd14 or cd14 , the original subdivision of monocytes into two subsets . therefore , this study highlights the need of further subdivision of monocytes in four subsets . aging is associated with the progressive decline in immune functions and increased susceptibility to microbial infections [ 13 ] . monocytes / macrophages play an important role in defense against microbes and express several tlrs . initially , two subpopulations of monocytes were described in human monocytes - based expression of cd14 and cd16 molecules , their migratory properties , and cytokine production [ 612 ] . in this study , we analyzed four subpopulations of human monocytes , and we observed that two subpopulations of cd14 monocytes ( cd14cd16 and cd14cd16 ) contained increased levels of tnf- ; however , we did not observe any significant difference for il-6 among any of the four subsets of monocytes . in our study , we analyzed intracellular cytokines ( synthesized ) in four subsets , whereas others have reported secreted cytokines in the culture supernatants of two purified subsets of monocytes [ 10 , 12 ] . in the present study , we also analyzed four subsets of human monocytes and their functions in aged humans . proportions and numbers of cd14cd16 and cd14cd16 monocytes were increased in aged subjects , whereas proportions and numbers of cd14cd16 monocytes were decreased in aged as compared to young subjects . there was no correlation between the changes in proportions and numbers of monocyte subsets and tlr1 expression , cytokine production , or erk1/2 phophorylation ( data not shown ) . aging is associated with increased levels of proinflammatory cytokines il-6 and tnf- [ 21 , 22 ] . since we observed an increased number of monocyte subsets in aging , we expected that monocytes may be a source of increased il-6 and tnf- in aging . however , our data show that pam3cys ( a ligand for tlr1 and tlr2)-induced il-6 and tnf--producing cells were significantly reduced in aged monocyte subsets as compared to that of young subjects . this is in agreement with published data for total or two subsets of monocytes in aged subjects in response to tlr2 and tlr4 activation [ 23 , 24 ] . furthermore , subpopulations of cd14 monocytes ( cd14cd16 ) had significantly fewer changes for il-6 or no change in tnf- production in aged as compared to other monocyte subsets . several investigators have reported decreased il-6 and tnf- production by splenic and activated peritoneal macrophages from aged mice activated by the ligands of tlrs as compared to young mice . however , no significant change was observed in the expression of tlr2 ad tlr4 in aged mice as compared to young mice . our study may have an advantage and perhaps reflect more in vivo events , since experiments were performed in whole blood and gated subpopulations of monocytes rather than isolated monocytes , as in the study of van duin et al . in the present study , we have measured the number of various monocyte subsets producing cytokines in whole blood samples rather than cytokines produced in culture supernatants . recently , we have reported that dendritic cells from aged subjects secrete increased amounts of proinflammatory cytokines , and may be the cell type contributing to the increased circulating levels of proinflammatory cytokines in aged subjects . a decreased cytokine production by monocyte subsets may result from decreased expression of tlr1 and/or tlr2 . however , we have observed that the proportion of cells expressing tlr2 , as well as the density of tlr2 in all four subsets of monocytes from aged subjects , was comparable to that of young subjects ( data not shown ) . in contrast , a significantly decreased expression of tlr1 was observed on cd14cd16 and cd14cd16 monocyte subsets from aged subjects . interestingly , expression of tlr1 and tlr2 on various subsets of monocytes from young subjects was different . tlr2 expression was significantly higher in cd14cd16 and cd14cd16 as compared to cd14cd16 and cd14cd16 monocytes ( data not shown ) . in contrast , tlr1 expression was predominantly on cd14cd16 and cd14cd16 as compared to cd14cd16 and cd14cd16 monocytes . reported that activation of dcs via tlr4 and tlr5 resulted in the phosphorylation of p38 and c - jun n - terminal kinase 1/2 ( jnk1/2 ) , whereas activation of dcs via pam3cys ( tlr2 ligand ) resulted in a sustained phosphorylation of erk1/2 . in this study , we observed that pam3cys - induced phosphorylation of erk1/2 was decreased in all subsets of monocytes in aging , suggesting that decreased il-6 and tnf- synthesis in aged humans appears to be in part due to impaired tlr1/2 signaling mediated via erk1/2 pathway . , tlr-2 expression is comparable between young and aged subjects ; therefore , it is likely that the defect in monocyte subsets in aged humans lies in tlr1-mediated signaling . this may also explain increased susceptibility to a number of microbes during aging , as well as poor response to vaccination . this would be consistent with impaired response to ospa vaccine ( ospa is a ligand for tlr1/2 ) against lyme disease in the elderly , which was associated with tlr1 defect but preserved tlr2 response . in summary , human aging is associated with alterations in the numbers of certain subpopulations of monocytes , and impaired secretion of il-6 and tnf- , which appear to be due to an impaired expression of tlr1 and signaling via erk1/2 pathway . furthermore , in aged subjects , greater and more significant changes were observed in cd16 + ( independent of level of cd14 expression , cd14 or cd14 ) monocytes as compared to cd16 monocyte subsets ( regardless of levels of cd14 expression ) . therefore , it is important to analyze monocytes into four subsets . these defects in monocytes may play a role in increased susceptibility to microbial pathogens and poor response to vaccines in aged humans .

from the earliest days of perutz crystal structures and the celebrated two - state ligation model of monod , changeux and wyman , hemoglobin ( hb ) has served as a paradigm for allosteric systems . our understanding of the complex mechanism whereby the high - affinity r structure shifts to the low - affinity t structure continues to advance through new experimental and computational developments . this shift accounts for the cooperative release of o2 from the four subunits in the hb tetramer , which is essential to efficient delivery of o2 from the lungs to tissues . hb is a tetrameric protein , built as a dimer of dimers , each containing two different , though structurally similar chains , and . superposition of crystal structures shows that the r t shift mainly involves a rotation of the 11 dimer against 22 , by 15. the main intersubunit contact changes are at the 12 interface , specifically contacts between opposing c helices and fg corners of the two chains ( figure 1 ) . these have been called the hinge and switch contacts , the former involving a reorientation and the latter a shifted interdigitation of the contacting residues . we note that a range of r structures are seen in different crystal structures of ligated hb , and the solution structure is a dynamical ensemble of these structures . however , the critical quaternary contacts are unaffected by these variations . left : view of the interdimer 12 interface of t - state hb ( pdb code 1a3n ) , emphasizing the allosteric core critical h - bonds ( highlighted dashes ) at tertiary ( a e and f h helices ) and quaternary ( hinge and switch ) contacts are labeled . top right : visible absorption spectrum in the soret band of pm and mp hybrids decomposed into protoheme and mesoheme contributions , showing the much greater absorptivity of protoheme at 440 nm ( contribution from vibronic side bands is in green ; data from ref ( 9 ) ) . lower right : chain - selective mesoheme substitution permits selective resonance raman spectroscopy ( 440 nm excitation ) of protoheme in the low - frequency region for deoxy hba ( black ) , mp ( red ) and pm ( blue ) hb sol gels . an important recent finding is that the r t shift is not concerted , but involves separate stages , with 3 and 20 s time constants , the first involving the hinge and the second the switch contacts , as monitored by uvrr and uv mcd spectroscopies . waxs ( wide - angle x - ray scattering ) measurements indicate that most of the molecular shape alteration occurs in the first transition . these experimental observations strongly support the two - stage quaternary pathway that has emerged from computational mapping of the hb energy landscape by karplus and co - workers . this pathway is not an interpolation between the end - states , but instead , a succession of two separate rotations , one involving the dimers , and the other involving mainly the chains . the first carries most of the conformational change , consistent with the waxs results , and involves the hinge contacts , while the second involves the switch . in addition to quaternary shifts , there are tertiary structural differences within ligated and unligated subunits . these can be seen in the crystal structures of the canonical r and t states , and also of tetramers that have been trapped , via chemical or mutational modifications , in partially ligated forms . the occurrence of tertiary changes along the allosteric pathway has been implicit in many models of binding cooperativity . filmer induced fit model for successive binding steps , and the molecular code model of ackers and co - workers , based on microstate thermodynamics derived from extensive measurements of tetramer - dimer dissociation equilibria . tertiary as well as quaternary changes have also been analyzed computationally , starting with gelin and karplus pioneering description of the allosteric core . however , the structures of tertiary intermediates remain to be defined , and are essential to a mechanistic understanding of cooperativity . an important experimental development is the finding that tertiary and quaternary changes can be separated in time by encapsulating hb in silica gel . the protein retains function in the gel , which permits access to buffer and to ligands , but greatly slows protein motion , especially quaternary rearrangement . the gels are transparent and permit application of optical spectroscopies . by monitoring both uvrr and visible rr spectra , histidine stretching frequency , fehis , undergoes evolution between high- and low - reactivity values prior to the r the fe his bond transmits force from the protein to the heme , and its stretching frequency has been shown to reflect heme reactivity . evolution of fehis prior to changes in the quaternary markers seen in uvrr spectra provided direct support for the tertiary two - state model of eaton and co - workers , in which both high- and low - affinity tertiary structures are accessible in either quaternary state . however , these results represented the average behavior of the and chains , which are known to have differing intrinsic fehis values . in the present work , we use a combined spectroscopic and computational approach to probe the distinct conformational transitions experienced by the and chains of hb . in the spectroscopic work , the two chains are monitored separately by using hybrid hb s , in which the native protoheme is replaced with mesoheme in either the or subunits . the shifted electronic transition energy of the mesoheme permits selective excitation of the protoheme rr spectrum . however , the extent of the fehis change is greater in the chains , while the chains evolve faster in the r t direction . in solution , the chain progress curve shows an inflection coincident with the hinge quaternary step , while the chains evolve in concert with the switch . these results are also consistent with the computational mapping of karplus and co - workers . in the computational work , we apply the pele method to predict the structural evolution of the heme and e and f helices of the and chains in response to removal of co from a single hb subunit , with the quaternary structure fixed in the r state . co is a chemically stable surrogate for o2 , inducing the same electronic and structural changes in the heme . we previously used this technique together with a qm / mm treatment of the heme and its surroundings , to successfully predict the helix displacements in myoglobin , mb , in response to heme deligation . fehis is calculated via qm / mm and normal - mode analysis , including benchmark testing on myoglobin . remarkably , pele predicts an evolution from high to low fehis , with the same shifts for each chain as those observed in the gel experiments . in addition , the pele structures showed similar heme perturbations to those observed in the deoxyhb ( t state ) crystal structure : out - of - plane distortions of the heme , and , in the chains , a tilted fe his bond . in other respects , however , the intermediate structure is distinct from the tertiary structure in deoxyhb . this result establishes the competency of computational techniques to monitor force transmission between the heme and the protein , and gives further insight into the allosteric mechanism . tetramethyl orthosilicate ( tmos ) , sodium phosphate buffers , and ihp were all purchased from sigma - aldrich and were of the highest purity available . sodium dithionite ( aldrich ) was of technical grade , was stored in a desiccator , and was used within six months of purchase . the purification of human hba and its chain - selective reconstitution with mesoheme has been described previously . briefly , hb was isolated from expired human blood and the heme was extracted by acetone - acid treatment . the apohb was then reconstituted and incubated for 3 days with synthetic iron(iii ) mesoporphyrin ix . the and chains of native hba and mesohb were then isolated and purified by published procedures , and (proto)-(meso ) ( pm ) and (meso)-(proto ) ( mp ) hb tetramers were recombined by incubating a 1.2-fold molar excess of the beta subunit with the corresponding alpha subunit in 100 mm phosphate buffer , ph 7.4 , overnight at 4 c in a co - containing environment , followed by cation exchange to remove excess monomeric chains . samples were then treated with dithionite to ensure complete reduction to the ferrous state , and were subject to gel - filtration chromatography to remove any residual porphyrin . the proto / mesoheme content and concentration of the hybrid hb solutions were established by uv vis absorbance , and the molecular weights of the alpha and beta chains were verified by electrospray ionization mass spectrometry . hybrid hb solutions were stored in the co - bound form at 80 c until use . cohb solutions were converted to the oxy form by photolysis on ice under 1 atm o2 . these oxyhb solutions were then used directly for oxyhb gels , or were converted to the deoxy form by purging with ar and adding 1 mm sodium dithionite for deoxyhb gels . equal volumes of tmos and 50 mm sodium phosphate buffer ( ph 6.5 ) were then mixed in an nmr tube and vortexed until cloudy , at which point a third volume ( i.e. , 1/3 of final total volume ) of hb solution was added and the solution vortexed until homogeneous . the sol gel emulsion was then rotated horizontally until hardened into a film on the inner surface of the sample tube , typically in 510 min . the gel was then bathed in o2- or ar - saturated phosphate buffer and stored in the dark at 4 c for 48 h prior to use , with one change of the bathing buffer ( 1 ml for 180 l of gel ) after 24 h to remove methanol and nonencapsulated protein . these steps were performed under ambient conditions for oxyhb gels and under 1 atm ar for deoxyhb gels . conversion of oxyhb gels to deoxyhb was initiated by adding sodium dithionite via needle to a final concentration of 2 mm in the bathing buffer . conversion of deoxyhb gels to the co - bound state was achieved by removing the degassed bathing buffer and replacing it with co - saturated buffer . hb solution samples were prepared as described previously , in 50 mm sodium phosphate , ph 7.4 , at a final hb concentration of 100 m tetramer . about 400 l of this solution was transferred by cannula to a sealed , purged nmr tube under 1 atm co or ar pressure for cohb or deoxyhb samples , respectively . resonance raman spectra of hb sol gels were obtained using an experimental apparatus described elsewhere . excitation ( 440 nm ) was obtained from the frequency - doubled output of a titanium - sapphire laser ( 1 khz , 20 ns pulse , 5 j / pulse ) pumped by a q - switched ( 1 khz ) , intracavity frequency - doubled nd : yttrium lithium fluoride laser ( photonics industries international ) . excitation light was focused through a spherical lens onto the surface of the spinning sample tube , and backscattered ( 135 ) light was collected through a camera lens and focused on the 0.200 mm entrance slit of a 0.8 m spectrometer ( spex 1401 ) equipped with a liquid n2-cooled charge - coupled device detector ( roper scientific ) . a 442 nm cutoff notch filter ( angle tuned ) was placed at the slit to minimize the spectral contribution of rayleigh scattering . for experiments on deoxyhb gels with added co , the sample tube was vertically translated back and forth through an 8 mm zone ( with a 100 m laser spot size ) at a rate of 1 mm / s during spectral acquisition , using a thorlabs apt motorized stage ( motor z812 ) . this step was found to be necessary to prevent probe - beam photolysis from suppressing the t r transition in hybrid hb sol gels ( data not shown ) . experiments were conducted at 22 0.5 c . spectra were calibrated with respect to standard raman spectra of dimethylformamide and carbon tetrachloride , and the fehis band was fit by a single gaussian function and a linear baseline in the 180260 cm region of the spectrum . error surface analysis on the fehis - derived time constants of tertiary relaxation was performed as described by beechem and haas and our previous paper . the experimental setup and photolysis pulses at 419 nm ( 20 ns , 65 j / pulse , 1 khz ) were generated by frequency doubling the output of a ti - sapphire laser , which was pumped ( 527 nm , 10 mj / pulse , 70 ns , 1 khz ) by an intracavity frequency - doubled nd : ylf laser ( gm30 , photonics international inc . ) . probe pulses ( 20 ns , 1 khz ) at 440 ( 0.5 j / pulse ) or 229 ( 0.51 j / pulse ) nm were obtained by frequency doubling or quadrupling a second ti - sapphire laser . about 0.4 ml of sample solution was contained in a co - saturated , septum - sealed 5 mm quartz or glass nmr tube . the solution was stirred via a stationary stirrer bar / magnet combination through spinning of the sample tube . the timing between pump and probe pulses was adjusted with a computer - controlled pulse generator ( dg 535 , stanford research systems ) . the data acquisition sequence subtracts averages of paired readings , pump probe minus probe pump , in order to cancel effects of slowly changing conditions . for uvrr , the pump and probe beams were spatially overlapped and focused to a rectangular spot on the sample , matched to the dimensions of the detector , through a pair of cylindrical quartz lenses . scattered light was collected at 135 with a pair of fused quartz lenses , f - matched to a 1.26 m spectrograph ( spex 1269 ) equipped with a holographic grating ( 3600 groove mm ) and uv enhanced liquid nitrogen cooled ccd ( back illuminated , spec-10 , roper scientific ) . spectra were obtained by averaging three 30 min measurements at each time delay . for visible rr , the pump and probe beams were spatially overlapped and focused at the sample using a spherical lens . scattered light was collected at 135 with a camera lens ( f # 1.2 ) and focused using an f - matched singlet lens at the entrance slit of the 0.8 m spectrograph equipped with a holographic grating ( 1800 groove mm ) and intensified ccd ( pi / max , princeton instruments ) . the probe pulse wavelength , 440 nm , was selected to provide selective resonance enhancement of protoheme in deoxy - hba and deoxy - proto / meso - hybrids ( figure 1 ) . the spectral data were processed with grams / ai ( 7.0 ) software ( thermo galactic ) . the fehis band near 220 cm was fit with a gaussian peak to obtain the center frequency . examples of the fehis spectra are presented in reference for seven of the 20 pump probe delay times plotted in figure 3 . the time dependence of these deconvoluted fe his stretching frequencies was modeled by a three - exponential function with fixed time constants obtained from uvrr spectroscopy , which tracks both tertiary and quaternary changes . computer simulations involved the following procedure : ( 1 ) systems setup for r , t and oxy / deoxymb , ( 2 ) initial conformational sampling using molecular dynamics ( md ) , ( 3 ) qm / mm geometry optimization and frequency calculations , ( 4 ) pele sampling for the r deligated species , and ( 5 ) qm / mm geometry optimization and frequency calculations for pele s intermediates . the following structures were taken from the protein data bank : 1a3n ( t , deoxyhb ) , 1bbb ( r2 , hbco ) , 2 v1k ( deoxymb ) and 1mbo ( oxymb ) , and prepared with protein preparation wizard in the schrdinger suite of programs . nacl addition , a short md simulation was performed with desmond for each structure , in order to obtain some conformational sampling . more in detail , the systems were equilibrated with desmond s default protocol followed by a 1 ns npt production phase at 300 k ( this short md was intended to keep the initial quaternary / tertiary arrangements ) . along equilibration and short production hoover chain thermostat with a relaxation time of 1.0 ps , and the pressure was controlled with the martyna the respa integrator was employed with bonded , near , and far time steps of 2.0 , 2.0 , and 6.0 fs , respectively . a 9 cutoff was used for nonbonded interactions together with the smooth particle mesh ewald method . for each system , six snapshots , at times 0 , 0.2 , 0.4 , 0.6 , 0.8 , and 1 ns were selected for the qm / mm study . the heme , the proximal histidine and the sixth ligand ( molecular oxygen or carbon monoxide , if present ) of a given chain subunit ( either of ) were included in the qm region . the rest of the subunit containing the qm region , the other chains and a 12 water layer were treated classically ( the remainder of the solvent was removed ) . the mm minimization included the chain under study , containing the qm region , plus all other residues within 5 . the density functional method with the b3lyp functional and the lacvp * basis set was used for the quantum region , while the opls - aa force field was used for the classical part . in the case of r and oxymb , the sixth ligand was removed after geometry optimization and reoptimized before frequency calculation . in doing so , the five - coordinated r , r , t , t , mb and oxy mb were obtained , allowing direct comparison with the raman data , which is limited to five - coordinate heme . quintet and singlet states were used for five- and six - coordinated heme , respectively . as expected , along the qm / mm optimizations , there were no substantial structural changes except for residues in direct contact with the qm region . thus , significant tertiary changes require a wider level of sampling , which can be achieved with pele as shown previously . additionally , a constrained r structure ( r * ) was modeled by keeping all backbone frozen along the mm and qm minimization . the five - coordinated r and r states obtained after qm / mm geometry optimizations were used as initial models for the pele simulation , aiming to reproduce tertiary changes that accompany deligation of hbco . pele involves three moves , a protein backbone perturbation , side chain sampling and minimization , in a monte carlo step . at the end of each step , a metropolis acceptance criterion is applied to accept or reject the new conformation . the initial backbone move is based on -carbon ( c ) displacements according to an anisotropic network model ( anm ) , a simple model for normal - mode analysis . during side chain sampling , side chains , those with the largest increase in energy as a result of the c-anm perturbation . finally a truncated newton minimization , using the opls all - atom force field and an implicit surface - generalized born continuum solvent , is performed to obtain a new local structure . along this minimization , c atoms are constrained to their final position obtained in the anm perturbation . additionally , side chain perturbation and the implicit solvent model introduce also entropic contributions , moving away from a minimum energy pathway . we have recently shown that this level of sampling provides accurate binding free energies . importantly , the c-anm perturbation was applied only to the chain under study . furthermore , only residues within 25 of the heme were minimized in order to avoid quaternary rearrangement of the tetramer , while allowing relaxation of the intersubunit interface . some intermediates generated by pele were than subjected to qm / mm geometry optimization and frequency calculation to track the dependence on heme reactivity on tertiary changes . a normalized covariance matrix , cij , of the c positional fluctuations along the pele trajectory was calculated to assess the extent of correlation between the residues within a given subunit . cij is given bywhere ri is the displacement vector of the i - th atom from its average position along the trajectory and the angle brackets represent an average over the ensemble of structures generated . positive values represent correlated motion ( i.e. , in the same direction ) , whereas negative values represent an anticorrelated motion ( i.e. , in opposite directions ) . while mesoheme is a close analogue of protoheme , the electronic and steric effects of saturating the vinyl substituents do have detectable effects on function in the reconstituted hybrid hemoglobins . relative to native hba , the meso / proto hybrids have slightly higher o2 affinities , and slightly lower cooperativity . consequently we were concerned about the integrity of the protein structure , and used uvrr spectroscopy to confirm that both hybrids in fact retain the key quaternary contacts , and also the conformational kinetics of hba . excitation at 229 nm produced spectra for deoxyhb and hbco with the same tyrosine and tryptophan band frequencies and intensities for the hybrids and hba , and subtraction gives identical difference spectra ( figure s1 , supporting information ) . this difference spectrum is known to result from the breaking of key h - bonds at the hinge and switch intersubunit contacts ( figure 1 ) when the t structure is converted to r. pump probe uvrr difference spectroscopy has shown that these quaternary contacts are formed in two successive time steps following ligand photodissociation from hbco , first the hinge ( 2 s ) and then the switch ( 20 s ) . we found ( figure 2 ) that the difference spectra were identical for the hybrids and hba at 3.0 and 50 s , before and after the switch contact is formed . likewise identical spectra were obtained at 0.15 s , following the first detectable tertiary transition ( 0.07 s ) and before the quaternary movements . this difference spectrum is associated with the breaking of interhelical h - bonds due to the concerted motion of the e and f helices . thus , despite the slight functional alteration associated with substitution of meso- for protoheme , the conformational dynamics , as monitored by uvrr spectroscopy , remain unaffected . probe difference spectra at the indicated delay times , reflecting alterations in tertiary ( 0.15 s ) and quaternary ( 3 and 50 s ) h - bonds , which reveal the same dynamics for hba and the hb hybrids . the mesoheme soret absorption band is blue - shifted relative to that of protoheme , so that in meso / proto hybrids , one can achieve selective excitation of protoheme rr modes ( figure 1 ) . histidine stretching mode frequency , fehis , separately in the and chains , after co photodissociation from aqueous hbco , using pump are shown for seven time points corresponding to important uvrr changes . in the present work , we show the full time course and reanalyzed these data , using curve - fitting to extract the band frequency at each time point . the fehis time - course is plotted in figure 3 , for comparison with the gel data ( see below ) . comparable data for native hb ( dotted line ) is seen to be an intensity - weighted average of the two chains , confirming that the hybrids provide a faithful representation of the fe the fehis intensity is greater for than for chains ( figure 1 ) ; thus the hb data are closer to those of the than the chains . time courses for fehis for the indicated subunit containing protoheme in the pm ( black ) and mp ( red ) hybrid hbs , following hbo2 deoxygenation in gels ( top ) or hbco photodissociation in solution ( bottom ) . the effect of ihp addition ( open squares ) in gels is also shown . time courses for hba ( pp ) under the same conditions are given as dashed lines for comparison . dotted horizontal lines indicate frequencies for unrelaxed deoxy heme , after photolysis of hbo2 in gels , or of hbco in solution ( bottom ) . marked at right are the chain frequencies for deoxyhb in gels ( top ) and solution ( bottom ) . dotted vertical lines are labeled along the bottom axis showing time constants for tertiary and quaternary contact changes . the chain value is nearly constant at early times , but drops dramatically at 20 s , coincident with the formation of the final quaternary contact ( switch ) , after which it descends to the value seen in deoxyhb ( figure 1 ; this value is somewhat arbitrary , as the deoxyhb band is asymmetric , and has two components 204 and 214 cm ) . the chain fehis begins its decline earlier than the chain , although the final value is considerably higher , as it is in deoxyhb ( figure 1 ) . there is a clear inflection in the chain time course at 3 s , coincident with formation of the first quaternary contact ( hinge ) . it has long been known that hbco photodissociation produces a prompt deoxy - heme rr spectrum with significantly elevated fehis , relative to that observed at later times . we have previously attributed this elevation to fe his bond compression due to protein resistance to heme relaxation after the fe co bond breaks , an effect confirmed by the computational results described below . however , the reported frequency of this prompt signal has been somewhat variable . we discovered that one reason for this variability is that the measured frequency decreases with the increasing incident laser power . figure 4 shows the power - dependent fehis values obtained from rr spectra produced by our 16 ns laser pulses ( which act as simultaneous pump and probe ) . at low power levels only a fraction of the hbco is photodissociated ; this fraction ( calculated from the normalized intensity ratio of the separate 4 porphyrin bands of deoxy and co - heme ) is also plotted in figure 4 . the fehis values were determined by guassian peak fitting , after subtraction of the co - heme spectrum ( there is a weak 238 cm co - heme band ) , using the isolated 508 cm fe co stretching band for normalization . at 1 j / pulse , where the photolyzed fraction is < 5% , fehis = 228.5 cm , but drops rapidly to a plateau value of 225.5 cm beyond 5 j / pulse . importantly , this effect is much weaker for myoglobin ( mb ) , which shows only a 1 cm initial drop , to 220.5 cm , which is the equilibrium deoxymb value . an even larger fehis difference between hb and mb was reported by mizutani and nagai , using picosecond pump a few ps after hbco photolysis , fehis was 232 cm for hb , while for mb it was 221 cm . fehis ( filled circles ) and co saturation ( open circles ) in hbco ( black ) and mbco ( red ) solutions , as functions of the incident 16 ns laser pulse energy . thus , there is a protein - specific fehis elevation in the prompt hbco photoproduct , which remains largely conserved out to 16 ns ( our laser pulse width ) . our nanosecond pump probe data gave fehis = 224 cm at the earliest time point , 70 ns ( figure 3 ) , slightly lower than the 225.5 cm , obtained within the 16 ns pump pulse at laser power levels above 5 j / pulse . the pump power exceeds this value in the pump probe experiments , in order to photolyze all of the hbco molecules . consequently , most of the frequency drop at early times is attributable to the laser power effect . we attribute this effect to relaxation of the heme pocket due to energy transfer from multiple excitations of the heme chromophore . the effect is not seen for mb because full relaxation of its heme occurs in picoseconds . we infer that the heme pocket is considerably more constrained in hb than in mb , retarding heme relaxation into the nanosecond time regime . as in our study of native hb embedded in silica gel , the protoheme fehis evolution of the hybrid hb s was monitored in the r t and t r directions by deoxygenating gel - encapsulated hbo2 ( r t ) , and by adding co to gel - encapsulated deoxyhb ( t r ) . t direction fehis falls to the final deoxyhb values within 2 days for both the and chains , whereas the uvrr markers of quaternary change were previously found to become detectable only after a week . the final values are , of course , quite different in the two chains , as they are in equilibrium deoxyhb . the corresponding gel data for native hb ( dashed line ) tracks the chain data more closely than the chain data , as in solution . in addition , the hb time course showed an early phase ( = 0.1 h ) , which is missing in the hybrids . the starting fehis in the hybrids is that of the second hb phase ( = 3 h ) . this difference between native hb and the hybrids is not seen in solution ( figure 3 , bottom ) . we infer that the gel slightly perturbs the local heme environment in the hybrids , allowing the hemes to relax further than they do in native hb at the earliest times . r direction ( figure 5 ) is induced in gels by adding co to encapsulated deoxyhb just prior to data collection . the rr probe beam transiently photodissociates bound co to produce the five - coordinate heme complex required for fehis detection . consequently , the 16 ns , 5 j laser pulses used in this experiment probe unrelaxed heme in the photoproduct , as discussed above , and therefore , the measured fehis values are expected to be elevated from those seen in the r r frequencies , then fehis evolution is seen to be just the reverse of the r t direction ( figure 3 ) , with chains starting out at somewhat lower frequencies than chains in deoxyhb , and converging to similar r state deoxy frequencies . these results are mirrored in encapsulated native hb , where fehis rises to values characteristic of the r state photoproduct ( measured in hbco gel ) within 2 days . in both hybrids and native hb , these events occur long before the full establishment of the uvrr markers of the t the effector molecule ihp ( inositol hexaphosphate ) was previously found to strongly affect fehis in the r t direction . the current results show that this effect is largely localized in the chains . the initial value of fehis is strongly depressed in the chains , and the depression is maintained throughout the time course , whereas the effect on the chains is minimal . in the t figure s2 ( supporting information ) shows the error surface analysis used to determine uncertainties . time courses for protoheme fe his in the pm ( top panel ) and mp ( bottom panel ) hybrid hbs following addition of co to deoxyhb gels . the addition of ihp ( open squares ) has only a minor effect , as shown . ( the first pm time point was excluded from the fit . ) . qm / mm computation on equilibrium deoxymb and deoxyhb structures ( table 2 ) , gave reasonable values for the fe his bond distances and the fehis frequencies . the computed frequencies are somewhat below the experimental values ( 4 cm for deoxymb , 14 cm for deoxyhb ) . importantly , however , the 7 cm frequency difference between the and chains in deoxyhb is in quantitative agreement with the experimental difference . values averaged over six snapshots taken from the 1 ns molecular dynamics simulation . the number in parentheses is the uncertainty of the mean , estimated as /n , where is the standard deviation and n is the number of values used to calculate the mean values ( 6 ) . experimental data from rr of sol gel encapsulated hbs . when the oxymb structure was reoptimized after removal of the ligand , the computed fehis was essentially the same as for the deoxymb structure . thus , the starting protein structure has no effect on the deoxy - heme in mb . both the and chains give elevated values , compared to those of deoxyhb , when the hbco structure is deligated . as observed experimentally , the elevation is much larger for the than for the chains , 16 vs 6 cm , respectively . these differences are similar to those seen for the meso / proto hybrid hb s over the deligation time course in the solution pump probe experiments ( figure 2 ; the differences seen in the gels are somewhat lower , because of prompt relaxation at early times , as discussed above ) . in order to verify that these results are not biased by the initial structure , we repeated the calculations with 2dn1 ( r , oxyhb ) and 2dn2 ( t , deoxyhb ) . as expected , the frequency values obtained with these crystals are comparable ( the r t shifts are 11 and 7 cm in and respectively ) . interestingly , when the protein backbone was kept frozen during geometry optimization of deligated hb , we obtained significantly higher frequencies in both chains , shown as r * in table 2 . this difference , 10 cm , is the same as that observed between hbco and mbco in the 2 ps photodissociation experiments of mizutani and nagai . the corresponding fe his distance is shorter than in the fully optimized structure , 2.13 vs 2.20 for the chains , and 2.14 vs 2.22 for the chains , and only slightly longer than in the initial 6-coordinate co - heme adduct , 2.12 . thus , modeling an unrelaxed subunit structure confirms our earlier conjecture : the elevated fehis observed for early hbco photoproduct results from bond compression associated with protein resistance to heme relaxation . together , these results show that the qm / mm methodology is sufficiently accurate to monitor the evolution of the individual chain frequencies following deligation . when pele is applied to a single deligated chain within the hbco structure the computed fehis decreases , just as it does in the time - resolved rr experiments . moreover , the effect is greater for the than the chains , again as observed experimentally . twenty steps are sufficient to reach a value around which it subsequently fluctuates . this value is essentially the same as that computed for the t structure , 197 and 204 cm for the and chains . thus , in striking agreement with the gel experiments , fehis evolves from the r - state to the t - state values , solely as a result of tertiary forces initiated by deligation , in the absence of quaternary change . heme - his tilting angle ( c pyrrole nitrogen iron his n angle in degrees ) . rmsd of carbon displacements ( ) between the pele intermediate and the deoxyhb crystal structure ( 1a3n ) for the 1 ( left ) or 1 ( right ) subunits . rmsd of carbon displacements ( ) between the pele intermediate and the initial r structure for the 1 ( left ) or 1 ( right ) subunits . however , the tertiary structure itself does not evolve very far in the direction of the tertiary structure found in the t state . for each chain , the root - mean square deviation ( rmsd ) between the evolving pele structures and the initial deligated structure never exceeds 0.94 ( table 3 ) . the rmsd between the initial structure and the structure in the t state is much larger , 1.59 and 3.68 in the and chains . more importantly , the rmsd of successive pele structures relative to the t state diminishes very little from these values , fluctuating around 1.47 ( ) and 3.67 ( ) ( table 3 ) . it is clear that the pele intermediates , although displaying fehis frequencies characteristics of the t state , have protein conformations that are distinct from the t conformations . for the same reason stated above , the calculations were repeated with 2dn1 and 2dn2 crystal structures . consistently , fehis shifted after pele from 210 to 199 cm for the chain , and from 212 to 206 cm in the chain . in searching for local determinants of fehis , we found that two structural parameters of the heme were most consistently correlated ( figure 6 ) in the successive pele steps : the fe his bond length , and the out - of - plane distortion of the heme , measured as the average displacement of heme s atoms from their least - squares plane . dependence of the computed fe his stretching frequency ( values in cm ) on the fe his bond distance and tilt angle ( nc fe n angle , where nc is in heme pyrrole c and n is in the proximal histidine ) , and with the heme out - of - plane distortion ( measured as the average displacement of heme s atoms from their least - squares plane ) , for hb chains ( red diamonds ) and chains ( blue circles ) . for the chains , the frequency also correlates with the tilt angle of the fe his bond with respect to the heme plane ( figure 6 , bottom ) . this angle is as large in the pele intermediate as it is in the t state , 102 , while the fe his bond is nearly normal to the heme ( 92 ) in hbco . his bond has long been noted as a feature of the t - state chain . however , this correlation does not hold for the chain , for which the tilt is lower along the pele trajectory . in the chain , the heme distortion , enforced by the packing interactions with nearby side chains , is the likely determinant of the slight fe his bond lengthening . figure 7 illustrates the differing heme - associated changes between the two chains , and the similar local structures in the pele intermediate and deoxyhb . overlay of heme , the proximal histidine , and relevant contacting residues for the initial ( light brown ) and intermediate ( green ) pele structures , and for deoxyhb ( pdb 1a3n ; cyan ) . to investigate the origin of these structural differences between the evolving and chains the c correlation maps between the pele intermediate and the initial structure show quite different patterns for the two chains ( figure 8) . in the chains , the displacements are positively correlated for the first 75 residues ( a , b , c and e helices and the ef corner ) , but they correlate poorly with the next 25 residues , which contain the f and part of the g helices . in particular the correlation between the e ( 5570 ) and f ( 7590 ) helices is essentially null , meaning that they move in orthogonal directions . in contrast , the e and f helix displacements are positively correlated in the chains , while the e and b helices are anticorrelated with the a helix . figure 9 illustrates the differing displacements of the e and f helices , which sandwich the heme group . two orientations are shown , in the ef plane ( left ) , and perpendicular to it ( right ) , with the hemes superposed . in the chain , the e and f helices move in the same direction between the initial ( light brown ) and intermediate ( green ) pele structures , toward the protein exterior ( right view ) and also toward the ef corner ( left view ) . however , they move oppositely in the chain , the f helix again moving out and toward the ef corner , while the e helix moves in and away from the ef corner . this difference may be partly due to packing constraints in the ef corner , which are more pronounced for the chain ( represented in figure 9 by the phe85 and leu81 side chains ) than for the chain . in addition , the h helix may play a role , via h - bonds from the penultimate residue , tyr140 and tyr145 to the f helix residues pro77 and val98 , respectively , and salt - bridges from glu116 and glu121 to the a helix residues lys16 and lys17 , respectively . indeed , these regions are significantly correlated in the chain ( figure 8) . the differing orientations of these connections ( figure 9 ) may guide the e and f helices in different directions , as reflected in the e and a helices being correlated for the chains but anticorrelated in the chains . covariance matrix for successive residue c displacements along the trajectory between initial and intermediate pele structures . the bar at the right shows the color scale from positive ( white ) to negative ( black ) correlation . successive helices are indicated by black bars with letter labels . in the chain , the concerted helix displacements lead to steric contacts with the heme that induce out - of - plane distortions , especially phe103 , which presses against one of the pyrrole rings ( figure 7 ) . this residue was identified in earlier qm / mm computations as making an important contribution to t corresponding contacts are attenuated in the chain , although the heme also distorts , perhaps due to contacts of the peripheral substituents . displacements of the heme are strongly correlated with those of the e helix in the chain , but less so for the chain ( figure s3 , supporting information ) . in the chain , the orthogonal motion of the e and f helices induces tilting of the fe his bond , due to the proximal his residue , his87 , becoming displaced relative to the heme . we found that the tilting is largely mediated by the h - bond connecting his87 with the backbone carbonyl of the upstream residue , leu83 ( see figure 7 ) . when the strength of this h - bond was lowered in silico , by reducing the atomic charge of the donor ( from 0.42 to 0.1 ) and increasing that of the acceptor ( from 0.5 to 0.1 ) , the fe his tilt was reduced by half . an important role of this h - bond in regulation of oxygen affinity has previously been found for other globins and on the basis of computational studies . ribbon diagram of the e and f helices for the pele intermediate ( green helices ; val62/67 in cyan ) and initial ( light brown helices ; val62/67 in orange ) structures with hemes superposed . also shown are packing residues ( blue ) at the ef corner , and residues ( magenta ) forming key interhelical h - bonds and salt - bridges . a central issue in understanding allostery is to establish the connection between function and structure , both tertiary and quaternary . for hemoglobin , crystallography has established allosteric end - state structures , as well as structures of variants and derivatives which plausibly represent intermediate states on the allosteric pathway . however , the details of this pathway , and the forces that guide the protein along it , remain to be determined . in this work , we focus on the frequency of the fe his stretching vibration as a monitor of heme reactivity . ligation of heme by o2 , or by its surrogate co , requires fe to be drawn into the heme plane from its out - of - plane position in deoxy - heme . they also weaken the fe his bond in deoxy - heme , and lower its vibrational frequency . fehis has been correlated with the affinities and binding rates of ligands in various hb preparations . in addition , we have shown that the hbco photolysis yield declines in parallel with increasing fehis of the photoproduct as t - state hb gels evolve toward the r state ; the decline in yield reflects a corresponding increase in the rate of geminate recombination . heme reactivity is the not the sole determinant of ligand affinity , as is clearly seen in the differing fehis for and chains , despite their having essentially the same o2 affinities . on the basis of early crystal structures , perutz proposed that oxygen affinity is reduced in t - state chains by movement of the e helix residues his(e7 ) and val(e11 ) over the heme iron . removal of steric hindrance with his(e7)gly and val(e11)ala mutations confirmed this idea for chains . these same mutations in chains showed that crowding of the binding site is relatively unchanged between the r and t states , indicating a dominant role for proximal control of affinity . a high level of fe his strain for t - state chains was initially found by the appearance of spectral signatures of pentacoordinate heme upon reaction of no , which exerts a strong trans effect , with hb stabilized in the t state . the assignment of -specific rupture of the fe his bond was later confirmed in crystallographic studies . in another approach , barrick et al . severed the heme - protein link by replacing the proximal histidine , his(f8 ) , with glycine and adding imidazole to serve as the proximal ligand . binding of co to these constructs fully disrupted the t - state quaternary contacts when the link was severed only in the chains , but not when it was severed only in the chains . thus , the proximal linkage in the but not the chains is critical to the t r transition , consistent with the greater proximal effect on heme reactivity in the chains . also reported that co affinity is controlled entirely by proximal effects in chains . the compensation of heme reactivity and steric factors produce similar oxygen equilibria for both chains ; chain equivalence is a fundamental requirement for maximum cooperativity in binding to the tetramer . this compensation is illuminated by our previous qm / mm computation of the structural factors contributing to the ligation energy differences between the r and t states . when protein constraints were relaxed in silico , the contribution of the proximal imidazole to t - state destabilization of the heme - co adduct was found to be 1.3 kcal / mol greater in the than the chains ( consistent with the fehis shifts reported here ) while the contribution of the heme plus the co ( reflecting nonbonded contacts ) was 1.4 kcal / mol greater in the than the chains , suggesting compensation between proximal strain and nonbonded forces ( see supporting information table s3 in ref ( 90 ) ) . here , we monitor the evolution of fehis , an indicator of heme reactivity , independently for each chain through the r / t conformational evolution and likewise find a larger effect in the chain . although we can not monitor steric factors spectroscopically , it is likely that they evolve in concert with the observed fehis shifts , due to the associated e helix motions , as discussed below . figure 10 provides a summary of fehis values in various deligated states : the photoproduct , prior to local relaxation ; the r state and the t state for each chain ; and the effect of adding ihp . the value is elevated in the prompt photoproduct ( r * , top right ) because of fehis bond compression . relaxation of the local environment permits heme relaxation in the r state ( lower left ) with similar fehis values in the two chains . in the t state ( lower right ) , fehis is lowered markedly in the chains due to fe his tilting . ihp has a large effect on fehis for the , but not the chains in the r state or either chain in the t state . our previous gel encapsulation study of native hba established that , following ligation or deligation , fehis evolved to values characteristic of the eventual quaternary structure , r or t , well before the quaternary transition itself took place . the present work extends these results to the individual chains , and , which evolve separately to the eventual r and t fehis values , prior to the quaternary transition . furthermore , these observations are supported by computational modeling where protein relaxation following deligation in one chain is coupled with qm / mm frequency calculations . along the simulations , the other three chains remain ligated , and the quaternary structure remains r. for both and chains , the qm / mm - computed fehis decreases by a similar amount to that observed in the gel experiments . together , these results establish that heme reactivity is controlled exclusively by tertiary forces within the hb tetramer . once the ligand is bound or released , the tertiary forces induce a structure change that switches the reactivity to the high and low values that have classically been associated with the r and t states . the local forces initiated by ligation and deligation of the heme are readily understood . the downward displacement of the fe atom as it loses the ligand and becomes 5-coordinate and high - spin , impels displacement of the f - helix , to which the proximal histidine ligand is attached . resistance to this displacement can be seen in the elevated fehis frequency of the immediate co - heme photoproduct ; this frequency elevation is well modeled in the present qm / mm calculation where we constrained the backbone motion . releasing this constraint lowers fehis by 10 cm , due to the relief of fe his bond compression . this lowering is the same as the fehis difference seen experimentally between the immediate photoproducts for hbco and mbco . for mb , the subsequent fehis evolution is very small , 1 cm , but in hb fehis drops by 6 cm within 70 ns , corresponding to the lifetime of the first tertiary intermediate detected by uvrr spectroscopy , designated rdeoxy . this intermediate is associated with the breaking of interhelical h - bonds involving the e and f helices . thus , hb has a built - in resistance to heme relaxation , which is absent in mb ( which does not have the same interhelical h - bonds ) . from previous studies , it is known that an invariant valine residue on the e helix ( val62 and val67 , figure 9 ) provides the main steric contact with ligands bound to the heme , and that loss of this contact upon deligation impels e helix displacement . our previous uvrr study in gels showed that the e helix displacement precedes the f helix displacement . thus , the deligation - induced fe out - of - plane movement begins in concert with the e helix displacement , and then increases as the f helix is displaced . while the helix displacements are common to both chains ( as evidenced by separate uvrr signal disappearances when the interhelical h - bond partners are separately mutated in the two chains ) , the pele computations reveal that they are guided in different directions within the tetramer . in the chain , the e and f helices move in the same direction , so that the fe his bond orientation is unaltered . the modest fehis lowering associated with this shift can be attributed to enhanced heme out - of - plane distortion , due to steric contacts . in contrast the e and f helices move in orthogonal directions for the chain , producing heme distortion and tilting of the fe his bond , and a more pronounced lowering of fehis . these differing relative displacements may be guided by differing packing arrangements in the ef corner , and with differing orientations of h - bonds and salt bridges connecting the h helix with the f and a helices ( figure 9 ) . gel encapsulation shows that the chain fehis evolves faster in the than in the chains , when the quaternary structure is frozen ( figure 3 ) . the two time constants are 5 and 2 shorter for than for ( table 1 ) . the solution pump probe measurements ( figure 3 ) show that , when the constraint on quaternary motion is removed , the chain fehis remains at its r state value until the final stage ( 20 s ) of the quaternary transition ( the switch ) , whereas the chain time course shows an inflection at the time ( 3 s ) of the first quaternary stage ( the hinge ) , before finishing the fehis shift to its low affinity value , in concert with the switch . these results are in remarkable accord with the allosteric pathway computed by fischer et al . , the quaternary transition occurs in two steps , as anticipated by the uvrr results . in the r t direction , the first step , q2 ( numbered in the reverse direction ) , produces a much larger change in quaternary structure than the second step , q1 . this prediction accords with the results of time - resolved waxs ( wide - angle x - ray scattering ) measurements , in which the main shape change of the hb tetramers was found at 3 s , coincident with the uvrr - detected hinge step . q2 is found by cpr to involve a 6 rotation of one dimer against the other , with altered 12 contacts in the hinge region . q2 is preceded by significant alteration in the chain tertiary structure , with little tertiary change in the chains . this prediction is in accord with our findings that in gels , fehis evolves faster in than in chains , while in solution , the fehis progress curve shows an inflection at 3 s ( q2 ) for but not for chains . in the second step , q1 , the chains move farther apart , and rotate 3 relative to the chains , bringing the switch contacts into register at the 12 interface , consistent with the uvrr switch signal at 20 s . q1 is accompanied by a major change in the chain tertiary structure , consistent with the large change in chain fehis at 20 s . the picture that emerges from these experimental and computational results is that deligation within the r structure induces a faster tertiary response in the than in the chains , whether the quaternary motion is constrained or not . a possible reason is that the chain has a longer cd loop at the end of the e helix ( figure 9 ) , providing flexibility , and perhaps allowing a faster e helix response to deligation than in the more restricted chain . the role of the cd loop in the dynamics of mb , whose chain movements are similar to those of the hb chain has been discussed in previous work . in solution , without external restraint on the quaternary motion , the chain tertiary motion drives rotation of the dimers around the hinge . subsequently , the slower tertiary motion of the chains drives reorientation of the chains , closing the switch and completing the r t transition . in the t r direction , the cpr - computed trajectory is reversed . experimentally , r sequence has not been monitored in solution , because ligation is slower than the protein structure changes . in gels , these changes are slowed sufficiently to allow spectroscopic observation following ligation of deoxyhb with co. there are small but prompt uvrr signal changes associated with both the switch and hinge contacts , indicating that ligation immediately loosens the t - state quaternary contacts . the small uvrr signals only approach the amplitude expected for the quaternary transition after about a week , while much earlier changes are seen in the photoproduct fehis , which evolve from low to high values on the same time scale as the opposite changes seen upon deligation of hbo2 gels . in the t r direction , however , there is no significant rate difference between the and chains . r changes in the gels are not quite the reverse of the r t changes . ligation - induced forces first weaken the quaternary contacts , then impel tertiary evolution within both chains , more - or - less simultaneously , after which the quaternary transition is completed . the discrepancy with the cpr trajectory is understandable , since the cpr method does not permit changes in heme ligation . the trajectory was computed for unligated hb transitioning between t and r quaternary structure . the gel experiments indicate that ligation within the t structure requires loosening of the quaternary contacts before evolution toward the r structure . this inference is strongly supported by the reported structure of a deoxyhb crystal that is oxygenated at low temperature . all four hemes are ligated by o2 , and quaternary contacts are strained at both the switch and the hinge . moreover , his bond is lengthened in the chains , while the heme is buckled in the chains , providing a structural explanation of the initially low photoproduct fehis values in the freshly ligated gels . the tertiary two - state ( tts ) model posits that low- and high - affinity tertiary structures , t and r , can coexist within each quaternary structure , which bias the tertiary populations . the r and t structures are favored in the r and t states , respectively , but both are present in each state , their populations determining the ligand binding partition function . the model was inspired by the finding that when co was bound to deoxyhb gels , recombination following photolysis showed two phases , with rates that matched those that had been associated with r- and t - state tetramers . since the gel kept the protein in the t state , the faster r - like rate must have resulted from a reactive tertiary structure . our finding that fehis , a marker of heme reactivity , evolves toward an r - like value while the gel - constrained protein remains in the t state , fully supports this view . likewise its evolution toward a t - like value while the protein remains in the r state supports the basic tenet of the tts model that both quaternary states harbor high- and low - reactivity tertiary structures . the and subunit fehis frequencies are essentially the same in the r state , and although they differ substantially in the t state , the ligation rate is similar , because of differing steric factors , as discussed above . consequently , it is not surprising that single r - like and t - like rates were observed in the gel co recombination experiments , despite differing subunit structures . however , our computations indicate that the intermediate tertiary structure resulting from deligation of a single subunit only partially resembles the tertiary structure found in the t state , even though the correct fehis frequency shift is predicted . thus , the r - state t structure ( tr ) of the tts model is quite different from the t - state t structure ( tt ) , although the two structures are functionally equivalent . that the complete tertiary change is not likely to occur within a given quaternary structure seems reasonable . the monomers are not independent but interact with intermolecular contacts ; therefore , their structural change should be correlated to the whole tetramer rearrangement in space . a recent md study showed that during the t r transition , the hb subunits approach the r - like conformation only in the final stage of the quaternary change . most likely the r- and t - state r ( rr and rt ) structures are also different . it appears that the hb architecture is designed to allow ligation and deligation to induce tertiary changes in the heme surroundings that shift the subunit reactivity between low and high values , while leaving the subunit as a whole in an intermediate tertiary configuration . these surroundings , the e and f helices and the fg corner , correspond to the allosteric core identified many years ago by gelin and karplus in their pioneering molecular mechanics study of hb . subsequent rearrangement of the tertiary configuration accompanies the quaternary shift between r and t , but is without further effect on heme reactivity . this separation of functional response between the two phases of tertiary change is what makes the tts model viable . polyanionic effectors like bpg ( 2,3-bisphosphoglycerate ) and ihp stabilize the t quaternary structure of hb by binding to its central cavity , which is lined with cationic sites . ihp addition to deoxyhb has little effect on fehis , in either the or chain . it also has minimal effects on the kinetics of fehis evolution for the hbco photoproduct in t - state gels , again for either chain . thus , in the t state , ihp does not alter heme reactivity and must exert its influence on ligand affinity through other structural mechanisms . ( viapianni et al . reported that a combination of strong effector molecules , ihp and bezafibrate , suppressed the faster r - like co recombination rate in t - state hbco gels . we were unable to explore this combination in our experiments because of strong raman scattering from bezafibrate . ) in the r state , however , ihp dramatically lowers fehis and accelerates its evolution following deligation . this effect is consistent with yonetani s observations of large effector - induced affinity lowering within the r state . long ago , marden et al . showed that effectors lower the last of the four ligand binding equilibrium constants when hb is clearly in the r state . marden concluded that while effectors influence the t - state affinity and the allosteric equilibrium , they have an even larger effect on r state affinity . we now find that the r state ihp effect on fehis is largely localized to the chains . consistent with this finding , an nmr study of hbco ( r state ) by ho and co - workers found that ihp greatly increases conformational dynamics around the interdimer interface , especially in the f helix and fg corner of the chains . no crystal structure is available for ihp bound near the subunits in an r state hb tetramer . however , we note that when hbo2 was crystallized from phosphate buffer at ph 8.5 , a phosphate ion was bound in the cleft between the chains , bridging the two lys99 side chains ( pdb 1hho ) , which are near the fg corner ( figure 11 ) . another crystal structure , in which lys99 has been derivatized with fructose ( pdb 3b75 ) , shows displacement of the fg corner and the f helix ( figure 11 ) , resulting in tilting of the fe his bond ( the c pyrrole nitrogen iron his n angle decreasing to 82 from 92 in the hbo2 structure ) and also of the proximal imidazole ring with respect to the fe his bond , the fe c(imidazole ) angle increasing from 127 in the hbo2 structure ( essentially its normal 126 value ) to 144 in the fructosyl derivative . we speculate that the combination of phosphate and a nearby fructose mimics binding of the bulky ihp molecule in the r state cleft , resulting in similar helix displacement and proximal his tilting , with consequent lowering of the chain fehis . thus , the availability of the inter - lys99 binding site may account for the strong -specific ihp influence on heme reactivity in the r state . superposition of the hb structures with bound phosphate , aligned via the c atoms of both chains , showing the displacement of the fg region and the f helix between native hbo2 ( orange ; pdb 1hho(113 ) ) and the lys99-fructosyl derivative ( cyan ; pdb 3b75(115 ) ) . combining the available experimental and computational results , we obtain the following view of the mechanism of hb allostery : ( 1 ) ligand detachment from r - state ligated protein produces unrelaxed deoxy - heme , with a compressed fe his bond in hb , but not in mb . the immediate protein environment relaxes in tens of nanoseconds in hb , but is already relaxed by 2 ps in mb . ( 2 ) subsequent evolution within the r state produces an intermediate tertiary structure , with functional properties that are associated with the t state . in the chains , the packing of surrounding residues enhances the out - of - plane distortion of the deoxy - heme , while in the chains , orthogonal motions of the e and f helices produce fe his bond tilting as well as heme distortion . ( 3 ) structural evolution is faster for the than the chains . in solution , the chain evolution induces the main quaternary transition , at 3 s , during which the dimers rotate , and establish the t hinge contacts between the 1 c helix and the 2 fg corner . the chain evolution induces the second transition , at 20 s , during which the chains rotate into their final position , establishing the t switch contacts between the 2 c helix and the 1 fg corner . ( 4 ) in the reverse direction , ligation in the gel - encapsulated t state requires loosening of quaternary contacts , but tertiary structures then evolve from low to high - affinity function , in this case at essentially the same rate for and chains , before the quaternary transitions . the structure sequence has not been monitored in solution because ligation is slower than protein structure change . ( 5 ) the finding that intermediate tertiary structures can have low or high heme reactivity within both quaternary states supports the tertiary two - state model of ligand binding , although it is clear that the intermediate structures are not the same as the end - state tertiary structures . in other word , ( 6 ) the allosteric effector ihp has a large effect on r state heme reactivity , specifically in the chains . this effect is suggested to result from ihp binding to the two facing lys99 residues in the cleft between the chains , resulting in displacement of the f helix and fg corner that tilt the fe ihp has little effect on heme reactivity in the t state , but instead stabilizes the t structure . many pieces of the allosteric puzzle are still missing , particularly the role of partially ligated states , and the mechanisms by which the intermediate tertiary structures induce the quaternary transitions . it may be that access to intermediate tertiary structures with functional properties characteristic of end - state quaternary structures is a general property of allosteric systems .

the use of hybrid hemoglobin ( hb ) , with mesoheme substituted for protoheme , allows separate monitoring of the or hemes along the allosteric pathway . using resonance raman ( rr ) spectroscopy in silica gel , which greatly slows protein motions , we have observed that the fe histidine stretching frequency , fehis , which is a monitor of heme reactivity , evolves between frequencies characteristic of the r and t states , for both or chains , prior to the quaternary r t and t r shifts . computation of fehis , using qm / mm and the conformational search program pele , produced remarkable agreement with experiment . analysis of the pele structures showed that the fehis shifts resulted from heme distortion and , in the chain , fe his bond tilting . these results support the tertiary two - state model of ligand binding ( henry et al . , biophys . chem.2002 , 98 , 149 ) . experimentally , the fehis evolution is faster for than for chains , and pump probe rr spectroscopy in solution reveals an inflection in the fehis time course at 3 s for but not for hemes , an interval previously shown to be the first step in the r t transition . in the chain fehis dropped sharply at 20 s , the final step in the r t transition . the time courses are fully consistent with recent computational mapping of the r t transition via conjugate peak refinement by karplus and co - workers ( fischer et al . , proc . natl . acad . sci . u. s. a.2011 , 108 , 5608 ) . the effector molecule ihp was found to lower fehis selectively for chains within the r state , and a binding site in the 12 cleft is suggested .

Introduction Methods Results Discussion Conclusions

from the earliest days of perutz crystal structures and the celebrated two - state ligation model of monod , changeux and wyman , hemoglobin ( hb ) has served as a paradigm for allosteric systems . an important recent finding is that the r t shift is not concerted , but involves separate stages , with 3 and 20 s time constants , the first involving the hinge and the second the switch contacts , as monitored by uvrr and uv mcd spectroscopies . these experimental observations strongly support the two - stage quaternary pathway that has emerged from computational mapping of the hb energy landscape by karplus and co - workers . these can be seen in the crystal structures of the canonical r and t states , and also of tetramers that have been trapped , via chemical or mutational modifications , in partially ligated forms . filmer induced fit model for successive binding steps , and the molecular code model of ackers and co - workers , based on microstate thermodynamics derived from extensive measurements of tetramer - dimer dissociation equilibria . by monitoring both uvrr and visible rr spectra , histidine stretching frequency , fehis , undergoes evolution between high- and low - reactivity values prior to the r the fe his bond transmits force from the protein to the heme , and its stretching frequency has been shown to reflect heme reactivity . evolution of fehis prior to changes in the quaternary markers seen in uvrr spectra provided direct support for the tertiary two - state model of eaton and co - workers , in which both high- and low - affinity tertiary structures are accessible in either quaternary state . in the spectroscopic work , the two chains are monitored separately by using hybrid hb s , in which the native protoheme is replaced with mesoheme in either the or subunits . however , the extent of the fehis change is greater in the chains , while the chains evolve faster in the r t direction . these results are also consistent with the computational mapping of karplus and co - workers . in the computational work , we apply the pele method to predict the structural evolution of the heme and e and f helices of the and chains in response to removal of co from a single hb subunit , with the quaternary structure fixed in the r state . in addition , the pele structures showed similar heme perturbations to those observed in the deoxyhb ( t state ) crystal structure : out - of - plane distortions of the heme , and , in the chains , a tilted fe his bond . spectra were calibrated with respect to standard raman spectra of dimethylformamide and carbon tetrachloride , and the fehis band was fit by a single gaussian function and a linear baseline in the 180260 cm region of the spectrum . computer simulations involved the following procedure : ( 1 ) systems setup for r , t and oxy / deoxymb , ( 2 ) initial conformational sampling using molecular dynamics ( md ) , ( 3 ) qm / mm geometry optimization and frequency calculations , ( 4 ) pele sampling for the r deligated species , and ( 5 ) qm / mm geometry optimization and frequency calculations for pele s intermediates . histidine stretching mode frequency , fehis , separately in the and chains , after co photodissociation from aqueous hbco , using pump are shown for seven time points corresponding to important uvrr changes . comparable data for native hb ( dotted line ) is seen to be an intensity - weighted average of the two chains , confirming that the hybrids provide a faithful representation of the fe the fehis intensity is greater for than for chains ( figure 1 ) ; thus the hb data are closer to those of the than the chains . the chain value is nearly constant at early times , but drops dramatically at 20 s , coincident with the formation of the final quaternary contact ( switch ) , after which it descends to the value seen in deoxyhb ( figure 1 ; this value is somewhat arbitrary , as the deoxyhb band is asymmetric , and has two components 204 and 214 cm ) . there is a clear inflection in the chain time course at 3 s , coincident with formation of the first quaternary contact ( hinge ) . the fehis values were determined by guassian peak fitting , after subtraction of the co - heme spectrum ( there is a weak 238 cm co - heme band ) , using the isolated 508 cm fe co stretching band for normalization . as in our study of native hb embedded in silica gel , the protoheme fehis evolution of the hybrid hb s was monitored in the r t and t r directions by deoxygenating gel - encapsulated hbo2 ( r t ) , and by adding co to gel - encapsulated deoxyhb ( t r ) . in addition , the hb time course showed an early phase ( = 0.1 h ) , which is missing in the hybrids . consequently , the 16 ns , 5 j laser pulses used in this experiment probe unrelaxed heme in the photoproduct , as discussed above , and therefore , the measured fehis values are expected to be elevated from those seen in the r r frequencies , then fehis evolution is seen to be just the reverse of the r t direction ( figure 3 ) , with chains starting out at somewhat lower frequencies than chains in deoxyhb , and converging to similar r state deoxy frequencies . in both hybrids and native hb , these events occur long before the full establishment of the uvrr markers of the t the effector molecule ihp ( inositol hexaphosphate ) was previously found to strongly affect fehis in the r t direction . the initial value of fehis is strongly depressed in the chains , and the depression is maintained throughout the time course , whereas the effect on the chains is minimal . qm / mm computation on equilibrium deoxymb and deoxyhb structures ( table 2 ) , gave reasonable values for the fe his bond distances and the fehis frequencies . thus , in striking agreement with the gel experiments , fehis evolves from the r - state to the t - state values , solely as a result of tertiary forces initiated by deligation , in the absence of quaternary change . in searching for local determinants of fehis , we found that two structural parameters of the heme were most consistently correlated ( figure 6 ) in the successive pele steps : the fe his bond length , and the out - of - plane distortion of the heme , measured as the average displacement of heme s atoms from their least - squares plane . dependence of the computed fe his stretching frequency ( values in cm ) on the fe his bond distance and tilt angle ( nc fe n angle , where nc is in heme pyrrole c and n is in the proximal histidine ) , and with the heme out - of - plane distortion ( measured as the average displacement of heme s atoms from their least - squares plane ) , for hb chains ( red diamonds ) and chains ( blue circles ) . for the chains , the frequency also correlates with the tilt angle of the fe his bond with respect to the heme plane ( figure 6 , bottom ) . this angle is as large in the pele intermediate as it is in the t state , 102 , while the fe his bond is nearly normal to the heme ( 92 ) in hbco . in the chain , the heme distortion , enforced by the packing interactions with nearby side chains , is the likely determinant of the slight fe his bond lengthening . in the chains , the displacements are positively correlated for the first 75 residues ( a , b , c and e helices and the ef corner ) , but they correlate poorly with the next 25 residues , which contain the f and part of the g helices . this residue was identified in earlier qm / mm computations as making an important contribution to t corresponding contacts are attenuated in the chain , although the heme also distorts , perhaps due to contacts of the peripheral substituents . in the chain , the orthogonal motion of the e and f helices induces tilting of the fe his bond , due to the proximal his residue , his87 , becoming displaced relative to the heme . in this work , we focus on the frequency of the fe his stretching vibration as a monitor of heme reactivity . in addition , we have shown that the hbco photolysis yield declines in parallel with increasing fehis of the photoproduct as t - state hb gels evolve toward the r state ; the decline in yield reflects a corresponding increase in the rate of geminate recombination . these same mutations in chains showed that crowding of the binding site is relatively unchanged between the r and t states , indicating a dominant role for proximal control of affinity . thus , the proximal linkage in the but not the chains is critical to the t r transition , consistent with the greater proximal effect on heme reactivity in the chains . this compensation is illuminated by our previous qm / mm computation of the structural factors contributing to the ligation energy differences between the r and t states . when protein constraints were relaxed in silico , the contribution of the proximal imidazole to t - state destabilization of the heme - co adduct was found to be 1.3 kcal / mol greater in the than the chains ( consistent with the fehis shifts reported here ) while the contribution of the heme plus the co ( reflecting nonbonded contacts ) was 1.4 kcal / mol greater in the than the chains , suggesting compensation between proximal strain and nonbonded forces ( see supporting information table s3 in ref ( 90 ) ) . here , we monitor the evolution of fehis , an indicator of heme reactivity , independently for each chain through the r / t conformational evolution and likewise find a larger effect in the chain . the present work extends these results to the individual chains , and , which evolve separately to the eventual r and t fehis values , prior to the quaternary transition . along the simulations , the other three chains remain ligated , and the quaternary structure remains r. for both and chains , the qm / mm - computed fehis decreases by a similar amount to that observed in the gel experiments . once the ligand is bound or released , the tertiary forces induce a structure change that switches the reactivity to the high and low values that have classically been associated with the r and t states . in the chain , the e and f helices move in the same direction , so that the fe his bond orientation is unaltered . in contrast the e and f helices move in orthogonal directions for the chain , producing heme distortion and tilting of the fe his bond , and a more pronounced lowering of fehis . the solution pump probe measurements ( figure 3 ) show that , when the constraint on quaternary motion is removed , the chain fehis remains at its r state value until the final stage ( 20 s ) of the quaternary transition ( the switch ) , whereas the chain time course shows an inflection at the time ( 3 s ) of the first quaternary stage ( the hinge ) , before finishing the fehis shift to its low affinity value , in concert with the switch . in the r t direction , the first step , q2 ( numbered in the reverse direction ) , produces a much larger change in quaternary structure than the second step , q1 . this prediction accords with the results of time - resolved waxs ( wide - angle x - ray scattering ) measurements , in which the main shape change of the hb tetramers was found at 3 s , coincident with the uvrr - detected hinge step . this prediction is in accord with our findings that in gels , fehis evolves faster in than in chains , while in solution , the fehis progress curve shows an inflection at 3 s ( q2 ) for but not for chains . in the second step , q1 , the chains move farther apart , and rotate 3 relative to the chains , bringing the switch contacts into register at the 12 interface , consistent with the uvrr switch signal at 20 s . q1 is accompanied by a major change in the chain tertiary structure , consistent with the large change in chain fehis at 20 s . a possible reason is that the chain has a longer cd loop at the end of the e helix ( figure 9 ) , providing flexibility , and perhaps allowing a faster e helix response to deligation than in the more restricted chain . subsequently , the slower tertiary motion of the chains drives reorientation of the chains , closing the switch and completing the r t transition . the tertiary two - state ( tts ) model posits that low- and high - affinity tertiary structures , t and r , can coexist within each quaternary structure , which bias the tertiary populations . the r and t structures are favored in the r and t states , respectively , but both are present in each state , their populations determining the ligand binding partition function . our finding that fehis , a marker of heme reactivity , evolves toward an r - like value while the gel - constrained protein remains in the t state , fully supports this view . the and subunit fehis frequencies are essentially the same in the r state , and although they differ substantially in the t state , the ligation rate is similar , because of differing steric factors , as discussed above . a recent md study showed that during the t r transition , the hb subunits approach the r - like conformation only in the final stage of the quaternary change . showed that effectors lower the last of the four ligand binding equilibrium constants when hb is clearly in the r state . consistent with this finding , an nmr study of hbco ( r state ) by ho and co - workers found that ihp greatly increases conformational dynamics around the interdimer interface , especially in the f helix and fg corner of the chains . another crystal structure , in which lys99 has been derivatized with fructose ( pdb 3b75 ) , shows displacement of the fg corner and the f helix ( figure 11 ) , resulting in tilting of the fe his bond ( the c pyrrole nitrogen iron his n angle decreasing to 82 from 92 in the hbo2 structure ) and also of the proximal imidazole ring with respect to the fe his bond , the fe c(imidazole ) angle increasing from 127 in the hbo2 structure ( essentially its normal 126 value ) to 144 in the fructosyl derivative . we speculate that the combination of phosphate and a nearby fructose mimics binding of the bulky ihp molecule in the r state cleft , resulting in similar helix displacement and proximal his tilting , with consequent lowering of the chain fehis . thus , the availability of the inter - lys99 binding site may account for the strong -specific ihp influence on heme reactivity in the r state . combining the available experimental and computational results , we obtain the following view of the mechanism of hb allostery : ( 1 ) ligand detachment from r - state ligated protein produces unrelaxed deoxy - heme , with a compressed fe his bond in hb , but not in mb . in the chains , the packing of surrounding residues enhances the out - of - plane distortion of the deoxy - heme , while in the chains , orthogonal motions of the e and f helices produce fe his bond tilting as well as heme distortion . in solution , the chain evolution induces the main quaternary transition , at 3 s , during which the dimers rotate , and establish the t hinge contacts between the 1 c helix and the 2 fg corner . ( 5 ) the finding that intermediate tertiary structures can have low or high heme reactivity within both quaternary states supports the tertiary two - state model of ligand binding , although it is clear that the intermediate structures are not the same as the end - state tertiary structures . in other word , ( 6 ) the allosteric effector ihp has a large effect on r state heme reactivity , specifically in the chains . this effect is suggested to result from ihp binding to the two facing lys99 residues in the cleft between the chains , resulting in displacement of the f helix and fg corner that tilt the fe ihp has little effect on heme reactivity in the t state , but instead stabilizes the t structure . many pieces of the allosteric puzzle are still missing , particularly the role of partially ligated states , and the mechanisms by which the intermediate tertiary structures induce the quaternary transitions .

in recent years , there has been a significant increase in the number of obese women who become pregnant ; in fact , obesity has currently become one of the most important gestational risk factors . gestational obesity is considered when body mass index ( bmi ) is 30 kg / m at the first obstetric evaluation . obesity should be considered a chronic disease , especially in the context of morbid obesity , and so therapeutic approaches should be directed to women of childbearing age before they become pregnant , during pregnancy , and in the postpartum period . so , women should schedule their pregnancies , and try to achieve an optimal body weight before conceiving , to avoid potential obstetric complications arising from obesity , including gestational diabetes mellitus ( gdm ) , hypertension , complications in delivery , increased rates of c - sections , macrosomia , and congenital defects , among others.1 management of obesity is complex and involves multiple variables , with sometimes limited efficacy of the conservative approach . in this setting , bariatric surgery ( bs ) emerged as a promising approach for those cases where previous treatments failed ; specifically , it implies a net negative energy balance , enabling subsequent effective and lasting weight loss . bariatric procedures can be classified into two groups depending on whether the main mechanism involved in weight loss is restriction or malabsorption . pure restrictive techniques include the adjustable gastric band ( agb ) and laparoscopic vertical sleeve gastrectomy . recently , endoscopic specialists and surgeons have developed new intraluminal / endoscopic techniques in the treatment of obesity , with less clinical risks and economic costs , while maintaining the benefits in terms of morbidity and mortality.2 roux - en - y gastric bypass ( rygb ) is still considered the gold standard , since it combines both restrictive and malabsorptive effects , allowing for optimum efficacy of combined weight loss , without severe nutrient and vitamin deficiencies , which can develop after pure malabsorptive techniques . with the increase in the number of bariatric procedures performed over the last decade , about half of them in women of childbearing age , a remarkable increase in the number of women who get pregnant after undergoing bs has also occurred.3 weight loss has been proven to reduce adverse maternal and perinatal outcomes , and so it could also be hypothesized that bs could achieve the same positive results . however , these procedures entail the risk of nutritional deficiencies , and nutrition is a crucial aspect during pregnancy . in this regard , it is important to be aware of the possible consequences of bs on maternal and fetal outcomes . furthermore , many women conceive while they are still overweight , so the potential adverse effects of the still existing obesity should be also considered . the quality assessment of studies evaluating the effect of bs on fertility , pregnancy , and maternal and perinatal outcomes is essential for proper understanding . however , these studies are scarce , and most of them are nonrandomized and based almost exclusively on the analysis of rygb and agb . in addition , the selection of study groups , comparability , and the determination of exposure for case control or interest outcome for the cohort studies remain key issues to properly evaluating the results of each publication and drawing valid conclusions . finally , there are currently no clinical practice guidelines specifically addressing this issue , so questions regarding the effectiveness , risks , and maternal and perinatal outcomes of bs in pregnant women remain unanswered so , conclusions should be taken with caution . general recommendations advocates46 for a prudent waiting period of at least two years after bs to become pregnant . this interval was chosen since the usual duration of the initial period in which a significant and rapid weight loss occurs , and the potential risk of developing nutritional deficiencies is increased , is approximately 1224 months . however , no high - quality evidence supported this recommendation . in recent years , several studies have reported that maternal and perinatal outcomes of pregnancies occurring earlier than 1218 months after bs were not inferior.4,79 but in general , in most publications , mean interval between surgery and pregnancy was greater than 18 months , ranging from 21 to 57.4 months.5,6,10,11 in this setting , most clinicians will recommend a minimum waiting period of 1 year after bs before attempting pregnancy , to allow stabilization of body weight and to allow correct identification and treatment of any possible nutritional deficiencies that may not be evident during the first months . however , recommendations should be individualized according to specific anthropometric , clinical , and analytical parameters , as well as the patient s reproductive will . despite undergoing bs , most women conceive while still being obese . in fact , pregestational bmi was reported to be between 30.3 and 32.4 kg / m in several recent studies,5,6,10,12 an issue which could negatively influence obstetric outcomes in comparison to the normal control population . however , results are greatly variable between studies , probably due to the heterogeneity of control groups and the small number of patients reported . in this regard , some studies found no differences in maternal and fetal outcomes , others found better results , and still others identified a poorer outcome regarding rates of gdm , c - sections , and newborn s weight.6,7,1315 nutritional status and potential adverse effects derived from nutritional deficiencies are two of the most crucial aspects during pregnancy . this is especially relevant in the setting of previous bs . in fact , because nutritional requirements increase during pregnancy , special attention should be paid to proteins , iron , calcium , and folic acid . even though routine practice after bs includes prescription of long - term vitamin and micronutrient supplements , rates of compliance in pregnant women may be variable , and they usually range between 84.3% and 93.7% , according to different publications.5,16,17 very few studies report on the rate of nutrient deficiencies observed during pregnancy in women who underwent bs . in those who do , for instance , rates of anemia were reported as being low , from 0% to 24.4%.7,17,18 however , no mention of oral supplements was made . in fact , if we consider the reported serum iron and ferritin levels , and if no specific prophylactic oral supplements were taken by women of childbearing age , a significant increase in anemia would be described.19,20 furthermore , some studies have reported the need for intravenous iron supplements , or even blood transfusion , although these cases were exceptional , since , in most situations , anemia was mild or moderate , and oral supplements were enough to restore body iron deposits and hemoglobin levels.7,21,22 in this context , the current american guidelines for the management of patients after bs recommend systematic oral administration of iron supplements to all women of childbearing age.23 regarding vitamin b12 and folic acid , a small study of 39 pregnant women who had undergone bs found a rate of deficiency of 53.4% and 16.1% , respectively.19 even though systematic supplementation of vitamin b12 is also advocated after most bariatric procedures , the authors of this study acknowledged that they only prescribed it if deficiency was evidenced in analytical workup . in another prospective study of pregnant women who had undergone bs , the evaluation of nutritional deficiencies before and during gestation showed that vitamin b12 was the most prevalent deficiency ( 45% ) , even greater than that of iron ( 35%).20 in a recent publication by our group , iron deficiency was the most prevalent one observed ( 60.7% of patients ) , and it required specific additional supplementation . this was followed by vitamin 25-oh - d3 deficiency in 40.5% of patients , b12 deficiency in 22.6% , vitamin a in 7.7% , calcium in 6.6% , vitamin e in 6% and folic acid in 5.4%.5 in another recent prospective study that followed 49 women who got pregnant after bs , progressively decreasing levels of vitamins a , d , b12 , and iron were observed over the course of pregnancy , despite correct compliance with oral supplements , before and during gestation . however , no increase in the rate of obstetric complications was reported.24 a major concern among clinicians is the potential development of teratogenesis due to fat - soluble vitamin overdose . in this regard , there have been reports of kidney abnormalities in the offspring of women who took between 40,000 and 50,000 u / d of vitamin a during pregnancy . however , daily ingestion of multivitamin tablets that include around 6,0008,000 u / d of vitamin a do not seem to increase the rate of malformations related to isotretinoine ( 13-cis retinoic ) exposure , and this result is from several long - term case - control studies.25,26 for vitamin d , exposure to high doses increased the rate of fetal cardiac malformations , especially aortic stenosis , which has been associated with an intake of 4,000 iu / d . a strict monitoring and biochemical follow - up of pregnant women taking high doses of vitamins a and d significantly minimizes the risk for developing adverse events . on the other hand , insufficient fetal levels of fat - soluble vitamins , although infrequent , should also be avoided , mainly by adequate control of maternal levels of the same . table 1 summarizes our current recommendations for vitamin and micronutrient supplements in pregnancy after bs and also in the general pregnant population . the risk of maternal undernutrition or malnutrition after bs should not be overlooked , especially since it involves an increased risk of fetal malnutrition . specifically , the functional and anatomical gastrointestinal modifications following bs , along with the increased frequency of nausea and vomiting during the first trimester of pregnancy , carry the risk of not being able to fulfill nutritional and caloric daily requirements to ensure an optimal fetal development . in fact , in certain situations , artificial nutritional supplements may be necessary . in this regard , in a study published over 20 years ago , total parenteral nutrition was required in as many as 21% of pregnancies.27 however , nowadays , this is usually not necessary , and enteral approach is preferred . for instance , there were 7.7% of patients requiring oral protein supplements.7 hypoalbuminemia has also been reported during pregnancy , apparently with no clinical consequences.28 standardized management of nutritional deficiencies in the case of a pregnancy occurring after bs is documented , so guidelines for screening and treatment are usually the same as in the conventional bs adjustment , but with special attention to the specific needs during normal pregnancy . in this regard , a complete blood test , including blood count , ferritin , b12 , and fat - soluble vitamins , should be performed at least once in each trimester , especially if the procedure included malabsorptive weight loss techniques . the prevalence of gdm varies depending on the population studied and the criteria used for its definition . in any case , a clear increase ( 10%100% ) has been observed worldwide over the last 20 years.29 the linear relationship between obesity and diabetes entails an increase in the incidence of gdm of up to three - fold with increasing bmi.3033 accordingly , numerous studies have reported a reduction in the rate of gdm following the evident weight loss occurring after bs ( 0%8.9% in pregnancies after bs versus 1.6%20.8% in the control group ) . however , there were no statistically significant differences in the prevalence of gdm when women with similar bmi were compared.68,1012 it is worth noting that not all publications explicitly describe how they screened for the presence of gdm . in fact , this is still a matter of debate , since the anatomical alterations secondary to bs themselves may lead to a poor tolerance of oral glucose overloads , absorption modifications , and changes in the preestablished timings for evaluating the response , making the traditional screening methods less reliable in this particular population . gestational hypertension is the most frequent cause of pregnancy - associated hypertensive disorders ( pahd ) , which affect around 6%17% of nulliparous women and 2%4% of multiparous women.34 obesity is a well - known associated risk factor for developing this complication , entailing a two to three - fold increased risk for hypertension and preeclampsia in women with a bmi > 30 kg / m.31,35,36 the incidence of pregnancy - associated hypertension and preeclampsia increases proportionately with maternal bmi , ranging from 1.4% to 2.4% in women with normal body weight , and reaching to 3.5%14.5% in women with morbid obesity.32,37 for women who underwent bs , studies regarding the prevalence of pahd are less consistent . although the odds ratio for pahd is two to three times lower in women who underwent surgery compared with women who have not undergone surgery , the incidence of pahd ranged widely between 0% in our own spanish multicentric cohort to 35% in other studies.4,5,7,911,38 in the series reported by bennett et al,38 there was a significantly lower incidence of hypertension in women who had undergone bs ( 2.5% of cases ) , compared with that reported in obese women who were managed conservatively ( 13% of patients ) . the same occurred for the prevalence of preeclampsia : 3% in women who underwent bs versus 15% in the control group . in another study , the incidence of pahd was 16.5% in the study group , compared with 31.9% in the control group , which was composed of the same women but with evaluations from a previous pregnancy , ie , before undergoing bs.11 however , in the cohort study by kjaer et al,6 no differences were found in the rate of preeclampsia among women who had undergone bs and those who had not . likewise , in the small study by patel et al,7 which evaluated 26 pregnant women after undergoing laparoscopic rygb surgery , there were no differences in rates of pahd compared with controls ; in addition , pahd was similar in pregnancies that occurred early some studies even reported a higher prevalence of hypertension in operated women , but in these cases there were up to three times more cases of bmi > 30 kg / m than in the control group.4 taking all this data into account , in a recent meta - analysis , the authors concluded that the risk of preeclampsia seems to reduce by about approximately half in women who underwent bs.39 finally , various gastrointestinal complications may develop during pregnancy because of the bariatric procedure itself , and although these complications are not frequent , they may be severe and entail a high morbidity . the associated risk factors are not fully elucidated , and diagnosis may be frequently delayed because symptoms are commonly masked due to the usual malaises that occur during normal pregnancy ( mainly abdominal pain , nausea , and vomiting ) . in a systematic review , 20 cases required urgent abdominal surgery during pregnancy , most of them due to internal hernia after laparoscopic rygb , and five neonatal and three maternal deaths were reported.40 in another more recent study , however , 23 pregnant women underwent urgent surgery due to internal hernias , but survival rates reached 100% for mother and fetuses , and there were no severe complications.41 spontaneous abortion may occur in around 10%20% of general pregnancies , and controversy exists regarding the potential influence of obesity on this rate . in general , obesity is usually considered as a risk factor for abortion ; in this regard , the risk increases proportionately to bmi42,43 and decreases following an optimal conservative or surgical weight loss.27,44,45 however , most of the evidence comes only from retrospective studies , so conclusions should be carefully derived . maternal obesity is also associated with an increase in perinatal mortality ; in fact , once again , this increase parallels maternal bmi values . in this regard , in three large studies performed in developed countries , there was a 1.42.6 increased risk of perinatal loss in obese women in comparison to controls.31,46 in the case of pregnant women with previous history of bs , increased intrauterine and neonatal mortality was paradoxically found , in comparison to control morbidly obese women ( 1.7% versus 0.7% , odds ratio : 2.39 ; 95% confidence interval : 0.985.85 , p=0.06 ) . however , subsequent subanalysis for each individual outcome did not confirm these differences , and the absolute number of adverse events was low.12 similarly , larger cohort studies reported no differences in perinatal or neonatal mortality between women with previous bs and the control group.6,47 in addition , in a study with no control group , sheiner et al9 reported that the incidence of perinatal mortality in operated pregnant women was 0.89% , regardless of the time that had elapsed between bs and pregnancy ( less than or more than 12 months ) . concerning the rate of spontaneous abortions , there are few studies that specifically address this issue , but the rate seems to be somewhat higher than in general population , ranging between 23% and 38.9%.11,19,27 in our own multicentric cohort , however , intrauterine fetal death occurred in 3.57% of cases , and the overall rate of fetal loss was 17.9%.5 several observational studies have reported an increased rate of intrapartum complications in women with prior bs . specifically , prolonged delivery , failure in the induction period , and failure in the efficacy of epidural anesthesia have all been described.4850 for the effect on preterm delivery , results from different studies have been rather controversial . in this regard , in a recent meta - analysis , the rate of spontaneous preterm delivery was similar between obese women and controls ; however , induced preterm delivery was in fact more frequent in the former , probably due to the associated comorbidities involved in these cases , such as gdm and pahd.51 reports of the follow - up of pregnant women with prior bs describe variable incidences of preterm delivery , but all below 10% ( range 1.9%9.7% ) . furthermore , most series show no differences regarding this adverse outcome between women who underwent bs and those who did not,12 although , in some cases , an overall lower mean gestational age was documented in operated women.12,52 several other studies without a control group found no differences in the rate of preterm delivery regarding the type of bariatric procedure , or the time lapse between surgery and conception.4,5,9,10 the study by roos et al,47 however , did find a greater incidence of preterm delivery in women who had undergone bs in comparison to the control group : 9.7% versus 6.7% ; interestingly , women with lower bmi ( below 30 kg / m ) were the ones with a greater incidence of this outcome . as previously outlined , obesity is a major risk factor for the need for c - sections , with this risk increasing for higher bmi values . for women with prior bs , however , rates for c - sections have been variable across the literature . for instance , in several recent studies , there were no statistically significant differences in rates of c - sections between operated women and bmi - matched controls , or between the operated group and conservatively managed obese women . rates for c - sections in these operated women ranged between 18.3% and 60% , while the rate in the control group was between 14.4% and 28.7%.4,6,52 there have been some further studies reporting that bs was a clear risk factor for c - section , even after adjusting for other potential confounding factors.9 a french study including 24 pregnancies following rygb surgery ( exposed group ) were compared with two different control groups : a normal bmi group and a group of 120 pregnancies matched by age , parity , and pregnancy bmi . the study showed increased numbers of reduced birth weight and c - section in the exposed group.13 there has also been several attempts to try to elucidate if this is due to a potential underlying cause involving the specific bariatric technique performed , or the period of time between bs and pregnancy , but no definite conclusion has been reached yet.4,9,10 in our own population , c - sections were performed in 19.4% of cases , but we did not observe any association with the type of bariatric technique performed.5 finally , obstetric outcomes were compared in another retrospective case control study with 427 obese women , 13 of whom underwent agb and 414 who did not . this study has shown a lower incidence of adverse obstetric outcomes in those in whom agb was performed compared with those who did not undergo agb , including a lower rate of c - section in the operated group.15 similarly , patel et al,7 in a population - based study comparing 259 pregnancies after bs with a normal pregnant population group , have found that previous bs was not associated with adverse perinatal outcomes.19 however , in this study , only 10.7% of the operated women remained obese before pregnancy.14 despite these controversial results , there is no presumable physiopathologic reason to hypothesize that c - sections could be more frequent after bs , and understanding this increased rate is somewhat difficult . several underlying reasons have been suggested to explain this increased rate of c - sections , including a history of a prior c - section , which is the most relevant issue , but also other variables such as maternal obesity , maternal choice , clinicians preconceptions regarding obstetric risk in obese women , and fetal positioning , among others . in fact , very few studies describe the specific reason for performing c - sections in their results . therefore , a thorough and complete objective evaluation should be carried out in these patients , balancing the risks and benefits for implementing this type of delivery , so as to be able to rationally recommend the best delivery approach for women with a prior history of bs . maternal obesity has long been associated with an increased rate of large - for - gestational - age ( lga ) newborns , while being a protective factor for small - for - gestational - age ( sga ) infants , even after adjusting for other maternal comorbidities.15,31,53 in addition , weight gain during pregnancy was found to positively correlate with the newborn s body weight.54 this influence of maternal weight on neonatal weight is important at birth time , but also , and probably most importantly , there is a great amount of scientific evidence supporting that it significantly influences intrauterine fetal development , portending an increased risk of overweight and obesity during adolescence and adulthood.5557 however , women with prior bs have been observed to have a lower rate of lga newborns compared with nonoperated control obese women ( ranging between 1.2% and 7.3% of cases).5,11,12,27,47,52 in fact , in recent years , many studies have reported that women who get pregnant after bs tend to deliver newborns with an overall lower body weight , and a higher rate of sga infants ( 5.2%27.8% ) , in comparison to nonoperated women.5,11,12,27,47,52 in a retrospective study by kjaer et al,6 339 women with a singleton delivery after bs were matched by pregnancy bmi , maternal age , parity , and date of delivery with 1,277 non - operated women . they found that babies born after maternal bs had lower birth weight , lower gestational age , 3.3-times lower risk of lga , and 2.3-times higher risk of sga than infants born to matched women without bs . in addition , a recent retrospective , matched - control cohort study has compared birth weights of babies born to women with pregnancies before and after rygb surgery.54 interestingly , the authors communicated that women who had undergone rygb had a significantly lower risk for having an lga newborn , but also a significantly increased risk for delivering an sga neonate , than nonoperated women closely matched by age and bmi before pregnancy.54 the variability and heterogeneity of the incidence reported may be due to differences in the definition of sga , and the type of bariatric procedure performed , among other reasons . however , a full explanation for this higher rate of sga infants after bs is still unknown . a possible mechanism could be that women with prior bs may present a higher risk for nutrient deficiency , and thus , favor fetal deficiencies . however , the negative influence of this issue in the long - term follow - up still deserves further investigation.5,11,12,27,47,52 however , the number of malabsorptive techniques performed has significantly reduced over the last couple of years , allowing other surgical approaches , with less adverse effects , to gain popularity ; thus , a reduction in the incidence of sga infants may be expected . there are also some publications reporting the outcomes of the offspring of women who had previously undergone bs . for instance , smith et al58 performed an interesting study in which they hypothesized that maternal weight loss due to bs could indeed affect the intrauterine environment and , consequently , affect the potential risk of children developing obesity during life . they observed that offspring born after maternal rygb surgery exhibited increased insulin sensitivity and improved lipid profiles compared with offspring born before maternal surgery , including a lower risk for obesity , even though neonatal body weight was lower and many women were still obese when they conceived.59 this finding suggests that intrauterine environment may be even more relevant for pregnancies in women with previous bs , because of its influence on epigenetics and subsequent development of obesity and other cardiovascular risk factors.58 in fact , kral et al59 evaluated the rates of overweight and obesity in children born to mothers with previous bs and found that they were lower . in another study , the authors investigated the body weight of siblings born of the same mother before and after bpd for obesity . at 1 and 6 years , the body weight was described as similar in both groups . however , at 12 years of age , a higher percentage of those born before bpd were considered overweight . the main strength of this study is that the influences of the genetic pattern and environmental and educational factors were minimized.60 in the same way , another study performed with 15 mothers with siblings born before and after bpd with duodenal switch has shown that the hostile dysmetabolic intrauterine environment modulates genotype and gene expression levels in the offspring , promoting the development of obesity and subsequent cardiometabolic risk factors in the offspring born from obese women.61 moreover , in another large and more recent study in which bmi was considered only until the age of 10 years in the children of women with prior bs , there were no differences or any specific association with pregestational maternal bmi . the authors acknowledged that they could not establish definite conclusions regarding the effect of bs in the offspring s long - term body weight.62 likewise , in a swedish cohort of women with at least one child born before and after bs , no differences in bmi score were observed between siblings when they arrive at preschool age.63 therefore , further long - term studies are needed to better understand this issue . finally , there are several reports of an increased incidence of congenital anomalies in children born after maternal bs . in this regard , the main alteration concerns neural tube defects , but cardiac abnormalities may also occur , particularly septal defects , as well as cleft lip and cleft palate , anorectal atresia , hydrocephalus , and limb shortening.64 this increased incidence of adverse congenital outcomes conveys an added difficulty to a correct identification of up to 15% of normal structures during the ultrasound at week 20 . in fact , the greater the maternal bmi is , the less visible the fetal structures may be , entailing a potential risk of an increased rate of unidentified congenital malformations and defects.65,66 in studies comparing the outcomes of operated pregnant women with those of a control group , in general , there were no differences in the prevalence of congenital malformations , although rates in both groups were diverse , ranging from 0 to 5.1%.7,9,12 conversely , in other studies without a control group , the prevalence was also variable , but low ( 0%2.2%).4,5,27,67 in addition , there have been several publications of isolated case reports or even case series , in which specific neonatal diseases were reported in association with maternal nutrient deficiencies . even though these cases are rare , they may be frequent in the setting of malabsorptive procedures , or poor compliance with medical follow - up and nutritional supplements . in this regard , the most frequently described maternal micronutrient deficiency is that of folic acid , which entails a high risk of fetal neural tube defects.68 moreover , there have been case reports of maternal and fetal hypercoagulability due to vitamin k deficiency following bpd , ophthalmic and renal malformations due to severe maternal and fetal vitamin a deficiency , and megaloblastic anemia due to vitamin b12 deficiency.6971 general recommendations advocates46 for a prudent waiting period of at least two years after bs to become pregnant . this interval was chosen since the usual duration of the initial period in which a significant and rapid weight loss occurs , and the potential risk of developing nutritional deficiencies is increased , is approximately 1224 months . however , no high - quality evidence supported this recommendation . in recent years , several studies have reported that maternal and perinatal outcomes of pregnancies occurring earlier than 1218 months after bs were not inferior.4,79 but in general , in most publications , mean interval between surgery and pregnancy was greater than 18 months , ranging from 21 to 57.4 months.5,6,10,11 in this setting , most clinicians will recommend a minimum waiting period of 1 year after bs before attempting pregnancy , to allow stabilization of body weight and to allow correct identification and treatment of any possible nutritional deficiencies that may not be evident during the first months . however , recommendations should be individualized according to specific anthropometric , clinical , and analytical parameters , as well as the patient s reproductive will . despite undergoing bs , most women conceive while still being obese . in fact , pregestational bmi was reported to be between 30.3 and 32.4 kg / m in several recent studies,5,6,10,12 an issue which could negatively influence obstetric outcomes in comparison to the normal control population . however , results are greatly variable between studies , probably due to the heterogeneity of control groups and the small number of patients reported . in this regard , some studies found no differences in maternal and fetal outcomes , others found better results , and still others identified a poorer outcome regarding rates of gdm , c - sections , and newborn s weight.6,7,1315 nutritional status and potential adverse effects derived from nutritional deficiencies are two of the most crucial aspects during pregnancy . in fact , because nutritional requirements increase during pregnancy , special attention should be paid to proteins , iron , calcium , and folic acid . even though routine practice after bs includes prescription of long - term vitamin and micronutrient supplements , rates of compliance in pregnant women may be variable , and they usually range between 84.3% and 93.7% , according to different publications.5,16,17 very few studies report on the rate of nutrient deficiencies observed during pregnancy in women who underwent bs . in those who do , for instance , rates of anemia were reported as being low , from 0% to 24.4%.7,17,18 however , no mention of oral supplements was made . in fact , if we consider the reported serum iron and ferritin levels , and if no specific prophylactic oral supplements were taken by women of childbearing age , a significant increase in anemia would be described.19,20 furthermore , some studies have reported the need for intravenous iron supplements , or even blood transfusion , although these cases were exceptional , since , in most situations , anemia was mild or moderate , and oral supplements were enough to restore body iron deposits and hemoglobin levels.7,21,22 in this context , the current american guidelines for the management of patients after bs recommend systematic oral administration of iron supplements to all women of childbearing age.23 regarding vitamin b12 and folic acid , a small study of 39 pregnant women who had undergone bs found a rate of deficiency of 53.4% and 16.1% , respectively.19 even though systematic supplementation of vitamin b12 is also advocated after most bariatric procedures , the authors of this study acknowledged that they only prescribed it if deficiency was evidenced in analytical workup . in another prospective study of pregnant women who had undergone bs , the evaluation of nutritional deficiencies before and during gestation showed that vitamin b12 was the most prevalent deficiency ( 45% ) , even greater than that of iron ( 35%).20 in a recent publication by our group , iron deficiency was the most prevalent one observed ( 60.7% of patients ) , and it required specific additional supplementation . this was followed by vitamin 25-oh - d3 deficiency in 40.5% of patients , b12 deficiency in 22.6% , vitamin a in 7.7% , calcium in 6.6% , vitamin e in 6% and folic acid in 5.4%.5 in another recent prospective study that followed 49 women who got pregnant after bs , progressively decreasing levels of vitamins a , d , b12 , and iron were observed over the course of pregnancy , despite correct compliance with oral supplements , before and during gestation . however , no increase in the rate of obstetric complications was reported.24 a major concern among clinicians is the potential development of teratogenesis due to fat - soluble vitamin overdose . in this regard , there have been reports of kidney abnormalities in the offspring of women who took between 40,000 and 50,000 u / d of vitamin a during pregnancy . however , daily ingestion of multivitamin tablets that include around 6,0008,000 u / d of vitamin a do not seem to increase the rate of malformations related to isotretinoine ( 13-cis retinoic ) exposure , and this result is from several long - term case - control studies.25,26 for vitamin d , exposure to high doses increased the rate of fetal cardiac malformations , especially aortic stenosis , which has been associated with an intake of 4,000 iu / d . a strict monitoring and biochemical follow - up of pregnant women taking high doses of vitamins a and d significantly minimizes the risk for developing adverse events . on the other hand , insufficient fetal levels of fat - soluble vitamins , although infrequent , should also be avoided , mainly by adequate control of maternal levels of the same . table 1 summarizes our current recommendations for vitamin and micronutrient supplements in pregnancy after bs and also in the general pregnant population . the risk of maternal undernutrition or malnutrition after bs should not be overlooked , especially since it involves an increased risk of fetal malnutrition . specifically , the functional and anatomical gastrointestinal modifications following bs , along with the increased frequency of nausea and vomiting during the first trimester of pregnancy , carry the risk of not being able to fulfill nutritional and caloric daily requirements to ensure an optimal fetal development . in fact , in certain situations , artificial nutritional supplements may be necessary . in this regard , in a study published over 20 years ago , total parenteral nutrition was required in as many as 21% of pregnancies.27 however , nowadays , this is usually not necessary , and enteral approach is preferred . for instance , there were 7.7% of patients requiring oral protein supplements.7 hypoalbuminemia has also been reported during pregnancy , apparently with no clinical consequences.28 standardized management of nutritional deficiencies in the case of a pregnancy occurring after bs is documented , so guidelines for screening and treatment are usually the same as in the conventional bs adjustment , but with special attention to the specific needs during normal pregnancy . in this regard , a complete blood test , including blood count , ferritin , b12 , and fat - soluble vitamins , should be performed at least once in each trimester , especially if the procedure included malabsorptive weight loss techniques . the prevalence of gdm varies depending on the population studied and the criteria used for its definition . in any case , a clear increase ( 10%100% ) has been observed worldwide over the last 20 years.29 the linear relationship between obesity and diabetes entails an increase in the incidence of gdm of up to three - fold with increasing bmi.3033 accordingly , numerous studies have reported a reduction in the rate of gdm following the evident weight loss occurring after bs ( 0%8.9% in pregnancies after bs versus 1.6%20.8% in the control group ) . however , there were no statistically significant differences in the prevalence of gdm when women with similar bmi were compared.68,1012 it is worth noting that not all publications explicitly describe how they screened for the presence of gdm . in fact , this is still a matter of debate , since the anatomical alterations secondary to bs themselves may lead to a poor tolerance of oral glucose overloads , absorption modifications , and changes in the preestablished timings for evaluating the response , making the traditional screening methods less reliable in this particular population . gestational hypertension is the most frequent cause of pregnancy - associated hypertensive disorders ( pahd ) , which affect around 6%17% of nulliparous women and 2%4% of multiparous women.34 obesity is a well - known associated risk factor for developing this complication , entailing a two to three - fold increased risk for hypertension and preeclampsia in women with a bmi > 30 kg / m.31,35,36 the incidence of pregnancy - associated hypertension and preeclampsia increases proportionately with maternal bmi , ranging from 1.4% to 2.4% in women with normal body weight , and reaching to 3.5%14.5% in women with morbid obesity.32,37 for women who underwent bs , studies regarding the prevalence of pahd are less consistent . although the odds ratio for pahd is two to three times lower in women who underwent surgery compared with women who have not undergone surgery , the incidence of pahd ranged widely between 0% in our own spanish multicentric cohort to 35% in other studies.4,5,7,911,38 in the series reported by bennett et al,38 there was a significantly lower incidence of hypertension in women who had undergone bs ( 2.5% of cases ) , compared with that reported in obese women who were managed conservatively ( 13% of patients ) . the same occurred for the prevalence of preeclampsia : 3% in women who underwent bs versus 15% in the control group . in another study , the incidence of pahd was 16.5% in the study group , compared with 31.9% in the control group , which was composed of the same women but with evaluations from a previous pregnancy , ie , before undergoing bs.11 however , in the cohort study by kjaer et al,6 no differences were found in the rate of preeclampsia among women who had undergone bs and those who had not . likewise , in the small study by patel et al,7 which evaluated 26 pregnant women after undergoing laparoscopic rygb surgery , there were no differences in rates of pahd compared with controls ; in addition , pahd was similar in pregnancies that occurred early ( < 12 months ) versus later ( > 18 months ) after bs . some studies even reported a higher prevalence of hypertension in operated women , but in these cases there were up to three times more cases of bmi > 30 kg / m than in the control group.4 taking all this data into account , in a recent meta - analysis , the authors concluded that the risk of preeclampsia seems to reduce by about approximately half in women who underwent bs.39 finally , various gastrointestinal complications may develop during pregnancy because of the bariatric procedure itself , and although these complications are not frequent , they may be severe and entail a high morbidity . the associated risk factors are not fully elucidated , and diagnosis may be frequently delayed because symptoms are commonly masked due to the usual malaises that occur during normal pregnancy ( mainly abdominal pain , nausea , and vomiting ) . in a systematic review , 20 cases required urgent abdominal surgery during pregnancy , most of them due to internal hernia after laparoscopic rygb , and five neonatal and three maternal deaths were reported.40 in another more recent study , however , 23 pregnant women underwent urgent surgery due to internal hernias , but survival rates reached 100% for mother and fetuses , and there were no severe complications.41 spontaneous abortion may occur in around 10%20% of general pregnancies , and controversy exists regarding the potential influence of obesity on this rate . in general , obesity is usually considered as a risk factor for abortion ; in this regard , the risk increases proportionately to bmi42,43 and decreases following an optimal conservative or surgical weight loss.27,44,45 however , most of the evidence comes only from retrospective studies , so conclusions should be carefully derived . maternal obesity is also associated with an increase in perinatal mortality ; in fact , once again , this increase parallels maternal bmi values . in this regard , in three large studies performed in developed countries , there was a 1.42.6 increased risk of perinatal loss in obese women in comparison to controls.31,46 in the case of pregnant women with previous history of bs , increased intrauterine and neonatal mortality was paradoxically found , in comparison to control morbidly obese women ( 1.7% versus 0.7% , odds ratio : 2.39 ; 95% confidence interval : 0.985.85 , p=0.06 ) . however , subsequent subanalysis for each individual outcome did not confirm these differences , and the absolute number of adverse events was low.12 similarly , larger cohort studies reported no differences in perinatal or neonatal mortality between women with previous bs and the control group.6,47 in addition , in a study with no control group , sheiner et al9 reported that the incidence of perinatal mortality in operated pregnant women was 0.89% , regardless of the time that had elapsed between bs and pregnancy ( less than or more than 12 months ) . concerning the rate of spontaneous abortions , there are few studies that specifically address this issue , but the rate seems to be somewhat higher than in general population , ranging between 23% and 38.9%.11,19,27 in our own multicentric cohort , however , intrauterine fetal death occurred in 3.57% of cases , and the overall rate of fetal loss was 17.9%.5 several observational studies have reported an increased rate of intrapartum complications in women with prior bs . specifically , prolonged delivery , failure in the induction period , and failure in the efficacy of epidural anesthesia have all been described.4850 for the effect on preterm delivery , results from different studies have been rather controversial . in this regard , in a recent meta - analysis , the rate of spontaneous preterm delivery was similar between obese women and controls ; however , induced preterm delivery was in fact more frequent in the former , probably due to the associated comorbidities involved in these cases , such as gdm and pahd.51 reports of the follow - up of pregnant women with prior bs describe variable incidences of preterm delivery , but all below 10% ( range 1.9%9.7% ) . furthermore , most series show no differences regarding this adverse outcome between women who underwent bs and those who did not,12 although , in some cases , an overall lower mean gestational age was documented in operated women.12,52 several other studies without a control group found no differences in the rate of preterm delivery regarding the type of bariatric procedure , or the time lapse between surgery and conception.4,5,9,10 the study by roos et al,47 however , did find a greater incidence of preterm delivery in women who had undergone bs in comparison to the control group : 9.7% versus 6.7% ; interestingly , women with lower bmi ( below 30 kg / m ) were the ones with a greater incidence of this outcome . as previously outlined , obesity is a major risk factor for the need for c - sections , with this risk increasing for higher bmi values . for women with prior bs , however , rates for c - sections have been variable across the literature . for instance , in several recent studies , there were no statistically significant differences in rates of c - sections between operated women and bmi - matched controls , or between the operated group and conservatively managed obese women . rates for c - sections in these operated women ranged between 18.3% and 60% , while the rate in the control group was between 14.4% and 28.7%.4,6,52 there have been some further studies reporting that bs was a clear risk factor for c - section , even after adjusting for other potential confounding factors.9 a french study including 24 pregnancies following rygb surgery ( exposed group ) were compared with two different control groups : a normal bmi group and a group of 120 pregnancies matched by age , parity , and pregnancy bmi . the study showed increased numbers of reduced birth weight and c - section in the exposed group.13 there has also been several attempts to try to elucidate if this is due to a potential underlying cause involving the specific bariatric technique performed , or the period of time between bs and pregnancy , but no definite conclusion has been reached yet.4,9,10 in our own population , c - sections were performed in 19.4% of cases , but we did not observe any association with the type of bariatric technique performed.5 finally , obstetric outcomes were compared in another retrospective case control study with 427 obese women , 13 of whom underwent agb and 414 who did not . this study has shown a lower incidence of adverse obstetric outcomes in those in whom agb was performed compared with those who did not undergo agb , including a lower rate of c - section in the operated group.15 similarly , patel et al,7 in a population - based study comparing 259 pregnancies after bs with a normal pregnant population group , have found that previous bs was not associated with adverse perinatal outcomes.19 however , in this study , only 10.7% of the operated women remained obese before pregnancy.14 despite these controversial results , there is no presumable physiopathologic reason to hypothesize that c - sections could be more frequent after bs , and understanding this increased rate is somewhat difficult . several underlying reasons have been suggested to explain this increased rate of c - sections , including a history of a prior c - section , which is the most relevant issue , but also other variables such as maternal obesity , maternal choice , clinicians preconceptions regarding obstetric risk in obese women , and fetal positioning , among others . in fact , very few studies describe the specific reason for performing c - sections in their results . therefore , a thorough and complete objective evaluation should be carried out in these patients , balancing the risks and benefits for implementing this type of delivery , so as to be able to rationally recommend the best delivery approach for women with a prior history of bs . maternal obesity has long been associated with an increased rate of large - for - gestational - age ( lga ) newborns , while being a protective factor for small - for - gestational - age ( sga ) infants , even after adjusting for other maternal comorbidities.15,31,53 in addition , weight gain during pregnancy was found to positively correlate with the newborn s body weight.54 this influence of maternal weight on neonatal weight is important at birth time , but also , and probably most importantly , there is a great amount of scientific evidence supporting that it significantly influences intrauterine fetal development , portending an increased risk of overweight and obesity during adolescence and adulthood.5557 however , women with prior bs have been observed to have a lower rate of lga newborns compared with nonoperated control obese women ( ranging between 1.2% and 7.3% of cases).5,11,12,27,47,52 in fact , in recent years , many studies have reported that women who get pregnant after bs tend to deliver newborns with an overall lower body weight , and a higher rate of sga infants ( 5.2%27.8% ) , in comparison to nonoperated women.5,11,12,27,47,52 in a retrospective study by kjaer et al,6 339 women with a singleton delivery after bs were matched by pregnancy bmi , maternal age , parity , and date of delivery with 1,277 non - operated women . they found that babies born after maternal bs had lower birth weight , lower gestational age , 3.3-times lower risk of lga , and 2.3-times higher risk of sga than infants born to matched women without bs . in addition , a recent retrospective , matched - control cohort study has compared birth weights of babies born to women with pregnancies before and after rygb surgery.54 interestingly , the authors communicated that women who had undergone rygb had a significantly lower risk for having an lga newborn , but also a significantly increased risk for delivering an sga neonate , than nonoperated women closely matched by age and bmi before pregnancy.54 the variability and heterogeneity of the incidence reported may be due to differences in the definition of sga , and the type of bariatric procedure performed , among other reasons . however , a full explanation for this higher rate of sga infants after bs is still unknown . a possible mechanism could be that women with prior bs may present a higher risk for nutrient deficiency , and thus , favor fetal deficiencies . however , the negative influence of this issue in the long - term follow - up still deserves further investigation.5,11,12,27,47,52 however , the number of malabsorptive techniques performed has significantly reduced over the last couple of years , allowing other surgical approaches , with less adverse effects , to gain popularity ; thus , a reduction in the incidence of sga infants may be expected . there are also some publications reporting the outcomes of the offspring of women who had previously undergone bs . for instance , smith et al58 performed an interesting study in which they hypothesized that maternal weight loss due to bs could indeed affect the intrauterine environment and , consequently , affect the potential risk of children developing obesity during life . they observed that offspring born after maternal rygb surgery exhibited increased insulin sensitivity and improved lipid profiles compared with offspring born before maternal surgery , including a lower risk for obesity , even though neonatal body weight was lower and many women were still obese when they conceived.59 this finding suggests that intrauterine environment may be even more relevant for pregnancies in women with previous bs , because of its influence on epigenetics and subsequent development of obesity and other cardiovascular risk factors.58 in fact , kral et al59 evaluated the rates of overweight and obesity in children born to mothers with previous bs and found that they were lower . in another study , the authors investigated the body weight of siblings born of the same mother before and after bpd for obesity . at 1 and 6 years however , at 12 years of age , a higher percentage of those born before bpd were considered overweight . the main strength of this study is that the influences of the genetic pattern and environmental and educational factors were minimized.60 in the same way , another study performed with 15 mothers with siblings born before and after bpd with duodenal switch has shown that the hostile dysmetabolic intrauterine environment modulates genotype and gene expression levels in the offspring , promoting the development of obesity and subsequent cardiometabolic risk factors in the offspring born from obese women.61 moreover , in another large and more recent study in which bmi was considered only until the age of 10 years in the children of women with prior bs , there were no differences or any specific association with pregestational maternal bmi . the authors acknowledged that they could not establish definite conclusions regarding the effect of bs in the offspring s long - term body weight.62 likewise , in a swedish cohort of women with at least one child born before and after bs , no differences in bmi score were observed between siblings when they arrive at preschool age.63 therefore , further long - term studies are needed to better understand this issue . finally , there are several reports of an increased incidence of congenital anomalies in children born after maternal bs . in this regard , the main alteration concerns neural tube defects , but cardiac abnormalities may also occur , particularly septal defects , as well as cleft lip and cleft palate , anorectal atresia , hydrocephalus , and limb shortening.64 this increased incidence of adverse congenital outcomes conveys an added difficulty to a correct identification of up to 15% of normal structures during the ultrasound at week 20 . in fact , the greater the maternal bmi is , the less visible the fetal structures may be , entailing a potential risk of an increased rate of unidentified congenital malformations and defects.65,66 in studies comparing the outcomes of operated pregnant women with those of a control group , in general , there were no differences in the prevalence of congenital malformations , although rates in both groups were diverse , ranging from 0 to 5.1%.7,9,12 conversely , in other studies without a control group , the prevalence was also variable , but low ( 0%2.2%).4,5,27,67 in addition , there have been several publications of isolated case reports or even case series , in which specific neonatal diseases were reported in association with maternal nutrient deficiencies . even though these cases are rare , they may be frequent in the setting of malabsorptive procedures , or poor compliance with medical follow - up and nutritional supplements . in this regard , the most frequently described maternal micronutrient deficiency is that of folic acid , which entails a high risk of fetal neural tube defects.68 moreover , there have been case reports of maternal and fetal hypercoagulability due to vitamin k deficiency following bpd , ophthalmic and renal malformations due to severe maternal and fetal vitamin a deficiency , and megaloblastic anemia due to vitamin b12 deficiency.6971 . however , an optimal and prudent waiting period between bs and pregnancy should be individually advised , at least until body weight stabilizes and potential nutritional deficiencies are overcome with targeted treatment . no significant differences in maternal and fetal outcomes have been found between pregnancies occurring before or after 12 months of bs . however , the number of cases reported in each study is too small to be able to draw definite conclusions . nutritional deficiencies that may be present before bs usually exacerbate during pregnancy , so it is highly recommended that strict medical monitoring and correct micronutrient and supplement compliance are observed . available scientific evidence suggests that the risk of gdm and pahd significantly decreases in women who previously underwent bs , in comparison to morbidly obese patients who were managed conservatively , and the risk may even be like that of women with a similar bmi in whom surgery was not performed . there is still insufficient evidence to suggest that bs reduces the risk of c - sections . on the other hand , there is enough data advising that women with prior bs have an increased risk of sga newborns in comparison to nonoperated obese pregnant women , but the relevance of this finding in the long - term follow - up still deserves further investigation.72

the significant increase in the prevalence of obesity has led to an increase in the number of obese women who become pregnant . in this setting , in recent years , there has been an exponential rise in the number of bariatric procedures , with approximately half of them performed in women of childbearing age , and a remarkable surge in the number of women who become pregnant after having undergone bariatric surgery ( bs ) . these procedures entail the risk of nutritional deficiencies , and nutrition is a crucial aspect during pregnancy . therefore , knowledge and awareness of the consequences of these techniques on maternal and fetal outcomes is essential . current evidence suggests a better overall obstetric outcome after bs , in comparison to morbid obese women managed conservatively , with a reduction in the prevalence of gestational diabetes mellitus , pregnancy - associated hypertensive disorders , macrosomia , and congenital defects . however , the risk of potential maternal nutritional deficiencies and newborns small for gestational age can not be overlooked . results concerning the incidence of preterm delivery and the number of c - sections are less consistent . in this paper , we review the updated evidence regarding the impact of bs on pregnancy .

Introduction Time lapse between BS and pregnancy Maternal nutritional issues Complications during pregnancy Fetal loss and perinatal deaths Peripartum issues Fetal outcomes Conclusion

in recent years , there has been a significant increase in the number of obese women who become pregnant ; in fact , obesity has currently become one of the most important gestational risk factors . so , women should schedule their pregnancies , and try to achieve an optimal body weight before conceiving , to avoid potential obstetric complications arising from obesity , including gestational diabetes mellitus ( gdm ) , hypertension , complications in delivery , increased rates of c - sections , macrosomia , and congenital defects , among others.1 management of obesity is complex and involves multiple variables , with sometimes limited efficacy of the conservative approach . with the increase in the number of bariatric procedures performed over the last decade , about half of them in women of childbearing age , a remarkable increase in the number of women who get pregnant after undergoing bs has also occurred.3 weight loss has been proven to reduce adverse maternal and perinatal outcomes , and so it could also be hypothesized that bs could achieve the same positive results . however , these procedures entail the risk of nutritional deficiencies , and nutrition is a crucial aspect during pregnancy . in this regard , it is important to be aware of the possible consequences of bs on maternal and fetal outcomes . in recent years , several studies have reported that maternal and perinatal outcomes of pregnancies occurring earlier than 1218 months after bs were not inferior.4,79 but in general , in most publications , mean interval between surgery and pregnancy was greater than 18 months , ranging from 21 to 57.4 months.5,6,10,11 in this setting , most clinicians will recommend a minimum waiting period of 1 year after bs before attempting pregnancy , to allow stabilization of body weight and to allow correct identification and treatment of any possible nutritional deficiencies that may not be evident during the first months . in this regard , some studies found no differences in maternal and fetal outcomes , others found better results , and still others identified a poorer outcome regarding rates of gdm , c - sections , and newborn s weight.6,7,1315 nutritional status and potential adverse effects derived from nutritional deficiencies are two of the most crucial aspects during pregnancy . in fact , if we consider the reported serum iron and ferritin levels , and if no specific prophylactic oral supplements were taken by women of childbearing age , a significant increase in anemia would be described.19,20 furthermore , some studies have reported the need for intravenous iron supplements , or even blood transfusion , although these cases were exceptional , since , in most situations , anemia was mild or moderate , and oral supplements were enough to restore body iron deposits and hemoglobin levels.7,21,22 in this context , the current american guidelines for the management of patients after bs recommend systematic oral administration of iron supplements to all women of childbearing age.23 regarding vitamin b12 and folic acid , a small study of 39 pregnant women who had undergone bs found a rate of deficiency of 53.4% and 16.1% , respectively.19 even though systematic supplementation of vitamin b12 is also advocated after most bariatric procedures , the authors of this study acknowledged that they only prescribed it if deficiency was evidenced in analytical workup . in any case , a clear increase ( 10%100% ) has been observed worldwide over the last 20 years.29 the linear relationship between obesity and diabetes entails an increase in the incidence of gdm of up to three - fold with increasing bmi.3033 accordingly , numerous studies have reported a reduction in the rate of gdm following the evident weight loss occurring after bs ( 0%8.9% in pregnancies after bs versus 1.6%20.8% in the control group ) . gestational hypertension is the most frequent cause of pregnancy - associated hypertensive disorders ( pahd ) , which affect around 6%17% of nulliparous women and 2%4% of multiparous women.34 obesity is a well - known associated risk factor for developing this complication , entailing a two to three - fold increased risk for hypertension and preeclampsia in women with a bmi > 30 kg / m.31,35,36 the incidence of pregnancy - associated hypertension and preeclampsia increases proportionately with maternal bmi , ranging from 1.4% to 2.4% in women with normal body weight , and reaching to 3.5%14.5% in women with morbid obesity.32,37 for women who underwent bs , studies regarding the prevalence of pahd are less consistent . likewise , in the small study by patel et al,7 which evaluated 26 pregnant women after undergoing laparoscopic rygb surgery , there were no differences in rates of pahd compared with controls ; in addition , pahd was similar in pregnancies that occurred early some studies even reported a higher prevalence of hypertension in operated women , but in these cases there were up to three times more cases of bmi > 30 kg / m than in the control group.4 taking all this data into account , in a recent meta - analysis , the authors concluded that the risk of preeclampsia seems to reduce by about approximately half in women who underwent bs.39 finally , various gastrointestinal complications may develop during pregnancy because of the bariatric procedure itself , and although these complications are not frequent , they may be severe and entail a high morbidity . in this regard , in three large studies performed in developed countries , there was a 1.42.6 increased risk of perinatal loss in obese women in comparison to controls.31,46 in the case of pregnant women with previous history of bs , increased intrauterine and neonatal mortality was paradoxically found , in comparison to control morbidly obese women ( 1.7% versus 0.7% , odds ratio : 2.39 ; 95% confidence interval : 0.985.85 , p=0.06 ) . in this regard , in a recent meta - analysis , the rate of spontaneous preterm delivery was similar between obese women and controls ; however , induced preterm delivery was in fact more frequent in the former , probably due to the associated comorbidities involved in these cases , such as gdm and pahd.51 reports of the follow - up of pregnant women with prior bs describe variable incidences of preterm delivery , but all below 10% ( range 1.9%9.7% ) . furthermore , most series show no differences regarding this adverse outcome between women who underwent bs and those who did not,12 although , in some cases , an overall lower mean gestational age was documented in operated women.12,52 several other studies without a control group found no differences in the rate of preterm delivery regarding the type of bariatric procedure , or the time lapse between surgery and conception.4,5,9,10 the study by roos et al,47 however , did find a greater incidence of preterm delivery in women who had undergone bs in comparison to the control group : 9.7% versus 6.7% ; interestingly , women with lower bmi ( below 30 kg / m ) were the ones with a greater incidence of this outcome . this study has shown a lower incidence of adverse obstetric outcomes in those in whom agb was performed compared with those who did not undergo agb , including a lower rate of c - section in the operated group.15 similarly , patel et al,7 in a population - based study comparing 259 pregnancies after bs with a normal pregnant population group , have found that previous bs was not associated with adverse perinatal outcomes.19 however , in this study , only 10.7% of the operated women remained obese before pregnancy.14 despite these controversial results , there is no presumable physiopathologic reason to hypothesize that c - sections could be more frequent after bs , and understanding this increased rate is somewhat difficult . maternal obesity has long been associated with an increased rate of large - for - gestational - age ( lga ) newborns , while being a protective factor for small - for - gestational - age ( sga ) infants , even after adjusting for other maternal comorbidities.15,31,53 in addition , weight gain during pregnancy was found to positively correlate with the newborn s body weight.54 this influence of maternal weight on neonatal weight is important at birth time , but also , and probably most importantly , there is a great amount of scientific evidence supporting that it significantly influences intrauterine fetal development , portending an increased risk of overweight and obesity during adolescence and adulthood.5557 however , women with prior bs have been observed to have a lower rate of lga newborns compared with nonoperated control obese women ( ranging between 1.2% and 7.3% of cases).5,11,12,27,47,52 in fact , in recent years , many studies have reported that women who get pregnant after bs tend to deliver newborns with an overall lower body weight , and a higher rate of sga infants ( 5.2%27.8% ) , in comparison to nonoperated women.5,11,12,27,47,52 in a retrospective study by kjaer et al,6 339 women with a singleton delivery after bs were matched by pregnancy bmi , maternal age , parity , and date of delivery with 1,277 non - operated women . however , the negative influence of this issue in the long - term follow - up still deserves further investigation.5,11,12,27,47,52 however , the number of malabsorptive techniques performed has significantly reduced over the last couple of years , allowing other surgical approaches , with less adverse effects , to gain popularity ; thus , a reduction in the incidence of sga infants may be expected . in this regard , the most frequently described maternal micronutrient deficiency is that of folic acid , which entails a high risk of fetal neural tube defects.68 moreover , there have been case reports of maternal and fetal hypercoagulability due to vitamin k deficiency following bpd , ophthalmic and renal malformations due to severe maternal and fetal vitamin a deficiency , and megaloblastic anemia due to vitamin b12 deficiency.6971 general recommendations advocates46 for a prudent waiting period of at least two years after bs to become pregnant . in recent years , several studies have reported that maternal and perinatal outcomes of pregnancies occurring earlier than 1218 months after bs were not inferior.4,79 but in general , in most publications , mean interval between surgery and pregnancy was greater than 18 months , ranging from 21 to 57.4 months.5,6,10,11 in this setting , most clinicians will recommend a minimum waiting period of 1 year after bs before attempting pregnancy , to allow stabilization of body weight and to allow correct identification and treatment of any possible nutritional deficiencies that may not be evident during the first months . in this regard , some studies found no differences in maternal and fetal outcomes , others found better results , and still others identified a poorer outcome regarding rates of gdm , c - sections , and newborn s weight.6,7,1315 nutritional status and potential adverse effects derived from nutritional deficiencies are two of the most crucial aspects during pregnancy . in fact , if we consider the reported serum iron and ferritin levels , and if no specific prophylactic oral supplements were taken by women of childbearing age , a significant increase in anemia would be described.19,20 furthermore , some studies have reported the need for intravenous iron supplements , or even blood transfusion , although these cases were exceptional , since , in most situations , anemia was mild or moderate , and oral supplements were enough to restore body iron deposits and hemoglobin levels.7,21,22 in this context , the current american guidelines for the management of patients after bs recommend systematic oral administration of iron supplements to all women of childbearing age.23 regarding vitamin b12 and folic acid , a small study of 39 pregnant women who had undergone bs found a rate of deficiency of 53.4% and 16.1% , respectively.19 even though systematic supplementation of vitamin b12 is also advocated after most bariatric procedures , the authors of this study acknowledged that they only prescribed it if deficiency was evidenced in analytical workup . in any case , a clear increase ( 10%100% ) has been observed worldwide over the last 20 years.29 the linear relationship between obesity and diabetes entails an increase in the incidence of gdm of up to three - fold with increasing bmi.3033 accordingly , numerous studies have reported a reduction in the rate of gdm following the evident weight loss occurring after bs ( 0%8.9% in pregnancies after bs versus 1.6%20.8% in the control group ) . gestational hypertension is the most frequent cause of pregnancy - associated hypertensive disorders ( pahd ) , which affect around 6%17% of nulliparous women and 2%4% of multiparous women.34 obesity is a well - known associated risk factor for developing this complication , entailing a two to three - fold increased risk for hypertension and preeclampsia in women with a bmi > 30 kg / m.31,35,36 the incidence of pregnancy - associated hypertension and preeclampsia increases proportionately with maternal bmi , ranging from 1.4% to 2.4% in women with normal body weight , and reaching to 3.5%14.5% in women with morbid obesity.32,37 for women who underwent bs , studies regarding the prevalence of pahd are less consistent . although the odds ratio for pahd is two to three times lower in women who underwent surgery compared with women who have not undergone surgery , the incidence of pahd ranged widely between 0% in our own spanish multicentric cohort to 35% in other studies.4,5,7,911,38 in the series reported by bennett et al,38 there was a significantly lower incidence of hypertension in women who had undergone bs ( 2.5% of cases ) , compared with that reported in obese women who were managed conservatively ( 13% of patients ) . some studies even reported a higher prevalence of hypertension in operated women , but in these cases there were up to three times more cases of bmi > 30 kg / m than in the control group.4 taking all this data into account , in a recent meta - analysis , the authors concluded that the risk of preeclampsia seems to reduce by about approximately half in women who underwent bs.39 finally , various gastrointestinal complications may develop during pregnancy because of the bariatric procedure itself , and although these complications are not frequent , they may be severe and entail a high morbidity . in this regard , in three large studies performed in developed countries , there was a 1.42.6 increased risk of perinatal loss in obese women in comparison to controls.31,46 in the case of pregnant women with previous history of bs , increased intrauterine and neonatal mortality was paradoxically found , in comparison to control morbidly obese women ( 1.7% versus 0.7% , odds ratio : 2.39 ; 95% confidence interval : 0.985.85 , p=0.06 ) . however , subsequent subanalysis for each individual outcome did not confirm these differences , and the absolute number of adverse events was low.12 similarly , larger cohort studies reported no differences in perinatal or neonatal mortality between women with previous bs and the control group.6,47 in addition , in a study with no control group , sheiner et al9 reported that the incidence of perinatal mortality in operated pregnant women was 0.89% , regardless of the time that had elapsed between bs and pregnancy ( less than or more than 12 months ) . in this regard , in a recent meta - analysis , the rate of spontaneous preterm delivery was similar between obese women and controls ; however , induced preterm delivery was in fact more frequent in the former , probably due to the associated comorbidities involved in these cases , such as gdm and pahd.51 reports of the follow - up of pregnant women with prior bs describe variable incidences of preterm delivery , but all below 10% ( range 1.9%9.7% ) . furthermore , most series show no differences regarding this adverse outcome between women who underwent bs and those who did not,12 although , in some cases , an overall lower mean gestational age was documented in operated women.12,52 several other studies without a control group found no differences in the rate of preterm delivery regarding the type of bariatric procedure , or the time lapse between surgery and conception.4,5,9,10 the study by roos et al,47 however , did find a greater incidence of preterm delivery in women who had undergone bs in comparison to the control group : 9.7% versus 6.7% ; interestingly , women with lower bmi ( below 30 kg / m ) were the ones with a greater incidence of this outcome . this study has shown a lower incidence of adverse obstetric outcomes in those in whom agb was performed compared with those who did not undergo agb , including a lower rate of c - section in the operated group.15 similarly , patel et al,7 in a population - based study comparing 259 pregnancies after bs with a normal pregnant population group , have found that previous bs was not associated with adverse perinatal outcomes.19 however , in this study , only 10.7% of the operated women remained obese before pregnancy.14 despite these controversial results , there is no presumable physiopathologic reason to hypothesize that c - sections could be more frequent after bs , and understanding this increased rate is somewhat difficult . maternal obesity has long been associated with an increased rate of large - for - gestational - age ( lga ) newborns , while being a protective factor for small - for - gestational - age ( sga ) infants , even after adjusting for other maternal comorbidities.15,31,53 in addition , weight gain during pregnancy was found to positively correlate with the newborn s body weight.54 this influence of maternal weight on neonatal weight is important at birth time , but also , and probably most importantly , there is a great amount of scientific evidence supporting that it significantly influences intrauterine fetal development , portending an increased risk of overweight and obesity during adolescence and adulthood.5557 however , women with prior bs have been observed to have a lower rate of lga newborns compared with nonoperated control obese women ( ranging between 1.2% and 7.3% of cases).5,11,12,27,47,52 in fact , in recent years , many studies have reported that women who get pregnant after bs tend to deliver newborns with an overall lower body weight , and a higher rate of sga infants ( 5.2%27.8% ) , in comparison to nonoperated women.5,11,12,27,47,52 in a retrospective study by kjaer et al,6 339 women with a singleton delivery after bs were matched by pregnancy bmi , maternal age , parity , and date of delivery with 1,277 non - operated women . however , the negative influence of this issue in the long - term follow - up still deserves further investigation.5,11,12,27,47,52 however , the number of malabsorptive techniques performed has significantly reduced over the last couple of years , allowing other surgical approaches , with less adverse effects , to gain popularity ; thus , a reduction in the incidence of sga infants may be expected . available scientific evidence suggests that the risk of gdm and pahd significantly decreases in women who previously underwent bs , in comparison to morbidly obese patients who were managed conservatively , and the risk may even be like that of women with a similar bmi in whom surgery was not performed .

fluorescent lighting technologies are undergoing rapid market growth as part of a resurgent societal interest in energy efficiency . much of current and projected growth is in the domestic use of compact fluorescent lamps ( cfls ) , which offer consumers approximately 75% reduction in energy usage and 10-fold increase in lifetime relative to incandescent bulbs . federal legislation in the u.s . will phase out incandescent bulbs by 2012 and likely cause their replacement by cfls . fluorescent lamps contain 0.7115 mg of hg per lamp ( 1 ) , and the subclass of cfls on average contain 35 mg per lamp . mercury is a well - known human toxicant that is of special concern for neural development in unborn and growing children . while most hg - containing products are being removed from homes and workplaces through substitution programs , cfl use is increasing sharply because the environmental benefits ( reduced energy consumption and coal combustion emissions ) ( 2 ) are widely recognized to outweigh the health risks . indeed an individual cfl contains much less hg than some older home devices ( e.g. , 500 mg for a typical older model fever thermometer ) , but the projected sales volumes for cfls are large . the association of lighting and mercury recyclers report that 700 million hg - containing lamps are discarded each year with only a 24% recycle rate . domestic cfl sales are likely to increase these numbers significantly , and currently 98% are not recycled . there is strong motivation to improve hg management over the life - cycle of these rapidly proliferating consumer products . our present work is motivated by two specific issues in the management of hg from cfls : direct exposure of consumers or workers to hg vapor from fractured or crushed lamps . some lamps are inevitably broken accidentally during shipping , retail sales , consumer use , and recycling and release a portion of their mercury inventory as volatile hg vapor , which is the dominant mercury form in the early stages of lamp life ( 3 ) . inhalation exposure is a concern because 80% of inhaled hg is physiologically absorbed ( 4 ) . the osha occupational exposure limit ( 8 h , 5-day week time average ) is 100 g / m . the niosh recommended exposure limit is 50 g / m , while american conference of governmental and industrial hygienists recommends 25 g / m under the same conditions ( 4 ) . because children are more susceptible , the agency for toxic substances and disease registry ( atsdr ) recommends 0.2 g / m level as a safe continual exposure limit for children ( 4 ) . as an illustration of the effects of cfl breakage , the release of only 1 mg of hg vapor ( 20% of the hg inventory in a single cfl ) into a 500 m room ( 10 10 5 m ) yields 2.0 g / m or ten times the atsdr - recommended level of 0.2 g / m in the absence of ventilation . there is limited information on the timing and extent of hg vapor release from fractured lamps ( 1,2,5 ) , especially the new cfls . jang et al . ( 1 ) report only 0.040.17% of the hg as vapor , but this was a study of the phase partitioning within the bulb volume , not a study of the gradual evaporation and release characteristics upon fracture under atmospheric conditions , where we find much larger amounts of hg vapor ( see below ) . following any mercury spill , hard surfaces can be cleaned , but in the absence of in situ treatment technologies , porous materials such as carpets or woodwork must be removed and discarded ( 4 ) . carpet vacuuming can release hg vapor when large gas volumes are forced across the hg - containing dust cake in the vacuum cleaner internal filter . if not removed , spilled hg liquid will continue to release vapor over time and can spread to other sites through foot traffic . most consumer information on cfls claim there is no significant health risk from small numbers of broken lamps , and indeed , since the 1960s , examples of hg poisoning from all sources have become rare ( 6 ) . there is one report of hg poisoning ( acrodynia ) in a child exposed to broken tube - type fluorescents in a detailed case study presented by tunnessen et al . overall , there is significant motivation to improve our management of hg exposures caused by accidental breakage of fluorescent lamps . the main route of human exposure to mercury is through environmental release followed by bacterial methylation , bioaccumulation in aquatic food webs , and fish consumption ( 7 ) . methyl - hg is listed by the international program of chemical safety as one of the most dangerous chemicals in the environment ( 8,9 ) , and one in twelve women of childbearing age are reported to have blood mercury levels above the epa reference dose ( 10 ) . methyl - hg not only crosses the human placenta but also accumulates at higher concentrations on the fetal side than on the maternal and crosses the blood - brain barrier , where it is retained ( 9,11 ) . currently 98% of cfls are not recycled , and there is concern about hg leaching from landfills . the epa has concluded that mercury can be present in significant concentrations in the leachate and groundwater at nonhazardous landfill sites and can migrate offsite to threaten drinking water supplies ( 2 ) . mercury in new lamps is primarily in elemental form , but over time interacts with the phosphor and glass to produce a more complex internal partitioning in spent lamps , which contain elemental , immobile ( glass matrix imbedded ) , and oxidized soluble forms ( 3,5,12 ) . landfill leaching can be minimized by avoiding or reducing the mercury to water - soluble oxidized forms . some manufacturers are reported to incorporate reducing agents in lamps to improve performance in tclp testing . this approach may protect local groundwater but would lead to formation of volatile elemental mercury and enhanced environmental release of the vapor in landfill gases . the largest source of anthropogenic mercury emission is now coal - fired power plants ( 48 ton / year ) , which is much larger than even the total annual inventory of spent lamps ( 700 million 8 mg = approximately 5.6 tons ) . new regulations on power plant emissions , however , should reduce coal - derived hg by 70% to 15 tons / year by 2018 ( 13 ) , which coupled with rapid projected growth in cfls may make lamp - derived mercury a more significant fraction of the total environmental burden . common to both issues ( direct exposure and environmental release ) is the motivation to develop better methods for hg vapor capture and stabilization at ambient temperatures . high - efficiency , low - temperature hg sorbents could be used in reactive barrier cloths to remediate carpets and porous substrates after cfl breakage or incorporated into disposal bags or modified retail package materials as receptacles for spent lamps to prevent end - of - life release . the goal of the present work is therefore 2-fold : ( i ) to characterize the release of hg vapor from cfls as a function of time since fracture and ( ii ) to identify and evaluate new high - efficiency sorbents for ambient temperature capture focusing on new methods of nanosynthesis . two different brands of compact fluorescent lamps were purchased commercially : 13 and 9 w devices containing 4.5 and 5.0 mg of mercury respectively . used bulbs were collected from local residences and retail recycling centers . to characterize the release of hg vapor under ambient conditions , the bulbs were catastrophically fractured inside a flexible teflon enclosure ( volume 2 l ) and the hg vapor transported away by a metered flow of nitrogen ( 1 lpm ) . a portion of the hg - contaminated flow was passed to a gold amalgamation atomic fluorescent vapor - phase mercury analyzer ( psa model 10.525 ) , and the concentration - time profiles were measured and integrated to obtain total sorbent capacity for mercury . the effectiveness of several sorbents in capturing mercury released from fractured cfls was tested in a proof - of - principle experiment using the flow system discussed above . the cfl was again fractured in the flexible 2 l teflon enclosure , which also contained sorbent as loose powder or impregnated cloth . after lamp fracture , the enclosure was isolated from the flow system for 24 h. at the end of this period , the enclosure was reintegrated into the flow system , the high purity nitrogen stream was initiated , and the effluent was analyzed for mercury content . a variety of carbon materials were used in this study ( see table 1 ) including darco fgl activated carbon , alfa aeser granulated activated carbon , cabot m-120 carbon black , sulfur impregnated carbon sample ( hgr ) , and a mesoporous carbon ( 14 ) . the origin , particle sizes , and surface areas of commercial sulfur used in this work are provided in table 1 , which also gives the mercury capture capacity of the sorbents in our standard gas environment ( 60 g hg / m argon at 20 c as described below ) . sulfur nanotubes were synthesized at brown by immersion of 200 nanometer channel aluminum templates in a 50 mass % solution of sigma aldrich 100 mesh commercial sulfur in cs2 . the loaded templates were dried , and excess sulfur was removed from the template top with a razor blade . the s - nanotube samples were washed twice with 1 m naoh , twice with 0.5 m naoh and four times with di water , followed by centrifugation and oven drying at 60 c . see table 1 and for the source , particle sizes , and surface areas of the metals and metal sulfides used as sorbents in this study . amorphous nano - selenium was prepared using a 4:1 molar mixture of glutathione ( gsh , reduced form , tci america ) and sodium selenite ( na2seo3 , alfa aesar ) solution . glutathione reduces sodium selenite to form seleno - diglutathione ( gssesg ) , which decomposes to elemental selenium as upon sodium hydroxide titration ( 15,16 ) . in the presence of bovine serum albumin ( bsa , sigma - aldrich ) , the reaction gives a stabilized nano - selenium dispersion ( 17 ) . for mercury capture experiments , the solutions of nano - selenium were divided in 1.52 ml aliquots and freeze - dried to prevent any thermal effects of heat drying . the nano - selenium samples were pelletized by centrifugation ( 13 000 rpm , 10 min ) before freeze - drying . these freeze - dried aliquots and se - impregnated cloth , which was prepared by soaking a 15 17 in . kimwipe in the amorphous nano - selenium solution and drying at room temperature , were used for the in situ mercury release experiments . ( 18 ) and the supporting information provide more detailed description of hg adsorption capacity measurement and analysis . the release is initially rapid producing vapor concentrations from 200800 g / m during the first hour , which far exceed the osha occupational limits . the release decays on a time scale of hours and continues at significant rate for at least four days ( data beyond 24 h not shown ) . the total hg released after 24 h is 504 ( 13 w model ) and 113 g ( for 9 w ) by integration , which are 11.1% and 1.9% of the total hg content specified by the vendors , respectively . over 4 days ( extended data not shown ) , the 13 w bulb released 1.34 mg or 30% of the total hg . in general , hg evaporation is known to be slow under ambient conditions , and our data suggest that much of the original mercury remains in the bulb debris after 96 h and will continue to evaporate slowly . saturated hg vapor ( 15 000 g / m ) in a typical lamp volume ( 50 ml ) corresponds to only 0.65 g of vapor phase hg , which is much less than the actual mercury release during the first hour , 1243 g . the majority of hg in a cfl must therefore be in a condensed phase originally , and the mercury release we observe must be primarily caused by desorption / evaporation phenomena . figure 1 also compares the actual cfl release with the evaporation of a free hg droplet under the same set of conditions . the actual cfl release exceeds the release from a free hg droplet of equal mass ( see figure 1 ) , which likely reflects the much larger surface area of the adsorbed phase ( on the phosphor , end caps , or glass ) relative to the single drop . similar release patterns but lower amounts were seen for spent bulbs ( example result 90 g in 24 h ) or from the fracture site of a new bulb after glass removal to simulate cleanup . removing large glass shards by hand after breakage on a carpet did not eliminate hg release , but reduced it by 67% relative to the data in figure 1 . the remaining ( 33% ) release from the fracture site is believed to be primarily associated with spilled phosphor powder , which is known to be the primary site for adsorbed hg partitioning in fresh bulbs ( 1 ) . mercury vapor release characteristics for two brands of compact fluorescent lamps following catastrophic fracture at room temperature . a : hg - vapor concentrations and release rates in a 2 l ptfe enclosure purged with a 1 l / min flow . for comparison , the plot shows the evaporation rate from a free hg drop corrected for differences in the hg mass between the drop and the bulb for two limiting cases : convective mass transfer at constant mass transfer coefficient ( rate area mass ) and diffusion dominated mass transfer from a drop ( rate k area mass ) . b : mercury evaporation rate as a function of gas flow rate over the broken lamp showing a weak influence of convection . because mercury vapor capture on solids occurs by adsorption or gassolid reaction where kinetics or capacities typically depend on surface area ( in addition to other factors such as composition ) , we hypothesized that high - area , nanoscale formulations of common mercury sorbents will show enhanced performance . this section evaluates a large set of new nanomaterial sorbents for ambient temperature hg vapor capture and compares their performance to conventional microscale formulations of the same materials . manchester et al . ( 18 ) shows an example breakthrough curve that is the raw output of the fixed - bed sorbent tests . integrating the area between the baseline inlet ( 60 g / m ) and the outlet concentration curve and dividing by sorbent mass yields a capacity reported in g - hg / g - sorbent ( 18 ) . table 1 is a complete list of the sorbents and their hg capacities under our standard conditions ( 60 g / m inlet stream ) , and the following sections discuss the results by sorbent class . zero - valent sulfur reacts with mercury to form stable mercuric sulfide in one of two crystal forms : red cinnabar ( hf = 58 kj / mol , gf = 49 kj / mol ) or black metacinnabar ( hf = 54 kj / mol , gf = 46 kj / mol ) and is thus attractive for waste or stockpile stabilization ( 20,21 ) . oji ( 20 ) discusses the advantages of hgs relative to zn amalgam for the stabilization and disposal of hg - containing mixed wastes , and svensson et al . ( 21 ) discuss favorable conditions for hgs formation from hg or hgo in geological repositories . surprisingly , there are few reports of nano - sulfur synthesis ( 2224 ) and to our knowledge no studies of nano - sulfur as a mercury sorbent . here we choose a convenient templating route to obtain small quantities of nanostructured sulfur for sorbent testing . figure 2 shows the morphology and sorption behavior of sulfur nanotubes fabricated by spontaneous infiltration of cs2/s solutions into nanochannel alumina templates followed by solvent evaporation and chemical etching of the template . the sulfur nanotubes show a 90-fold increase in surface area and a 24-fold increase in hg capacity over conventional powdered sulfur . the total captured hg is much less than the hgs stoichiometric limit and much less than even surface monolayer capacity , and the capacities increase with increasing temperature . these results indicate a kinetically limited chemisorption / reaction on active sites that represent a small fraction of the nanotube surfaces . standard hg adsorption capacities for elemental sulfur nanotubes and conventional sulfur powder as a function of adsorption reaction temperature . there is an extensive literature on hg interaction with metals ( 2528 ) , much of it focused on elevated temperatures using conventional film or microparticle formulations . here we investigate newly available nanoparticles as room - temperature hg sorbents and compare them to conventional microscale powders . table 1 shows that mercury capacities vary greatly with chemistry ( ag > cu > ni > zn ) and , for each metal , are significantly enhanced by nanosynthesis . the rank order parallels the standard free energies for metal oxidation , nm + /2o2 mno2 ( ag2o , gf = 9.3 kj / mol ; cuo , gf = 133.5 kj / mol ; nio , gf = 216 kj / mol ; zno , gf = 318.5 kj / mol ) , and ( complete ) oxidation of copper is shown to greatly reduce its sorption activity ( 31.8 to 4.3 g / g ) . interestingly , copper metal activity is observed to increase modestly as the fresh metal nanoparticles age in the atmosphere , which may suggest elevated activity for partially oxidized surfaces . the nanometal capacities represent from about 10 ( zn ) to 35% ( ag ) of theoretical monolayer coverage on the nominal outer surfaces indicating that the process is far from reaching stoichiometric alloy formation , even in an outer shell , and the reactions are limited to specific active surface sites under these low temperature conditions . among these metal sorbents , nano - silver is potentially attractive as a high - capacity sorbent ( capacities up to 8510 g / g ) for room temperature applications like cfl capture . annealing nano - silver reduces both its surface area and hg capture capacity ( table 1 ) . ( 28 ) investigated metal sulfides mos2 and fes2 as hg sorbents at elevated temperature and report a high capacity for mos2 . in preliminary experiments , we found ws2 to be significantly more reactive than mos2 ( both conventional powders ) and therefore were motivated to test ws2 nanoparticles as potential high - capacity sorbents . in this case , nanosynthesis offered no significant advantage , and none of the metal sulfides appear among the most active and useful low - temperature sorbents in table 1 . activated carbons are widely used to capture mercury vapor , and their performance can be enhanced by surface modification with sulfur , halogen , or oxygen - containing functional groups ( 18,2833 ) . because carbons are capable of developing extensive internal surface area , there is little motivation to enhance the external surface area through nanosynthesis methods . here we evaluate carbons as readily available reference materials that are market - relevant benchmarks for the new nanosorbents . table 1 shows low to modest capacities on carbons ( 0.45115 g / g ) with the exception of the s - impregnated material ( 2600 g / g ) , which is one of best commercially available sorbents in this study . , it sequesters mercury into insoluble and metabolically inactive mercury selenides and by this mechanism is protective against mercury neurotoxicity ( 9,34 ) . its antioxidant nature helps to protect against mercury - induced dna damage ( 35 ) . in the environment the stable sequestration of mercury by selenium may reduce its mobility , bioavailability , and ecotoxicity ( 9,36,37 ) . strong hg / se binding may be key to understanding the biological and environmental behavior of both mercury and selenium ( 3840 ) . there are few published studies of selenium - based mercury vapor capture , although selenium has been used in hg removal from off gases in sulfide ore processing ( 41 ) and is being considered for hg stockpile stabilization and long - term storage ( 42 ) . the presumed capture mechanism is reaction to hgse ( gf = 38.1 kj / mol ) ( 43 ) . here we focus on amorphous nanoselenium , which has received recent attention in chemoprevention ( 17 ) but has not to our knowledge been used for low - temperature hg vapor capture . figure 3 shows the colloidal synthesis of nanoselenium , the particle size distributions , and the mercury capture behavior of competing se forms . the original synthesis method uses glutathione ( gsh ) as a reductant and bovine serum albumin ( bsa ) as a surface stabilizing agent to achieve very small particles in colloidal suspension ( 17 ) as shown in figure 3a , left . surprisingly the bsa - stabilized nano - se has a lower capacity than conventional se powder despite the much smaller particle size ( 660 nm vs 10200 m ) . we hypothesized that the protein stabilizer ( bsa ) either blocked hg access to the se surfaces or chemically passivated the surfaces through sethiol interactions . we therefore removed the bsa , as shown in figure 3a , right , to make unstabilized nano - se , which figure 3c shows to have a remarkably high hg sorption capacity and much faster kinetics than conventional micro - se . mercury uptake continues over very long times , and a 184 h experiment was necessary to approach the end state , at which point the unstabilized nano - se had adsorbed 188 000 g hg / g or approximately 20% hg / se mass ratio . x - ray diffraction analysis shows both the micro - se and unstabilized nano - se are amorphous , as is the stabilized nano - se ( 45 ) . synthesis , particle size distributions , and hg - uptake kinetics of competing forms of selenium . a : colloidal synthesis of bsa - stabilized ( left ) and unstabilized ( right ) nano - se . b : particle - size distributions in aqueous media by dynamic light scattering ( 44 ) . c : hg - uptake kinetics under standard conditions ( 60 g / m ) . figure 4 shows a comparison of the new and reference sorbents in this study . the right - hand axis gives the amount of sorbent required to capture 1 mg of hg vapor , typical of cfl release . surprisingly , some common sorbents such as powdered s or zn require enormous amounts of material ( > 10 kg ) to treat the vapor released from a single cfl and most of the sorbents require amounts that are not attractive for incorporation into consumer packaging ( > 10 g ) . a small number of sorbents ( nano - ag , s - impregnated activated carbon , and two selenium forms ) have capacities that should allow < 1 g of sorbent to be used . the most effective sorbent is unstabilized nano - se , which can capture the contents of a cfl with amounts less than 10 mg . this capacity corresponds to about five monolayer equivalents indicating significant subsurface penetration of mercury into selenium nanoparticles ( unlike the other sorbents ) . the capacity is still only about 7% of the bulk stoichiometric conversion to hgse , however , indicating the potential for further capacity improvement by sorbent optimization . comparison of the sorbents in this study . left axis : standard hg adsorption capacity . right axis : amount of sorbent required for capture of 1 mg of hg vapor typical of the total release from a single cfl over a three - day period . although the amount of hg released from cfls on fracture is small ( typically < 1 mg ) , only a few sorbents have sufficient capacity to sequester it all at room temperature for practical application ( see figure 5 ) . for in situ capture , where the sorbent is supplied to consumers in the form of a safe disposal bag , impregnated cloth , or modified retail package , only nano - ag , selenium forms , or sulfur - impregnated activated carbon could be used in reasonable quantities . the concept of in situ capture is demonstrated below , here treatment is defined as sealing the fractured cfl and sorbent in a confined space for 24 h , then removing the sorbent and measuring the residual vapor release . effect of sorbents applied in situ on mercury vapor release following catastrophic fracture of a cfl at room temperature . bottom curves : same cfl broken in presence of sulfur - impregnated activated carbon ( 1 g of hgr ) and unstabilized nano - selenium ( 10 mg ) either as dry nanopowder or impregnated cloth . the integrated mercury released over the course of this experiment is 113 ( untreated lamp ) , 20 ( 1 g of hgr treatment ) , 1.6 ( se in vials ) , and 1.2 g ( se - impregnated cloth ) . the commercial sulfur - impregnated activated carbon reduced the mercury release by 83% over the untreated bulb , making it a viable candidate for in situ capture of mercury vapor . moreover , the low cost and low toxicity of this material make it an attractive option for consumer use . even better performance was exhibited by the unstabilized nano - selenium , which decreased the mercury release by 99% over an untreated bulb , regardless of the application method , and with 100-fold less sorbent mass . nearly complete suppression of mercury vapor from fractured lamps can be achieved by sealing the lamp in a confined space with 10 mg of unstabilized nano - selenium for 24 h , either as an impregnated cloth draped over the fractured bulb or as a loose powder in vials . the present article provides sufficient motivation to pursue further development of sorbent - based technologies for suppressing mercury vapor release from broken fluorescent lamps . work is underway to engineer ( i ) sorbent - impregnated reactive barrier cloths for remediation of porous substrates such as carpets at break sites and ( ii ) sorbent - containing disposal bags or recycle boxes to allow safe handling and stable disposition in the environment . important issues in this development include reaction kinetics , landfill stability , impregnated cloth design , bag design , and management of secondary risks to both human health and the environment associated with possible release or and exposure to the nanomaterial sorbents themselves .

the projected increase in the use of compact fluorescent lamps ( cfls ) motivates the development of methods to manage consumer exposure to mercury and its environmental release at the end of lamp life . this work characterizes the time - resolved release of mercury vapor from broken cfls and from underlying substrates after removal of glass fragments to simulate cleanup . in new lamps , mercury vapor is released gradually in amounts that reach 1.3 mg or 30% of the total lamp inventory after four days . similar time profiles but smaller amounts are released from spent lamps or from underlying substrates . nanoscale formulations of s , se , cu , ni , zn , ag , and ws2 are evaluated for capture of hg vapor under these conditions and compared to conventional microscale formulations . adsorption capacities range over 7 orders of magnitude , from 0.005 ( zn micropowder ) to 188 000 g / g ( unstabilized nano - se ) , depending on sorbent chemistry and particle size . nanosynthesis offers clear advantages for most sorbent chemistries . unstabilized nano - selenium in two forms ( dry powder and impregnated cloth ) was successfully used in a proof - of - principle test for the in situ , real - time suppression of hg vapor escape following cfl fracture .

Introduction Materials and Methods Results and Discussion

much of current and projected growth is in the domestic use of compact fluorescent lamps ( cfls ) , which offer consumers approximately 75% reduction in energy usage and 10-fold increase in lifetime relative to incandescent bulbs . fluorescent lamps contain 0.7115 mg of hg per lamp ( 1 ) , and the subclass of cfls on average contain 35 mg per lamp . , 500 mg for a typical older model fever thermometer ) , but the projected sales volumes for cfls are large . our present work is motivated by two specific issues in the management of hg from cfls : direct exposure of consumers or workers to hg vapor from fractured or crushed lamps . some lamps are inevitably broken accidentally during shipping , retail sales , consumer use , and recycling and release a portion of their mercury inventory as volatile hg vapor , which is the dominant mercury form in the early stages of lamp life ( 3 ) . the osha occupational exposure limit ( 8 h , 5-day week time average ) is 100 g / m . the niosh recommended exposure limit is 50 g / m , while american conference of governmental and industrial hygienists recommends 25 g / m under the same conditions ( 4 ) . as an illustration of the effects of cfl breakage , the release of only 1 mg of hg vapor ( 20% of the hg inventory in a single cfl ) into a 500 m room ( 10 10 5 m ) yields 2.0 g / m or ten times the atsdr - recommended level of 0.2 g / m in the absence of ventilation . there is limited information on the timing and extent of hg vapor release from fractured lamps ( 1,2,5 ) , especially the new cfls . ( 1 ) report only 0.040.17% of the hg as vapor , but this was a study of the phase partitioning within the bulb volume , not a study of the gradual evaporation and release characteristics upon fracture under atmospheric conditions , where we find much larger amounts of hg vapor ( see below ) . following any mercury spill , hard surfaces can be cleaned , but in the absence of in situ treatment technologies , porous materials such as carpets or woodwork must be removed and discarded ( 4 ) . carpet vacuuming can release hg vapor when large gas volumes are forced across the hg - containing dust cake in the vacuum cleaner internal filter . most consumer information on cfls claim there is no significant health risk from small numbers of broken lamps , and indeed , since the 1960s , examples of hg poisoning from all sources have become rare ( 6 ) . there is one report of hg poisoning ( acrodynia ) in a child exposed to broken tube - type fluorescents in a detailed case study presented by tunnessen et al . overall , there is significant motivation to improve our management of hg exposures caused by accidental breakage of fluorescent lamps . the main route of human exposure to mercury is through environmental release followed by bacterial methylation , bioaccumulation in aquatic food webs , and fish consumption ( 7 ) . methyl - hg is listed by the international program of chemical safety as one of the most dangerous chemicals in the environment ( 8,9 ) , and one in twelve women of childbearing age are reported to have blood mercury levels above the epa reference dose ( 10 ) . currently 98% of cfls are not recycled , and there is concern about hg leaching from landfills . mercury in new lamps is primarily in elemental form , but over time interacts with the phosphor and glass to produce a more complex internal partitioning in spent lamps , which contain elemental , immobile ( glass matrix imbedded ) , and oxidized soluble forms ( 3,5,12 ) . this approach may protect local groundwater but would lead to formation of volatile elemental mercury and enhanced environmental release of the vapor in landfill gases . the largest source of anthropogenic mercury emission is now coal - fired power plants ( 48 ton / year ) , which is much larger than even the total annual inventory of spent lamps ( 700 million 8 mg = approximately 5.6 tons ) . new regulations on power plant emissions , however , should reduce coal - derived hg by 70% to 15 tons / year by 2018 ( 13 ) , which coupled with rapid projected growth in cfls may make lamp - derived mercury a more significant fraction of the total environmental burden . common to both issues ( direct exposure and environmental release ) is the motivation to develop better methods for hg vapor capture and stabilization at ambient temperatures . high - efficiency , low - temperature hg sorbents could be used in reactive barrier cloths to remediate carpets and porous substrates after cfl breakage or incorporated into disposal bags or modified retail package materials as receptacles for spent lamps to prevent end - of - life release . the goal of the present work is therefore 2-fold : ( i ) to characterize the release of hg vapor from cfls as a function of time since fracture and ( ii ) to identify and evaluate new high - efficiency sorbents for ambient temperature capture focusing on new methods of nanosynthesis . two different brands of compact fluorescent lamps were purchased commercially : 13 and 9 w devices containing 4.5 and 5.0 mg of mercury respectively . to characterize the release of hg vapor under ambient conditions , the bulbs were catastrophically fractured inside a flexible teflon enclosure ( volume 2 l ) and the hg vapor transported away by a metered flow of nitrogen ( 1 lpm ) . a portion of the hg - contaminated flow was passed to a gold amalgamation atomic fluorescent vapor - phase mercury analyzer ( psa model 10.525 ) , and the concentration - time profiles were measured and integrated to obtain total sorbent capacity for mercury . the effectiveness of several sorbents in capturing mercury released from fractured cfls was tested in a proof - of - principle experiment using the flow system discussed above . the cfl was again fractured in the flexible 2 l teflon enclosure , which also contained sorbent as loose powder or impregnated cloth . after lamp fracture , the enclosure was isolated from the flow system for 24 h. at the end of this period , the enclosure was reintegrated into the flow system , the high purity nitrogen stream was initiated , and the effluent was analyzed for mercury content . a variety of carbon materials were used in this study ( see table 1 ) including darco fgl activated carbon , alfa aeser granulated activated carbon , cabot m-120 carbon black , sulfur impregnated carbon sample ( hgr ) , and a mesoporous carbon ( 14 ) . the origin , particle sizes , and surface areas of commercial sulfur used in this work are provided in table 1 , which also gives the mercury capture capacity of the sorbents in our standard gas environment ( 60 g hg / m argon at 20 c as described below ) . the loaded templates were dried , and excess sulfur was removed from the template top with a razor blade . see table 1 and for the source , particle sizes , and surface areas of the metals and metal sulfides used as sorbents in this study . amorphous nano - selenium was prepared using a 4:1 molar mixture of glutathione ( gsh , reduced form , tci america ) and sodium selenite ( na2seo3 , alfa aesar ) solution . glutathione reduces sodium selenite to form seleno - diglutathione ( gssesg ) , which decomposes to elemental selenium as upon sodium hydroxide titration ( 15,16 ) . in the presence of bovine serum albumin ( bsa , sigma - aldrich ) , the reaction gives a stabilized nano - selenium dispersion ( 17 ) . for mercury capture experiments , the solutions of nano - selenium were divided in 1.52 ml aliquots and freeze - dried to prevent any thermal effects of heat drying . the nano - selenium samples were pelletized by centrifugation ( 13 000 rpm , 10 min ) before freeze - drying . these freeze - dried aliquots and se - impregnated cloth , which was prepared by soaking a 15 17 in . kimwipe in the amorphous nano - selenium solution and drying at room temperature , were used for the in situ mercury release experiments . ( 18 ) and the supporting information provide more detailed description of hg adsorption capacity measurement and analysis . the release decays on a time scale of hours and continues at significant rate for at least four days ( data beyond 24 h not shown ) . the total hg released after 24 h is 504 ( 13 w model ) and 113 g ( for 9 w ) by integration , which are 11.1% and 1.9% of the total hg content specified by the vendors , respectively . over 4 days ( extended data not shown ) , the 13 w bulb released 1.34 mg or 30% of the total hg . in general , hg evaporation is known to be slow under ambient conditions , and our data suggest that much of the original mercury remains in the bulb debris after 96 h and will continue to evaporate slowly . saturated hg vapor ( 15 000 g / m ) in a typical lamp volume ( 50 ml ) corresponds to only 0.65 g of vapor phase hg , which is much less than the actual mercury release during the first hour , 1243 g . the majority of hg in a cfl must therefore be in a condensed phase originally , and the mercury release we observe must be primarily caused by desorption / evaporation phenomena . the actual cfl release exceeds the release from a free hg droplet of equal mass ( see figure 1 ) , which likely reflects the much larger surface area of the adsorbed phase ( on the phosphor , end caps , or glass ) relative to the single drop . similar release patterns but lower amounts were seen for spent bulbs ( example result 90 g in 24 h ) or from the fracture site of a new bulb after glass removal to simulate cleanup . mercury vapor release characteristics for two brands of compact fluorescent lamps following catastrophic fracture at room temperature . a : hg - vapor concentrations and release rates in a 2 l ptfe enclosure purged with a 1 l / min flow . because mercury vapor capture on solids occurs by adsorption or gassolid reaction where kinetics or capacities typically depend on surface area ( in addition to other factors such as composition ) , we hypothesized that high - area , nanoscale formulations of common mercury sorbents will show enhanced performance . this section evaluates a large set of new nanomaterial sorbents for ambient temperature hg vapor capture and compares their performance to conventional microscale formulations of the same materials . ( 18 ) shows an example breakthrough curve that is the raw output of the fixed - bed sorbent tests . integrating the area between the baseline inlet ( 60 g / m ) and the outlet concentration curve and dividing by sorbent mass yields a capacity reported in g - hg / g - sorbent ( 18 ) . table 1 is a complete list of the sorbents and their hg capacities under our standard conditions ( 60 g / m inlet stream ) , and the following sections discuss the results by sorbent class . oji ( 20 ) discusses the advantages of hgs relative to zn amalgam for the stabilization and disposal of hg - containing mixed wastes , and svensson et al . surprisingly , there are few reports of nano - sulfur synthesis ( 2224 ) and to our knowledge no studies of nano - sulfur as a mercury sorbent . figure 2 shows the morphology and sorption behavior of sulfur nanotubes fabricated by spontaneous infiltration of cs2/s solutions into nanochannel alumina templates followed by solvent evaporation and chemical etching of the template . the total captured hg is much less than the hgs stoichiometric limit and much less than even surface monolayer capacity , and the capacities increase with increasing temperature . these results indicate a kinetically limited chemisorption / reaction on active sites that represent a small fraction of the nanotube surfaces . standard hg adsorption capacities for elemental sulfur nanotubes and conventional sulfur powder as a function of adsorption reaction temperature . there is an extensive literature on hg interaction with metals ( 2528 ) , much of it focused on elevated temperatures using conventional film or microparticle formulations . here we investigate newly available nanoparticles as room - temperature hg sorbents and compare them to conventional microscale powders . the rank order parallels the standard free energies for metal oxidation , nm + /2o2 mno2 ( ag2o , gf = 9.3 kj / mol ; cuo , gf = 133.5 kj / mol ; nio , gf = 216 kj / mol ; zno , gf = 318.5 kj / mol ) , and ( complete ) oxidation of copper is shown to greatly reduce its sorption activity ( 31.8 to 4.3 g / g ) . interestingly , copper metal activity is observed to increase modestly as the fresh metal nanoparticles age in the atmosphere , which may suggest elevated activity for partially oxidized surfaces . the nanometal capacities represent from about 10 ( zn ) to 35% ( ag ) of theoretical monolayer coverage on the nominal outer surfaces indicating that the process is far from reaching stoichiometric alloy formation , even in an outer shell , and the reactions are limited to specific active surface sites under these low temperature conditions . among these metal sorbents , nano - silver is potentially attractive as a high - capacity sorbent ( capacities up to 8510 g / g ) for room temperature applications like cfl capture . in this case , nanosynthesis offered no significant advantage , and none of the metal sulfides appear among the most active and useful low - temperature sorbents in table 1 . activated carbons are widely used to capture mercury vapor , and their performance can be enhanced by surface modification with sulfur , halogen , or oxygen - containing functional groups ( 18,2833 ) . table 1 shows low to modest capacities on carbons ( 0.45115 g / g ) with the exception of the s - impregnated material ( 2600 g / g ) , which is one of best commercially available sorbents in this study . in the environment the stable sequestration of mercury by selenium may reduce its mobility , bioavailability , and ecotoxicity ( 9,36,37 ) . there are few published studies of selenium - based mercury vapor capture , although selenium has been used in hg removal from off gases in sulfide ore processing ( 41 ) and is being considered for hg stockpile stabilization and long - term storage ( 42 ) . here we focus on amorphous nanoselenium , which has received recent attention in chemoprevention ( 17 ) but has not to our knowledge been used for low - temperature hg vapor capture . figure 3 shows the colloidal synthesis of nanoselenium , the particle size distributions , and the mercury capture behavior of competing se forms . surprisingly the bsa - stabilized nano - se has a lower capacity than conventional se powder despite the much smaller particle size ( 660 nm vs 10200 m ) . we therefore removed the bsa , as shown in figure 3a , right , to make unstabilized nano - se , which figure 3c shows to have a remarkably high hg sorption capacity and much faster kinetics than conventional micro - se . mercury uptake continues over very long times , and a 184 h experiment was necessary to approach the end state , at which point the unstabilized nano - se had adsorbed 188 000 g hg / g or approximately 20% hg / se mass ratio . x - ray diffraction analysis shows both the micro - se and unstabilized nano - se are amorphous , as is the stabilized nano - se ( 45 ) . synthesis , particle size distributions , and hg - uptake kinetics of competing forms of selenium . a : colloidal synthesis of bsa - stabilized ( left ) and unstabilized ( right ) nano - se . c : hg - uptake kinetics under standard conditions ( 60 g / m ) . figure 4 shows a comparison of the new and reference sorbents in this study . the right - hand axis gives the amount of sorbent required to capture 1 mg of hg vapor , typical of cfl release . surprisingly , some common sorbents such as powdered s or zn require enormous amounts of material ( > 10 kg ) to treat the vapor released from a single cfl and most of the sorbents require amounts that are not attractive for incorporation into consumer packaging ( > 10 g ) . a small number of sorbents ( nano - ag , s - impregnated activated carbon , and two selenium forms ) have capacities that should allow < 1 g of sorbent to be used . the most effective sorbent is unstabilized nano - se , which can capture the contents of a cfl with amounts less than 10 mg . the capacity is still only about 7% of the bulk stoichiometric conversion to hgse , however , indicating the potential for further capacity improvement by sorbent optimization . right axis : amount of sorbent required for capture of 1 mg of hg vapor typical of the total release from a single cfl over a three - day period . although the amount of hg released from cfls on fracture is small ( typically < 1 mg ) , only a few sorbents have sufficient capacity to sequester it all at room temperature for practical application ( see figure 5 ) . for in situ capture , where the sorbent is supplied to consumers in the form of a safe disposal bag , impregnated cloth , or modified retail package , only nano - ag , selenium forms , or sulfur - impregnated activated carbon could be used in reasonable quantities . the concept of in situ capture is demonstrated below , here treatment is defined as sealing the fractured cfl and sorbent in a confined space for 24 h , then removing the sorbent and measuring the residual vapor release . effect of sorbents applied in situ on mercury vapor release following catastrophic fracture of a cfl at room temperature . bottom curves : same cfl broken in presence of sulfur - impregnated activated carbon ( 1 g of hgr ) and unstabilized nano - selenium ( 10 mg ) either as dry nanopowder or impregnated cloth . the integrated mercury released over the course of this experiment is 113 ( untreated lamp ) , 20 ( 1 g of hgr treatment ) , 1.6 ( se in vials ) , and 1.2 g ( se - impregnated cloth ) . the commercial sulfur - impregnated activated carbon reduced the mercury release by 83% over the untreated bulb , making it a viable candidate for in situ capture of mercury vapor . even better performance was exhibited by the unstabilized nano - selenium , which decreased the mercury release by 99% over an untreated bulb , regardless of the application method , and with 100-fold less sorbent mass . nearly complete suppression of mercury vapor from fractured lamps can be achieved by sealing the lamp in a confined space with 10 mg of unstabilized nano - selenium for 24 h , either as an impregnated cloth draped over the fractured bulb or as a loose powder in vials . the present article provides sufficient motivation to pursue further development of sorbent - based technologies for suppressing mercury vapor release from broken fluorescent lamps . important issues in this development include reaction kinetics , landfill stability , impregnated cloth design , bag design , and management of secondary risks to both human health and the environment associated with possible release or and exposure to the nanomaterial sorbents themselves .

the mechanism of semiconductor - assisted photocatalytic processes is based on the principle that colloidal semiconductor nanoparticles behave as miniature photoelectrochemical cells [ 1 - 3 ] . the overall photoexcitation process of semiconductor nanoparticles by ultra bandgap energies involve the photogeneration of electron hole pairs within particle , followed by the competition between recombination , interfacial charge transfer to adsorbed compounds , and migration into midgap surface states . nanocrystalline tio2 has attracted widespread attention as a photocatalyst in various practical applications [ 5 - 7 ] as well as the part of photoelectrochemical systems , such as grtzel cells . although nanoparticulate tio2 is very effective from an energetic point of view , it is a relatively inefficient photocatalyst . due to its large bandgap ( eg = 3.2 ev ) , tio2 absorbs less than 5% of the available solar light photons . the main energy loss is due to the process of radiative or nonradiative recombination of charges generated upon photoexcitation of tio2 , which is manifested as the relatively low efficiency of long - lived charge separation . hence , the successful photochemical energy conversion and subsequent chemical reactions require both the extended separation of photogenerated charges and the response in the visible spectral region . to achieve larger separation distances , preventing the hole electron recombination before desired redox reaction occurs , reconstructed surface of tio2 nanoparticle surface was employed for establishing a strong coupling with electron - accepting and/or electron - donating species . consequently , the lifetime of charge separation and photocatalytic activity of tio2 nanoparticles are increased . the origin of the unique photocatalytic activities of tio2 nanoparticles comparing to the bulk is found in larger surface area and the existence of surface sites with distorted coordination . owing to large curvature of tio2 particles in the nanosize regime , the surface reconstructs in such manner that distorts the crystalline environment of surface ti atoms forming coordinatively unsaturated ti atoms . the changes in the pre - edge structure of ti k - edge spectrum ( xanes ) of tio2 nanoparticles ( d < 20 nm ) revealed the existence of square pyramidal coordination of surface ti atoms ( pentacoordinate ) . the results obtained from measurements of xafs spectra of tio2 nanoparticles proved the existence of shorter ti o bond lengths ( 1.71 ) as compared to bulk anatase tio2 ( 1.96 ) . it was reported , on a whole class of electron - donating enediol ligands , benzene derivatives , or mercapto - carboxylic acids that binding to coordinatively unsaturated ti atoms simultaneously adjusts their coordination to octahedral geometry at the surface of nanocrystallites and changes the electronic properties of tio2 . in such hybrid structures localized orbitals of surface - attached ligands , are electronically coupled with the delocalized electron levels from the conduction band of a tio2 semiconductor . as a consequence , absorption of light by the charge transfer ( ct ) complex yields to the excitation of electrons from the chelating ligand directly into the conduction band of tio2 nanocrystallites . this results in a red shift of the semiconductor absorption compared to that of unmodified nanocrystallites and enables efficient harvesting of solar photons . additionally , this type of electronic coupling yields to instantaneous separation of photogenerated charges into two phases , the holes localize on the donating organic modifier , and the electrons delocalize in the conduction band of tio2 . moreover , the enediol ligands were found to act as conductive leads , allowing wiring of oligonucleotides and proteins resulting in enhanced charge separation and ensuing chemical transformations . in last two decades the surface modification of commercial tio2(degussa p25,d = 30 nm ) with benzene derivatives ( mainly catechol and salicylic acid ) was studied [ 19 - 34 ] . just few articles [ 9,13 - 15,35 ] investigated ct complex formation between enediol ligands and colloidal tio2 nanoparticles ( d = 45 ) , where binding of modifiers is stabilized by ligand - induced surface reconstruction of the nanoparticles . herein , we report surface modification of tio2 nanoparticles with enediol ligands ( catechol , pyrogallol , and gallic acid ) that are able to adjust the coordination geometry of the surface ti atoms inducing shift of the absorption onset toward the visible region of the spectrum , compared to unmodified nanocrystallites . since these novel ct semiconducting materials exhibit optical properties that are distinct from their constituents , not absorbing in the visible region , benesi hildebrand analysis for molecular complexes was employed to determine the stability constants from the absorption spectra . all the chemicals were of the highest purity available and were used without further purification ( aldrich , fluka ) . milli - q deionized water ( resistivity 18.2 m cm ) was used as solvent . the colloidal tio2 dispersions were prepared by the dropwise addition of titanium(iv ) chloride to cooled water . the ph of the solution was between 0 and 1 , depending on the ticl4 concentration . slow growth of the particles was achieved by using dialysis at 4 c against water until the ph = 3.5 was reached . the concentration of tio2 ( 0.2 m ) was determined from the concentration of the peroxide complex obtained after dissolving the colloid in concentrated h2so4 . surface modification of tio2 resulting in the formation of a ct complex was achieved by the addition of surface - active ligands up to concentrations required to cover all surface sites ( [ tisurf ] = [ tio2]12.5/d , where tisurf is the molar concentration of surface ti sites , [ tio2 ] is the molar concentration of tio2 in molecular units , and d is the diameter of the particle in angstroms ) . as the consequence of enhanced particle particle interaction , upon surface modification that eliminates the surface charge , precipitation , and/or gelling of the solution may occur . in order to avoid these problems ph of the solution for the determination of ct complex binding constants the absorption spectra were recorded at room temperature in cells with 1 cm optical path length using thermo scientific evolution 600 uv / vis spectrophotometer . the experiments determining the composition of complexes by job s method were conducted by using 7.2 mm tio2 and 2 mm modifier solutions . volumes of tio2 solution used varied from 1 to 9 ml and those of modifiers solutions from 9 to 1 ml ; and total volume was always 10 ml . infrared spectra were taken in attenuated total reflection ( atr ) mode using a nicolet 380 ftir spectrometer equipped with a smart orbit atr attachment containing a single - reflection diamond crystal . the angle of incidence was 45. in order to avoid the precipitation of modified tio2 and the excess of unbound modifier , in the preparation of samples for ftir measurements the quantity of ligands added was 50% of all tisurf sites ( [ tio2 ] = 0.2 m , cligand = 25 mm ) . the dispersions containing surface modified tio2 nanoparticles were dried under argon at room temperature , and powders obtained were placed into the vacuum oven for 8 h to get to complete dryness . before measuring ftir spectra , powders were triturated in the agate mortar . the spectrum of the dried tio2 aqueous slurry ( not containing modifier ) was used as the background . when tio2 particles are in the nanocrystalline regime , a large fraction of the atoms that constitute the nanoparticle is located at the surface with significantly altered electrochemical properties . as the size of nanocrystalline tio2 becomes smaller than 20 nm the surface ti atoms adjust their coordination environment from hexacoordinated ( octahedral ) to pentacoordinated ( square pyramidal ) , which is followed by the compression of the ti o bond to accommodate for the curvature of the nanoparticle . these undercoordinated defect sites are the source of novel enhanced and selective reactivity of nanoparticles toward bidentate ligand binding . all of the investigated ligands listed in table 1 were found to undergo binding at the surface ( see inset in fig . these hybrid properties arise from the ligand - to - metal ct interaction coupled with electronic properties of the core of semiconductor nanoparticles . ligands used for modification of tio2 nanoparticles , benesi hildebrand binding constants , and bandgaps upon binding the absorption wavelengths at which the stability constants were determined are 400 nm ( catechol ) , 475 nm ( gallic acid ) , and 425 nm ( pyrogallol ) absorption spectra of surface modified 45 tio2 nanoparticles ( 0.09 m , ph 2 ) with different ligands ( 2.5 mm ) : ( a ) bare tio2 ; ( b ) catechol ; ( c ) gallic acid ; and ( d ) pyrogallol . molecular structures of ligands are shown in table 1 consequently , the onset of absorption of these ct nanocrystallites is red shifted compared to unmodified tio2(fig . the shift in the absorption edge in the modified semiconductor nanoparticles is attributed to the excitation of localized electrons from the surface modifier into the conduction band continuum states of the semiconductor particle . similar position of the absorption threshold for surface - modified nanoparticles with catechol , pyrogallol , and gallic acid ( bandgaps are presented in table 1 ) is probably the consequence of very similar dipole moments of various surface bound ti ligand complexes formed . apart from the shift in the absorption edge , the optical properties of surface modified semiconductor nanoparticles , having a continuous rise of absorption toward higher energies , paralleled the absorption properties characteristic of the band structure in bare semiconductor nanoparticles . a similar red shift , but in localized ct complex resulting in a pronounced absorption maximum , it should be noted that all investigated enediol ligands are by themselves extremely susceptible to oxidation . apparently , because of the bidentate binding to nanoparticles , enediol ligands gain stability and are not easily oxidized . enediol - modified tio2 colloids preserved their optical properties even after exposure to daylight for 23 months . due to the existence of undercoordinated surface defect sites and their lower efficiency of covalent bonding with solvent molecules in comparison with covalent bonding between atoms within the tio2 lattice , the surface species posses energy level in the midgap region . apart from red shift of the absorption onset of surface modified tio2 nanoparticles , ct interaction between the molecule of modifier and surface ti atoms also induces fine - tuning of the electrochemical potential of semiconductor nanocrystals indicating changes in oxidizing abilities . upon surface modification , the effective bandgap of catechol- , gallic acid- , and pyrogallol - modified tio2 nanoparticles at ph = 2 were determined to be : 1.96 , 1.92 , and 1.89 ev , respectively . these results indicate similar electrochemical potentials of semiconducting nanocrystals modified with chosen class of enediol ligands indicating similar binding structures and effective electronic coupling . tio2 nanoparticles modified with this type of bidentate benzene derivatives can be used for development of type ii dye - sensitized nanoporous titania solar cells where the dyes bind to the particle surface through enediol groups . also , blends of surface modified tio2 nanoparticles and organic semiconductors cast from co - solutions can be used for synthesis of hybrid solar cells combining the unique properties of inorganic semiconductor and film - forming properties of polymers . since these novel ct semiconducting materials exhibit optical properties that are distinct from their constituents , not absorbing in the visible region , benesi hildebrand analysis for molecular complexes can be employed to determine the stability constant of ct complex . hildebrand analysis can be used for small particles since the same relationship is obtained between the stability constant and ligand concentration from langmuir isotherm used for bulk compounds . for a colloidal solution of 45 tio2 one can consider the formation of an inner - sphere ct complex with the stability constant kb expressed as since the absorption in the visible region originates solely from the complex formed it is obvious that [ ctcomplex ] = a/l and the eq . 2can be rearranged to the following linearized form where [ l ] is the concentration of ligand , a and amax the absorbances of a ct complex for a given concentration of ligand l , and saturation concentration corresponding to the full coverage of tio2 surface , respectively . stability constants kb were determined from the absorbances of a series of solutions ( fig . 2 ) containing a fixed concentration of tio2 nanoparticles ( = 3.5 mm , i.e. , 0.97 mm tisurf according to equation in experimental section ) and increasing concentrations of ligands ( cligand = 0.061.23 mm ) . in order to avoid great errors in kb determination , the wavelength of complex absorption is chosen for each ligand to correspond to the requested absorption range ( 0.1 < a < 0.9 ) . by plotting 1/a vs. 1/[l ] the straight lines were obtained and from the ratio of the intercept and the slope , kb were determined and presented in table 1 . from the molecular structures of ligands used it can be concluded that two types of binding to tisurf may appear catecholate type ( oh , oh ) or carboxylate ( cooh ) in the case of gallic acid solely . however , it is quite obvious that by comparing the measured values of kb one can not differentiate between these two types of binding since these values are similar , of the order 10 m. in the literature , to determine the ct complex stability constants for catechol and/or gallic acid the adsorption approach after filtration method or ftir measurements have been used and two to three times larger kb values were reported . we believe that lower kb values we reported are more precisely determined since they are obtained from the absorbance measurements directly . absorption spectra of 3.5 mm tio2 nanoparticles before and after surface modification with pyrogallol ( 01.23 mm in 0.06 mm steps ) . inset : absorption at 425 nm of tio2pyrogallol ct complex versus pyrogallol concentration ( data recorded 20 h after surface modification ) . solutions of tio2 nanoparticles used for modification were freshly prepared ( a ) and 4-years - old ( b ) it must be pointed out that very interesting phenomenon was observed when tio2 solutions of the same molecular concentration ( 3.5 mm ) freshly prepared and 4-years - old were used for modification ( inset of fig . 2).a425 versus pyrogallol concentration curve for freshly prepared tio2 shows much higher saturation value of a425 than was observed in the case of 4-years - old tio2 colloidal solution ( a425 = 0.65 a425 ) . it is known that crystallinity increases with aging of tio2 nanoparticles inducing the decrease in concentration of coordinatively unsaturated surface defect sites . in order to eliminate the possibility that decrease in number of tisurf sites is simply the consequence of agglomeration or oswald ripening we performed the measurements of the particle size diameter using dynamic light scattering ( dls ) . in both cases ( freshly prepared and 4-years - old tio2 solutions ) the effective diameter was found to be the same . additionally , the stoichiometric ratio between tisurf atoms and modifiers in the ct complexes was checked by job s method of continuous variation assuming that only one type of complex is present in solution . job s plots for both complexes reached a maximum value at a mole fraction of [ tisurf]/[tisurf ] + [ l ] = 0.7 , confirming that molar ratio between tisurf atoms and ligands in the complex is 2:1 . job s curve of equimolar solutions for ligand tisurf complex ( acatechol , at 400 nm;agallic acid at 475 nm ) ; [ tisurf ] + [ l ] = 2 mm the way ligands bind to tio2 surface was investigated by using atr ftir spectroscopy . since the infrared spectrum of dried tio2 has only the characteristic broadband in 37002000 cm region , we were able to measure spectra of modified colloids in 17501000 cm region where the characteristic bands of modifiers exist . spectra of adsorbed ligands were obtained by subtracting the spectrum of bare tio2 nanoparticles from the spectrum of surface modified tio2 nanoparticles . the atr ftir spectra of catechol , free and adsorbed on tio2 nanoparticles were presented in fig . the main bands and their assignments in free catechol ( curve a ) are as follows : stretching vibrations of the aromatic ring (c c)/(c = c ) at 1618 , 1594 , 1512 , and 1468 cm , stretching vibrations of the phenolic group (c oh ) at 1279 , 1254 and 1239 cm , bending vibrations of the phenolic group (c oh ) at 1365 , 1184 , 1163 , and 1149 cm and bending (c h ) at 1039 and 1093 cm . upon adsorption of catechol onto tio2 ( curve b ) the difference between ftir spectra of free and adsorbed modifier appears , indicating surface complexation with catechol bound to the oxide surface in bidentate form . oh ) vibrations in the region below 1200 cm loose their hyperfine structure , while the pronounced band at 1365 cm nearly disappears and a very weak and broad feature centered at 1354 cm appears . three bands of stretching vibrations (c oh ) merge to one prominent band at 1249 cm . the binding of catechol to tio2 via two adjacent phenolic groups even affects the stretching of the aromatic ring ( bands above 1400 cm ) . ( b ) catecholate type of binding inherent to catechol molecule adsorption to metal - oxide surfaces , with two adjacent phenolic oh groups taking part in complexation , was reported to result in the formation of both bidentate mononuclear chelating and/or bidentate binuclear bridging complexes . there are two opinions dealing with catecholate binding : some authors claim that five - membered ring coordination complexes predominate , while the others find bridging complexes energetically more favorable . since according to job s curve the molar ratio between tisurf atoms and catechol in the complex is 2:1 ( fig . 3 ) , the ct complex formed is most likely bidentate binuclear ( bridging ) complex ( scheme 1 ) . coordination mode of tisurf catechol complex the atr ftir spectra of pyrogallol , free and adsorbed on tio2 nanoparticles were presented in fig . the main bands and their assignments in free pyrogallol ( curve a ) are as follows : stretching vibrations of the aromatic ring (c c)/(c = c ) at 1619 , 1518 , 1479 , 1402 , and 1383 cm , stretching vibrations of the phenolic group (c oh ) at 1518 , 1402 , 1315 , 1285 , 1242 , and 999 cm , bending vibrations of the phenolic group (c oh ) at 1479 , 1383 , 1350 , 1189 , and 1156 cm and bending (c h ) at 1150 and 1064 cm . the adsorption of pyrogallol onto tio2 nanoparticles ( curve b ) leads to obvious changes in ftir spectra : complete disappearance of the bands at 1518 , 1402 , 1383 , 1350 , and 1285 cm , shift of the band at 999 to 1032 cm , and the intensity attenuation of the bands at 1315 , 1189 , and 1156 cm . the results obtained are quite expected since these bands are assigned to stretching and bending vibrations of phenolic oh groups that participate in the formation of ct complex with ti surface atoms . the binding of pyrogallol to tio2 , like in the case of catechol , also shifts the bands assigned to stretching of the aromatic ring in the region above 1400 cm . bands at 1315 , 1242 , 1189 , and 1157 cm , being changed in intensity , are probably the vibrations of the unbound , third phenolic oh group of pyrogallol , since just two adjacent phenolic groups participate in binding ( scheme 1 ) . ( b ) the atr ftir spectra of gallic acid , free and adsorbed on tio2 nanoparticles were presented in fig . 6 . the main bands and their assignments in free protonated gallic acid ( curve a ) are as follows : stretching vibrations of the aromatic ring (c c)/(c = c ) at 1612 , 1539 , 1469 , and 1437 cm , stretching vibrations of the phenolic group (c oh ) at 1372 , 1305 , 1240 , and 1020 cm , bending vibrations of the phenolic group (c oh ) at 1336 , 1240 , and 1100 cm , stretching and/or bending vibrations of co or oh in cooh at 1199 , 1100 , and 1046 cm and pronounced stretching vibration of the carbonyl group (c = o ) at 1698 cm . the adsorption of gallic acid onto tio2 nanoparticles ( curve b ) leads to complete disappearance of the bands at 1372 and 1305 cm , while the band at 1020 cm disappears with the formation of broad intensive band at 1070 cm . the band at 1240 cm , these bands , assigned to stretching and bending vibrations of phenolic oh groups ( (c oh ) and (c oh ) ) , are probably those participating in the formation of ct complex with ti surface atoms . since upon binding the third phenolic group remains unbound , bands at 1336 and 1240 cm remain in spectrum , although slightly shifted and with decreased intensity . the pronounced bands assigned to the vibrations of the carboxylic group ( 1199 and 1698 cm ) remain in the spectrum suggesting that this group is not involved in complexation . the binding of gallic acid to tio2 , like in the case of catechol and pyrogallol , also shifts the bands assigned to stretching of the aromatic ring in the region above 1400 cm . ( b ) according to the molecular structure of gallic acid two types of binding to tisurf may appear catecholate ( oh , oh ) or carboxylate ( cooh ) type . literature data are quite controversial whether or not benzoic acid , having only cooh functional group , adsorbs on tio2 surface . tunesi and anderson claim that no adsorption was observed , while the others confirmed the formation of bidentate chelate or bridging complexes , pointing out weak adsorption . according to ftir results obtained we may conclude that in the case of gallic acid binding through carboxylate group does not exist . possible reason for that is as follows : in the carboxylate type of binding formation of chelated four - membered ring is proposed being energetically less favorable than the ring formed through catecholate group ( scheme 1 ) . hence , in all three ligands investigated ( catechol , pyrogallol , and gallic acid ) catecholate type of binding is obvious , since carboxylate type of binding takes no part in the binding of gallic acid to tio2 nanoparticles . when tio2 particles are in the nanocrystalline regime , a large fraction of the atoms that constitute the nanoparticle is located at the surface with significantly altered electrochemical properties . as the size of nanocrystalline tio2 becomes smaller than 20 nm the surface ti atoms adjust their coordination environment from hexacoordinated ( octahedral ) to pentacoordinated ( square pyramidal ) , which is followed by the compression of the ti o bond to accommodate for the curvature of the nanoparticle . these undercoordinated defect sites are the source of novel enhanced and selective reactivity of nanoparticles toward bidentate ligand binding . all of the investigated ligands listed in table 1 were found to undergo binding at the surface ( see inset in fig . these hybrid properties arise from the ligand - to - metal ct interaction coupled with electronic properties of the core of semiconductor nanoparticles . ligands used for modification of tio2 nanoparticles , benesi hildebrand binding constants , and bandgaps upon binding the absorption wavelengths at which the stability constants were determined are 400 nm ( catechol ) , 475 nm ( gallic acid ) , and 425 nm ( pyrogallol ) absorption spectra of surface modified 45 tio2 nanoparticles ( 0.09 m , ph 2 ) with different ligands ( 2.5 mm ) : ( a ) bare tio2 ; ( b ) catechol ; ( c ) gallic acid ; and ( d ) pyrogallol . molecular structures of ligands are shown in table 1 consequently , the onset of absorption of these ct nanocrystallites is red shifted compared to unmodified tio2(fig . the shift in the absorption edge in the modified semiconductor nanoparticles is attributed to the excitation of localized electrons from the surface modifier into the conduction band continuum states of the semiconductor particle . similar position of the absorption threshold for surface - modified nanoparticles with catechol , pyrogallol , and gallic acid ( bandgaps are presented in table 1 ) is probably the consequence of very similar dipole moments of various surface bound ti ligand complexes formed . apart from the shift in the absorption edge , the optical properties of surface modified semiconductor nanoparticles , having a continuous rise of absorption toward higher energies , paralleled the absorption properties characteristic of the band structure in bare semiconductor nanoparticles . a similar red shift , but in localized ct complex resulting in a pronounced absorption maximum , it should be noted that all investigated enediol ligands are by themselves extremely susceptible to oxidation . apparently , because of the bidentate binding to nanoparticles , enediol ligands gain stability and are not easily oxidized . enediol - modified tio2 colloids preserved their optical properties even after exposure to daylight for 23 months . due to the existence of undercoordinated surface defect sites and their lower efficiency of covalent bonding with solvent molecules in comparison with covalent bonding between atoms within the tio2 lattice , the surface species posses energy level in the midgap region . apart from red shift of the absorption onset of surface modified tio2 nanoparticles , ct interaction between the molecule of modifier and surface ti atoms also induces fine - tuning of the electrochemical potential of semiconductor nanocrystals indicating changes in oxidizing abilities . upon surface modification , the effective bandgap of catechol- , gallic acid- , and pyrogallol - modified tio2 nanoparticles at ph = 2 were determined to be : 1.96 , 1.92 , and 1.89 ev , respectively . these results indicate similar electrochemical potentials of semiconducting nanocrystals modified with chosen class of enediol ligands indicating similar binding structures and effective electronic coupling . tio2 nanoparticles modified with this type of bidentate benzene derivatives can be used for development of type ii dye - sensitized nanoporous titania solar cells where the dyes bind to the particle surface through enediol groups . also , blends of surface modified tio2 nanoparticles and organic semiconductors cast from co - solutions can be used for synthesis of hybrid solar cells combining the unique properties of inorganic semiconductor and film - forming properties of polymers . since these novel ct semiconducting materials exhibit optical properties that are distinct from their constituents , not absorbing in the visible region , benesi hildebrand analysis for molecular complexes can be employed to determine the stability constant of ct complex . hildebrand analysis can be used for small particles since the same relationship is obtained between the stability constant and ligand concentration from langmuir isotherm used for bulk compounds . for a colloidal solution of 45 tio2 one can consider the formation of an inner - sphere ct complex with the stability constant kb expressed as since the absorption in the visible region originates solely from the complex formed it is obvious that [ ctcomplex ] = a/l and the eq . 2can be rearranged to the following linearized form where [ l ] is the concentration of ligand , a and amax the absorbances of a ct complex for a given concentration of ligand l , and saturation concentration corresponding to the full coverage of tio2 surface , respectively . stability constants kb were determined from the absorbances of a series of solutions ( fig . 2 ) containing a fixed concentration of tio2 nanoparticles ( = 3.5 mm , i.e. , 0.97 mm tisurf according to equation in experimental section ) and increasing concentrations of ligands ( cligand = 0.061.23 mm ) . in order to avoid great errors in kb determination , the wavelength of complex absorption is chosen for each ligand to correspond to the requested absorption range ( 0.1 < a < 0.9 ) . by plotting 1/a vs. 1/[l ] the straight lines were obtained and from the ratio of the intercept and the slope , kb were determined and presented in table 1 . from the molecular structures of ligands used it can be concluded that two types of binding to tisurf may appear catecholate type ( oh , oh ) or carboxylate ( cooh ) in the case of gallic acid solely . however , it is quite obvious that by comparing the measured values of kb one can not differentiate between these two types of binding since these values are similar , of the order 10 m. in the literature , to determine the ct complex stability constants for catechol and/or gallic acid the adsorption approach after filtration method or ftir measurements have been used and two to three times larger kb values were reported . we believe that lower kb values we reported are more precisely determined since they are obtained from the absorbance measurements directly . absorption spectra of 3.5 mm tio2 nanoparticles before and after surface modification with pyrogallol ( 01.23 mm in 0.06 mm steps ) . inset : absorption at 425 nm of tio2pyrogallol ct complex versus pyrogallol concentration ( data recorded 20 h after surface modification ) . solutions of tio2 nanoparticles used for modification were freshly prepared ( a ) and 4-years - old ( b ) it must be pointed out that very interesting phenomenon was observed when tio2 solutions of the same molecular concentration ( 3.5 mm ) freshly prepared and 4-years - old were used for modification ( inset of fig . 2).a425 versus pyrogallol concentration curve for freshly prepared tio2 shows much higher saturation value of a425 than was observed in the case of 4-years - old tio2 colloidal solution ( a425 = 0.65 a425 ) . it is known that crystallinity increases with aging of tio2 nanoparticles inducing the decrease in concentration of coordinatively unsaturated surface defect sites . in order to eliminate the possibility that decrease in number of tisurf sites is simply the consequence of agglomeration or oswald ripening we performed the measurements of the particle size diameter using dynamic light scattering ( dls ) . in both cases ( freshly prepared and 4-years - old tio2 solutions ) additionally , the stoichiometric ratio between tisurf atoms and modifiers in the ct complexes was checked by job s method of continuous variation assuming that only one type of complex is present in solution . job s plots for both complexes reached a maximum value at a mole fraction of [ tisurf]/[tisurf ] + [ l ] = 0.7 , confirming that molar ratio between tisurf atoms and ligands in the complex is 2:1 . job s curve of equimolar solutions for ligand tisurf complex ( acatechol , at 400 nm;agallic acid at 475 nm ) ; [ tisurf ] + [ l ] = 2 mm the way ligands bind to tio2 surface was investigated by using atr ftir spectroscopy . since the infrared spectrum of dried tio2 has only the characteristic broadband in 37002000 cm region , we were able to measure spectra of modified colloids in 17501000 cm region where the characteristic bands of modifiers exist . spectra of adsorbed ligands were obtained by subtracting the spectrum of bare tio2 nanoparticles from the spectrum of surface modified tio2 nanoparticles . the atr ftir spectra of catechol , free and adsorbed on tio2 nanoparticles were presented in fig . the main bands and their assignments in free catechol ( curve a ) are as follows : stretching vibrations of the aromatic ring (c c)/(c = c ) at 1618 , 1594 , 1512 , and 1468 cm , stretching vibrations of the phenolic group (c oh ) at 1279 , 1254 and 1239 cm , bending vibrations of the phenolic group (c oh ) at 1365 , 1184 , 1163 , and 1149 cm and bending (c h ) at 1039 and 1093 cm . upon adsorption of catechol onto tio2 ( curve b ) the difference between ftir spectra of free and adsorbed modifier appears , indicating surface complexation with catechol bound to the oxide surface in bidentate form . oh ) vibrations in the region below 1200 cm loose their hyperfine structure , while the pronounced band at 1365 cm nearly disappears and a very weak and broad feature centered at 1354 cm appears . three bands of stretching vibrations (c oh ) merge to one prominent band at 1249 cm . the binding of catechol to tio2 via two adjacent phenolic groups even affects the stretching of the aromatic ring ( bands above 1400 cm ) . ftir spectra of catechol : free ( a ) and adsorbed on tio2 nanoparticles ( b ) catecholate type of binding inherent to catechol molecule adsorption to metal - oxide surfaces , with two adjacent phenolic oh groups taking part in complexation , was reported to result in the formation of both bidentate mononuclear chelating and/or bidentate binuclear bridging complexes . there are two opinions dealing with catecholate binding : some authors claim that five - membered ring coordination complexes predominate , while the others find bridging complexes energetically more favorable . since according to job s curve the molar ratio between tisurf atoms and catechol in the complex is 2:1 ( fig . 3 ) , the ct complex formed is most likely bidentate binuclear ( bridging ) complex ( scheme 1 ) . coordination mode of tisurf catechol complex the atr ftir spectra of pyrogallol , free and adsorbed on tio2 nanoparticles were presented in fig . the main bands and their assignments in free pyrogallol ( curve a ) are as follows : stretching vibrations of the aromatic ring (c c)/(c = c ) at 1619 , 1518 , 1479 , 1402 , and 1383 cm , stretching vibrations of the phenolic group (c oh ) at 1518 , 1402 , 1315 , 1285 , 1242 , and 999 cm , bending vibrations of the phenolic group (c oh ) at 1479 , 1383 , 1350 , 1189 , and 1156 cm and bending (c h ) at 1150 and 1064 cm . the adsorption of pyrogallol onto tio2 nanoparticles ( curve b ) leads to obvious changes in ftir spectra : complete disappearance of the bands at 1518 , 1402 , 1383 , 1350 , and 1285 cm , shift of the band at 999 to 1032 cm , and the intensity attenuation of the bands at 1315 , 1189 , and 1156 cm . the results obtained are quite expected since these bands are assigned to stretching and bending vibrations of phenolic oh groups that participate in the formation of ct complex with ti surface atoms . the binding of pyrogallol to tio2 , like in the case of catechol , also shifts the bands assigned to stretching of the aromatic ring in the region above 1400 cm . bands at 1315 , 1242 , 1189 , and 1157 cm , being changed in intensity , are probably the vibrations of the unbound , third phenolic oh group of pyrogallol , since just two adjacent phenolic groups participate in binding ( scheme 1 ) . ftir spectra of pyrogallol : free ( a ) and adsorbed on tio2 nanoparticles ( b ) the atr ftir spectra of gallic acid , free and adsorbed on tio2 nanoparticles were presented in fig . the main bands and their assignments in free protonated gallic acid ( curve a ) are as follows : stretching vibrations of the aromatic ring (c c)/(c = c ) at 1612 , 1539 , 1469 , and 1437 cm , stretching vibrations of the phenolic group (c oh ) at 1372 , 1305 , 1240 , and 1020 cm , bending vibrations of the phenolic group (c oh ) at 1336 , 1240 , and 1100 cm , stretching and/or bending vibrations of co or oh in cooh at 1199 , 1100 , and 1046 cm and pronounced stretching vibration of the carbonyl group (c = o ) at 1698 cm . the adsorption of gallic acid onto tio2 nanoparticles ( curve b ) leads to complete disappearance of the bands at 1372 and 1305 cm , while the band at 1020 cm disappears with the formation of broad intensive band at 1070 cm . the band at 1240 cm , although slightly shifted , is preserved with decreased intensity . these bands , assigned to stretching and bending vibrations of phenolic oh groups ( (c oh ) and (c oh ) ) , are probably those participating in the formation of ct complex with ti surface atoms . since upon binding the third phenolic group remains unbound , bands at 1336 and 1240 cm remain in spectrum , although slightly shifted and with decreased intensity . the pronounced bands assigned to the vibrations of the carboxylic group ( 1199 and 1698 cm ) remain in the spectrum suggesting that this group is not involved in complexation . the binding of gallic acid to tio2 , like in the case of catechol and pyrogallol , also shifts the bands assigned to stretching of the aromatic ring in the region above 1400 cm . ( b ) according to the molecular structure of gallic acid two types of binding to tisurf may appear catecholate ( oh , oh ) or carboxylate ( cooh ) type . literature data are quite controversial whether or not benzoic acid , having only cooh functional group , adsorbs on tio2 surface . tunesi and anderson claim that no adsorption was observed , while the others confirmed the formation of bidentate chelate or bridging complexes , pointing out weak adsorption . according to ftir results obtained we may conclude that in the case of gallic acid binding through carboxylate group does not exist . possible reason for that is as follows : in the carboxylate type of binding formation of chelated four - membered ring is proposed being energetically less favorable than the ring formed through catecholate group ( scheme 1 ) . hence , in all three ligands investigated ( catechol , pyrogallol , and gallic acid ) catecholate type of binding is obvious , since carboxylate type of binding takes no part in the binding of gallic acid to tio2 nanoparticles . all investigated ligands ( catechol , pyrogallol , and gallic acid ) form inner - sphere ct complexes with tio2 nanoparticles ( d = 45 ) . binding of the modifier molecules to undercoordinated surface ti atoms ( defect sites ) results in a significant change in the onset of absorption and the effective bandgap . from the benesi hildebrand plot , the stability constants at ph 2 of the order 10 m have been determined . for chosen enediol modifiers binding was found to be through bidentate binuclear ( bridging ) complexes leading to restoration of six - coordinated octahedral geometry of surface ti atoms . stabilized charge separation , being important feature of these systems opens - up possibility for using modifier molecules as conductive leads that allow electronic linking of the nanoparticle into molecular circuits providing further extension of photoinduced electron transfer . financial support for this study was granted by the ministry of science and technological development of the republic of serbia ( project 142066 ) .

surface modification of nanocrystalline tio2 particles ( 45 ) with bidentate benzene derivatives ( catechol , pyrogallol , and gallic acid ) was found to alter optical properties of nanoparticles . the formation of the inner - sphere charge transfer complexes results in a red shift of the semiconductor absorption compared to unmodified nanocrystallites . the binding structures were investigated by using ftir spectroscopy . the investigated ligands have the optimal geometry for chelating surface ti atoms , resulting in ring coordination complexes ( catecholate type of binuclear bidentate binding bridging ) thus restoring in six - coordinated octahedral geometry of surface ti atoms . from the benesi hildebrand plot , the stability constants at ph 2 of the order 103 m1 have been determined .

Introduction Experimental Section Results and Discussion Optical Properties of Surface Modified Nanocrystalline TiO Determination of Stability (Binding) Constants Binding Structure of Ligands at Nanoparticle Surface Conclusions Acknowledgment

the overall photoexcitation process of semiconductor nanoparticles by ultra bandgap energies involve the photogeneration of electron hole pairs within particle , followed by the competition between recombination , interfacial charge transfer to adsorbed compounds , and migration into midgap surface states . owing to large curvature of tio2 particles in the nanosize regime , the surface reconstructs in such manner that distorts the crystalline environment of surface ti atoms forming coordinatively unsaturated ti atoms . the changes in the pre - edge structure of ti k - edge spectrum ( xanes ) of tio2 nanoparticles ( d < 20 nm ) revealed the existence of square pyramidal coordination of surface ti atoms ( pentacoordinate ) . it was reported , on a whole class of electron - donating enediol ligands , benzene derivatives , or mercapto - carboxylic acids that binding to coordinatively unsaturated ti atoms simultaneously adjusts their coordination to octahedral geometry at the surface of nanocrystallites and changes the electronic properties of tio2 . this results in a red shift of the semiconductor absorption compared to that of unmodified nanocrystallites and enables efficient harvesting of solar photons . additionally , this type of electronic coupling yields to instantaneous separation of photogenerated charges into two phases , the holes localize on the donating organic modifier , and the electrons delocalize in the conduction band of tio2 . moreover , the enediol ligands were found to act as conductive leads , allowing wiring of oligonucleotides and proteins resulting in enhanced charge separation and ensuing chemical transformations . in last two decades the surface modification of commercial tio2(degussa p25,d = 30 nm ) with benzene derivatives ( mainly catechol and salicylic acid ) was studied [ 19 - 34 ] . herein , we report surface modification of tio2 nanoparticles with enediol ligands ( catechol , pyrogallol , and gallic acid ) that are able to adjust the coordination geometry of the surface ti atoms inducing shift of the absorption onset toward the visible region of the spectrum , compared to unmodified nanocrystallites . since these novel ct semiconducting materials exhibit optical properties that are distinct from their constituents , not absorbing in the visible region , benesi hildebrand analysis for molecular complexes was employed to determine the stability constants from the absorption spectra . slow growth of the particles was achieved by using dialysis at 4 c against water until the ph = 3.5 was reached . the concentration of tio2 ( 0.2 m ) was determined from the concentration of the peroxide complex obtained after dissolving the colloid in concentrated h2so4 . surface modification of tio2 resulting in the formation of a ct complex was achieved by the addition of surface - active ligands up to concentrations required to cover all surface sites ( [ tisurf ] = [ tio2]12.5/d , where tisurf is the molar concentration of surface ti sites , [ tio2 ] is the molar concentration of tio2 in molecular units , and d is the diameter of the particle in angstroms ) . as the consequence of enhanced particle particle interaction , upon surface modification that eliminates the surface charge , precipitation , and/or gelling of the solution may occur . as the size of nanocrystalline tio2 becomes smaller than 20 nm the surface ti atoms adjust their coordination environment from hexacoordinated ( octahedral ) to pentacoordinated ( square pyramidal ) , which is followed by the compression of the ti o bond to accommodate for the curvature of the nanoparticle . all of the investigated ligands listed in table 1 were found to undergo binding at the surface ( see inset in fig . these hybrid properties arise from the ligand - to - metal ct interaction coupled with electronic properties of the core of semiconductor nanoparticles . ligands used for modification of tio2 nanoparticles , benesi hildebrand binding constants , and bandgaps upon binding the absorption wavelengths at which the stability constants were determined are 400 nm ( catechol ) , 475 nm ( gallic acid ) , and 425 nm ( pyrogallol ) absorption spectra of surface modified 45 tio2 nanoparticles ( 0.09 m , ph 2 ) with different ligands ( 2.5 mm ) : ( a ) bare tio2 ; ( b ) catechol ; ( c ) gallic acid ; and ( d ) pyrogallol . molecular structures of ligands are shown in table 1 consequently , the onset of absorption of these ct nanocrystallites is red shifted compared to unmodified tio2(fig . the shift in the absorption edge in the modified semiconductor nanoparticles is attributed to the excitation of localized electrons from the surface modifier into the conduction band continuum states of the semiconductor particle . similar position of the absorption threshold for surface - modified nanoparticles with catechol , pyrogallol , and gallic acid ( bandgaps are presented in table 1 ) is probably the consequence of very similar dipole moments of various surface bound ti ligand complexes formed . apart from the shift in the absorption edge , the optical properties of surface modified semiconductor nanoparticles , having a continuous rise of absorption toward higher energies , paralleled the absorption properties characteristic of the band structure in bare semiconductor nanoparticles . a similar red shift , but in localized ct complex resulting in a pronounced absorption maximum , it should be noted that all investigated enediol ligands are by themselves extremely susceptible to oxidation . apart from red shift of the absorption onset of surface modified tio2 nanoparticles , ct interaction between the molecule of modifier and surface ti atoms also induces fine - tuning of the electrochemical potential of semiconductor nanocrystals indicating changes in oxidizing abilities . upon surface modification , the effective bandgap of catechol- , gallic acid- , and pyrogallol - modified tio2 nanoparticles at ph = 2 were determined to be : 1.96 , 1.92 , and 1.89 ev , respectively . tio2 nanoparticles modified with this type of bidentate benzene derivatives can be used for development of type ii dye - sensitized nanoporous titania solar cells where the dyes bind to the particle surface through enediol groups . since these novel ct semiconducting materials exhibit optical properties that are distinct from their constituents , not absorbing in the visible region , benesi hildebrand analysis for molecular complexes can be employed to determine the stability constant of ct complex . for a colloidal solution of 45 tio2 one can consider the formation of an inner - sphere ct complex with the stability constant kb expressed as since the absorption in the visible region originates solely from the complex formed it is obvious that [ ctcomplex ] = a/l and the eq . from the molecular structures of ligands used it can be concluded that two types of binding to tisurf may appear catecholate type ( oh , oh ) or carboxylate ( cooh ) in the case of gallic acid solely . however , it is quite obvious that by comparing the measured values of kb one can not differentiate between these two types of binding since these values are similar , of the order 10 m. in the literature , to determine the ct complex stability constants for catechol and/or gallic acid the adsorption approach after filtration method or ftir measurements have been used and two to three times larger kb values were reported . job s curve of equimolar solutions for ligand tisurf complex ( acatechol , at 400 nm;agallic acid at 475 nm ) ; [ tisurf ] + [ l ] = 2 mm the way ligands bind to tio2 surface was investigated by using atr ftir spectroscopy . ( b ) catecholate type of binding inherent to catechol molecule adsorption to metal - oxide surfaces , with two adjacent phenolic oh groups taking part in complexation , was reported to result in the formation of both bidentate mononuclear chelating and/or bidentate binuclear bridging complexes . the adsorption of pyrogallol onto tio2 nanoparticles ( curve b ) leads to obvious changes in ftir spectra : complete disappearance of the bands at 1518 , 1402 , 1383 , 1350 , and 1285 cm , shift of the band at 999 to 1032 cm , and the intensity attenuation of the bands at 1315 , 1189 , and 1156 cm . the binding of pyrogallol to tio2 , like in the case of catechol , also shifts the bands assigned to stretching of the aromatic ring in the region above 1400 cm . bands at 1315 , 1242 , 1189 , and 1157 cm , being changed in intensity , are probably the vibrations of the unbound , third phenolic oh group of pyrogallol , since just two adjacent phenolic groups participate in binding ( scheme 1 ) . the main bands and their assignments in free protonated gallic acid ( curve a ) are as follows : stretching vibrations of the aromatic ring (c c)/(c = c ) at 1612 , 1539 , 1469 , and 1437 cm , stretching vibrations of the phenolic group (c oh ) at 1372 , 1305 , 1240 , and 1020 cm , bending vibrations of the phenolic group (c oh ) at 1336 , 1240 , and 1100 cm , stretching and/or bending vibrations of co or oh in cooh at 1199 , 1100 , and 1046 cm and pronounced stretching vibration of the carbonyl group (c = o ) at 1698 cm . the adsorption of gallic acid onto tio2 nanoparticles ( curve b ) leads to complete disappearance of the bands at 1372 and 1305 cm , while the band at 1020 cm disappears with the formation of broad intensive band at 1070 cm . the band at 1240 cm , these bands , assigned to stretching and bending vibrations of phenolic oh groups ( (c oh ) and (c oh ) ) , are probably those participating in the formation of ct complex with ti surface atoms . the binding of gallic acid to tio2 , like in the case of catechol and pyrogallol , also shifts the bands assigned to stretching of the aromatic ring in the region above 1400 cm . possible reason for that is as follows : in the carboxylate type of binding formation of chelated four - membered ring is proposed being energetically less favorable than the ring formed through catecholate group ( scheme 1 ) . hence , in all three ligands investigated ( catechol , pyrogallol , and gallic acid ) catecholate type of binding is obvious , since carboxylate type of binding takes no part in the binding of gallic acid to tio2 nanoparticles . when tio2 particles are in the nanocrystalline regime , a large fraction of the atoms that constitute the nanoparticle is located at the surface with significantly altered electrochemical properties . as the size of nanocrystalline tio2 becomes smaller than 20 nm the surface ti atoms adjust their coordination environment from hexacoordinated ( octahedral ) to pentacoordinated ( square pyramidal ) , which is followed by the compression of the ti o bond to accommodate for the curvature of the nanoparticle . all of the investigated ligands listed in table 1 were found to undergo binding at the surface ( see inset in fig . these hybrid properties arise from the ligand - to - metal ct interaction coupled with electronic properties of the core of semiconductor nanoparticles . ligands used for modification of tio2 nanoparticles , benesi hildebrand binding constants , and bandgaps upon binding the absorption wavelengths at which the stability constants were determined are 400 nm ( catechol ) , 475 nm ( gallic acid ) , and 425 nm ( pyrogallol ) absorption spectra of surface modified 45 tio2 nanoparticles ( 0.09 m , ph 2 ) with different ligands ( 2.5 mm ) : ( a ) bare tio2 ; ( b ) catechol ; ( c ) gallic acid ; and ( d ) pyrogallol . molecular structures of ligands are shown in table 1 consequently , the onset of absorption of these ct nanocrystallites is red shifted compared to unmodified tio2(fig . the shift in the absorption edge in the modified semiconductor nanoparticles is attributed to the excitation of localized electrons from the surface modifier into the conduction band continuum states of the semiconductor particle . similar position of the absorption threshold for surface - modified nanoparticles with catechol , pyrogallol , and gallic acid ( bandgaps are presented in table 1 ) is probably the consequence of very similar dipole moments of various surface bound ti ligand complexes formed . apart from the shift in the absorption edge , the optical properties of surface modified semiconductor nanoparticles , having a continuous rise of absorption toward higher energies , paralleled the absorption properties characteristic of the band structure in bare semiconductor nanoparticles . a similar red shift , but in localized ct complex resulting in a pronounced absorption maximum , it should be noted that all investigated enediol ligands are by themselves extremely susceptible to oxidation . apart from red shift of the absorption onset of surface modified tio2 nanoparticles , ct interaction between the molecule of modifier and surface ti atoms also induces fine - tuning of the electrochemical potential of semiconductor nanocrystals indicating changes in oxidizing abilities . upon surface modification , the effective bandgap of catechol- , gallic acid- , and pyrogallol - modified tio2 nanoparticles at ph = 2 were determined to be : 1.96 , 1.92 , and 1.89 ev , respectively . tio2 nanoparticles modified with this type of bidentate benzene derivatives can be used for development of type ii dye - sensitized nanoporous titania solar cells where the dyes bind to the particle surface through enediol groups . since these novel ct semiconducting materials exhibit optical properties that are distinct from their constituents , not absorbing in the visible region , benesi hildebrand analysis for molecular complexes can be employed to determine the stability constant of ct complex . for a colloidal solution of 45 tio2 one can consider the formation of an inner - sphere ct complex with the stability constant kb expressed as since the absorption in the visible region originates solely from the complex formed it is obvious that [ ctcomplex ] = a/l and the eq . from the molecular structures of ligands used it can be concluded that two types of binding to tisurf may appear catecholate type ( oh , oh ) or carboxylate ( cooh ) in the case of gallic acid solely . however , it is quite obvious that by comparing the measured values of kb one can not differentiate between these two types of binding since these values are similar , of the order 10 m. in the literature , to determine the ct complex stability constants for catechol and/or gallic acid the adsorption approach after filtration method or ftir measurements have been used and two to three times larger kb values were reported . job s curve of equimolar solutions for ligand tisurf complex ( acatechol , at 400 nm;agallic acid at 475 nm ) ; [ tisurf ] + [ l ] = 2 mm the way ligands bind to tio2 surface was investigated by using atr ftir spectroscopy . the binding of catechol to tio2 via two adjacent phenolic groups even affects the stretching of the aromatic ring ( bands above 1400 cm ) . ftir spectra of catechol : free ( a ) and adsorbed on tio2 nanoparticles ( b ) catecholate type of binding inherent to catechol molecule adsorption to metal - oxide surfaces , with two adjacent phenolic oh groups taking part in complexation , was reported to result in the formation of both bidentate mononuclear chelating and/or bidentate binuclear bridging complexes . there are two opinions dealing with catecholate binding : some authors claim that five - membered ring coordination complexes predominate , while the others find bridging complexes energetically more favorable . the adsorption of pyrogallol onto tio2 nanoparticles ( curve b ) leads to obvious changes in ftir spectra : complete disappearance of the bands at 1518 , 1402 , 1383 , 1350 , and 1285 cm , shift of the band at 999 to 1032 cm , and the intensity attenuation of the bands at 1315 , 1189 , and 1156 cm . the binding of pyrogallol to tio2 , like in the case of catechol , also shifts the bands assigned to stretching of the aromatic ring in the region above 1400 cm . bands at 1315 , 1242 , 1189 , and 1157 cm , being changed in intensity , are probably the vibrations of the unbound , third phenolic oh group of pyrogallol , since just two adjacent phenolic groups participate in binding ( scheme 1 ) . the main bands and their assignments in free protonated gallic acid ( curve a ) are as follows : stretching vibrations of the aromatic ring (c c)/(c = c ) at 1612 , 1539 , 1469 , and 1437 cm , stretching vibrations of the phenolic group (c oh ) at 1372 , 1305 , 1240 , and 1020 cm , bending vibrations of the phenolic group (c oh ) at 1336 , 1240 , and 1100 cm , stretching and/or bending vibrations of co or oh in cooh at 1199 , 1100 , and 1046 cm and pronounced stretching vibration of the carbonyl group (c = o ) at 1698 cm . the adsorption of gallic acid onto tio2 nanoparticles ( curve b ) leads to complete disappearance of the bands at 1372 and 1305 cm , while the band at 1020 cm disappears with the formation of broad intensive band at 1070 cm . these bands , assigned to stretching and bending vibrations of phenolic oh groups ( (c oh ) and (c oh ) ) , are probably those participating in the formation of ct complex with ti surface atoms . the binding of gallic acid to tio2 , like in the case of catechol and pyrogallol , also shifts the bands assigned to stretching of the aromatic ring in the region above 1400 cm . hence , in all three ligands investigated ( catechol , pyrogallol , and gallic acid ) catecholate type of binding is obvious , since carboxylate type of binding takes no part in the binding of gallic acid to tio2 nanoparticles . all investigated ligands ( catechol , pyrogallol , and gallic acid ) form inner - sphere ct complexes with tio2 nanoparticles ( d = 45 ) . binding of the modifier molecules to undercoordinated surface ti atoms ( defect sites ) results in a significant change in the onset of absorption and the effective bandgap . from the benesi hildebrand plot , the stability constants at ph 2 of the order 10 m have been determined . for chosen enediol modifiers binding was found to be through bidentate binuclear ( bridging ) complexes leading to restoration of six - coordinated octahedral geometry of surface ti atoms .

five young related male labrador retrievers with a rapidly progressive disorder causing muscle atrophy and weakness were evaluated at the veterinary teaching hospital at the western college of veterinary medicine ( vthwcvm ) , university of saskatchewan between august 2006 and march 2009 . all clinical examinations , diagnostic procedures and testing performed on these puppies were in accordance with guidelines established by the university of saskatchewan 's animal care committee . clinical and historical information regarding these puppies and 2 additional related puppies were obtained by medical record review and consultation with owners and referring veterinarians . all affected puppies were tested for the mutation in the ptpla gene causing autosomal recessive cnm in labrador retrievers2 1 and 1 was tested for the mutation causing dystrophindeficient muscular dystrophy ( dmd ) in golden retrievers.2 complete blood counts and routine serum chemistry profiles were performed on all dogs and toxoplasma gondii and neospora caninum serology were performed on the first two dogs evaluated . histologic , histochemical , and immunohistochemical studies of muscle and peripheral nerve biopsy specimens were performed at the comparative neuromuscular laboratory , university of california san diego ( la jolla , california , usa ) . litter 1 . seven male and 4 female puppies were born to a clinically normal 4yearold female chocolate labrador retriever bred to a clinically normal male chocolate labrador retriever ( fig . 1 ) . as reported previously,5 , 10 5 of the male puppies developed signs of muscle weakness and atrophy between 12 and 17 weeks of age . four were euthanized without further evaluation ; 1 pup ( pup 1 ) was referred to the vthwcvm . muscles and peripheral nerves were collected from this pup for evaluation and a congenital myopathy was diagnosed . dna tests were submitted for the cnm mutation and for the golden retriever dmd mutation , which were not found . according to the owner , the dam of this litter had 2 previous litters , sired by different males , each producing multiple male pups with early onset progressive muscle atrophy and weakness . this dam was presented to the vthwcvm for euthanasia because of behavioral problems a few months after evaluation of pup 1 . a litter of 5 male and 3 female chocolate labrador retriever puppies was born to a clinically normal 3yearold female chocolate labrador retriever bred to a clinically normal male chocolate labrador retriever.5 two male pups from this litter developed signs of progressive muscular weakness and atrophy and were presented for veterinary evaluation between 3 and 4 months of age . muscle specimens and cheek swabs for dna analysis were collected from 1 of the affected male puppies ( pup 2 ) immediately after humane euthanasia by the general veterinarian . the second affected puppy ( pup 3 ) was referred to the vthwcvm . when questioned , the breeder indicated that 3 of the dam 's male littermates had been euthanized at a young age for progressive muscle weakness ( fig . 1 ) . this dam and 1 female puppy from the litter subsequently were examined at the wcvm and determined to be clinically and neurologically normal . litter 3 . a litter of 9 male ( 8 black and 1 chocolate ) and 2 female ( 1 black and 1 chocolate ) puppies was born to a clinically normal 5yearold female black labrador retriever bred to a clinically normal 3.5yearold male black labrador retriever.5 in this litter , 4 black male puppies showed signs of muscle weakness and atrophy at 7 weeks of age ( fig . 1 ) the affected puppies seemed to improve , but then were reevaluated for progressive muscle weakness and atrophy at 1318 weeks of age . limited clinical information was available from 1 of the affected male puppies ( pup 4 ) . the remaining 3 affected male puppies were evaluated at the vthwcvm ( pups 5 , 6 , and 7 ) . one additional male puppy from this litter reportedly died shortly after birth from accidental trauma , and it was uncertain whether that puppy was affected . this dam had 1 previous litter that was the result of an accidental breeding to a nonpurebred male dog , and the breeder reported no problems with any of the puppies . in total , 5 affected male puppies from 3 separate litters , ranging from 14 to 26 weeks of age were evaluated over a 3year period at the vthwcvm . clinical information , tissues , or both also were available from 2 affected puppies not evaluated at the wcvm ( pup 2 from litter 2 and pup 4 from litter 3 ; fig . 1 ) . all affected puppies were considered small for their age and all were thin to emaciated with body condition scores ( bs ) 0.51.5/5 when they were evaluated at the vthwcvm . although most owners and breeders did not notice clinical abnormalities until the puppies were approximately 14 weeks of age ( range , 818 weeks ; median , 14 weeks ) , the veterinarian who examined litter 3 at 7 weeks of age reported that the affected male puppies were abnormal at that time . those 4 puppies ( pups 47 ) were small for their age ( 2.02.7 kg ) and thin ( bcs 2 ) , and they all had pelvic limb weakness , generalized muscle atrophy , and masticatory muscle weakness leading to a dropped jaw . deworming , hand feeding , and physiotherapy reportedly resulted in improvement and the puppies were sent to their new owners . all of these pups were represented to a veterinarian between 13 and 18 weeks of age because of progressive weakness and muscle atrophy . clinical abnormalities in all affected puppies progressed rapidly once they were severe enough to be noted by owners . by the age of 1523 weeks , most puppies were unable to stand and walk . the owners of pup 6 were convinced that muscular weakness became much more pronounced during cold weather when the ambient temperature remained below 30c for several consecutive days . pups 1 and 2 had been treated with corticosteroids ( prednisone 1.52.0 mg / kg bw q24h ) for presumed juvenile cellulitis early in the course of their weakness and pup 7 had been treated with corticosteroids after becoming nonambulatory . pups 1 and 6 had been treated with nonsteroidal antiinflammatory drugs ( meloxicam 0.1 mg / kg bw po q24 h ) . there was no noticeable improvement or slowing of disease progression with either of these treatments . body temperature , pulse , and respiratory rate were normal in all dogs evaluated at the vthwcvm with the exception of the oldest and most severely affected puppy ( pup 7 ; 26 weeks of age ) that exhibited rapid , shallow , abdominal respirations ( 60/min ) , suggesting intercostal muscle weakness . complete ophthalmologic examination including fundoscopic evaluation was normal in the 2 dogs in which this was evaluated . orthopedic examinations were unremarkable in all dogs ; no bone , joint , or spinal pain was identified . only 3 of the puppies ( pups 1 , 3 , and 6 ) could still walk with assistance when they were evaluated at the vthwcvm . they stood with an arched spine and were unable to lift their heads , resulting in low head carriage ( fig . 2 ) . when they walked , they exhibited severe weakness , with a short , choppy stride , and collapsing after only a few steps . within 1 week image of pup 1 ( left ) showing an arched spine and typical low head carriage resulting from neck muscle weakness . proprioception , as assessed by response to knuckling and hopping , was normal in all affected pups that could be evaluated . pup 7 did not have voluntary movement in any limbs , and postural reactions could not be assessed . generalized muscle atrophy was pronounced and muscle tone was decreased in all affected pups , with flaccid muscles reported in the most severely affected pups . although paravertebral skin pinch elicited vocalization in pup 7 , there was no detectable twitch of the cutaneous trunci muscle . skin sensation , tail function , anal tone , and the perineal reflex were normal in all affected pups . mentation was assessed to be normal in all pups and the palpebral reflex was present and was not fatigable . the most severely affected pups ( pups 5 , 6 , and 7 ) had dropped jaws attributed to severe atrophy and weakness of the muscles of mastication . laryngeal , pharyngeal , and esophageal dysfunction were suspected or confirmed in all 5 affected dogs evaluated at the vthwcvm . laryngeal dysfunction was suspected based on a hoarse bark and confirmed by visual observation of failure of the arytenoid cartilages to abduct during inspiration in unsedated pups . fluoroscopic swallowing and esophagram studies in 2 pups ( pups 5 and 6 ) confirmed pharyngeal and esophageal dysfunction . laboratory results were available from the 5 affected pups evaluated at the vthwcvm and from 1 other affected pup from litter 3 ( pup 4 ) . none of the cbc findings were remarkable ; 1 affected pup ( pup 1 ) had a mild neutropenia ( 2.295 10/l ; reference range , 3.010 10/l ) and a few pups had reactive lymphocytes thought to be secondary to recent vaccination . serum biochemical abnormalities included mild increases in alkaline phosphatase activity ( 103172 u / l ; reference range , 990 u / l ) in 3 pups and mild increases in alanine aminotransferase activity ( 136211 u / l ; reference range , 1959 u / l ) in all pups that had been treated previously with glucocorticoids . mild hyperphosphatemia ( range , 2.252.72 mmol / l ; reference range , 0.632.41 mmol / l ) was evident in 4 of the affected pups consistent with agerelated bone turnover . serum creatinine concentration was slightly decreased in the affected pups ( range , 1541 mol / l ; reference range , 6197 mol / l ) with the decrease most marked in the oldest and most severely affected pup ( pup 7 ) suggesting that decreased muscle mass was the cause . serum creatine kinase ( ck ) activity was either normal ( n = 3 ) or only slightly increased ( thoracic radiographs were evaluated in 2 pups ( pups 1 and 7 ) and were normal . after pain was identified during deep palpation of the limbs of pup 2 , radiographs of all limbs were performed and findings were normal . fluoroscopic swallowing studies and esophagrams were performed in 2 pups ( pups 5 and 6 ) using liquid barium and then multiple consistencies of barium coated food . pup 5 was unable to form a bolus with liquid barium , and showed impaired bolusforming ability with canned food and dry kibble until a large volume of food had accumulated in the oropharynx , but then esophageal transit was normal . pup 6 exhibited normal swallowing and esophageal transit with canned food , but slightly delayed bolus formation and decreased primary and secondary esophageal contractions with liquid and with dry kibble . echocardiography was performed in pup 7 , the oldest and most severely affected dog , with normal cardiac dimensions and blood flow identified with slightly decreased fractional shortening ( 29.9% ) , suggesting mild myocardial dysfunction . electromyographic studies ( emgs ) performed under general anesthesia showed nonspecific abnormalities suggesting destabilization of the sarcolemmal membrane in the 3 pups tested ( pups 2 , 5 , and 6 ) . positive sharp waves were identified in the neck and shoulder girdle muscles of pup 2 . moderate to severe fibrillation potentials were recorded in the left digastricus , masseter , and temporalis muscles of pup 5 and positive sharp waves were found in the left cranial tibial muscle . positive sharp waves were recorded in the cervical , temporalis , lingual , gastrocnemius , lumbar muscles , and extensor carpi radialis muscles in pup 6 ( fig . electromyographic tracings from pup 6 showing positive sharp waves recorded from the left extensor carpi radialis and lingual muscles . edrophonium chloride3 ( 0.2 mg / kg bw iv ) was administered to pups 1 and 5 with neither showing any improvement in muscle strength . circulating serum antibodies against acetylcholine receptors were measured and were normal ( 0.21 nmol / l ; reference range , < 0.6 nmol / l ) in pup 1 . serological testing for antibodies to toxoplasma gondii and neospora caninum were negative in the 2 pups tested ( pups 1 and 2 ) and cerebrospinal fluid ( csf ) cytology and protein content were normal in these 2 pups as well . dna testing for the ptpla exon 2 insertion associated with autosomal recessive cnm of labrador retrievers was negative for affected and carrier states in all puppies . the dam of pup 1 was tested for cnm and also for the dmd gene mutation causing xlinked dystrophindeficient muscular dystrophy in golden retrievers and was negative for both . comprehensive descriptions of histopathology , histochemistry , immunohistochemistry , and ultrastructural analysis of muscle biopsies from these affected pups and controls have been reported previously.5 pathologic changes were diagnostic of myotubular myopathy . typical pathologic changes included excessive variability in myofiber size , type 1 fiber predominance , centrally placed nuclei in most myofibers , and subsarcolemmal ringed and central dense areas highlighted with mitochondrial specific reactions typical of necklace fibers.11 by immunohistochemical and ultrastructural analysis , centrally located muscle nuclei were confirmed , and muscle triads were disorganized and exhibited an abnormal orientation of t tubules . none of these changes were evident in similarly processed muscle biopsy specimens obtained from the dam of litter 1 and a normal female puppy from litter 2 . peroneal nerve biopsies from pups 1 , 5 , and 7 were evaluated in frozen and resin sections . the nerve biopsies were normal without evidence of axonal degeneration , demyelination , or abnormalities of supporting structures . as previously published , inspection of the pedigrees confirmed that the myopathy in these dogs was inherited as an xlinked recessive trait ( fig . 1).5 all affected dogs evaluated were male and each had multiple affected male siblings and all female siblings were normal . all affected puppies resulted from the mating of a clinically normal dam and sire . breeding a carrier female to multiple normal sires resulted in affected puppies , typical of xlinked inheritance . subsequent genetic analysis confirmed that each of the affected pups described above carried a pathogenic mtm1 gene mutation that was considered responsible for their severe skeletal muscle myopathy.5 litter 1 . seven male and 4 female puppies were born to a clinically normal 4yearold female chocolate labrador retriever bred to a clinically normal male chocolate labrador retriever ( fig . 1 ) . as reported previously,5 , 10 5 of the male puppies developed signs of muscle weakness and atrophy between 12 and 17 weeks of age . four were euthanized without further evaluation ; 1 pup ( pup 1 ) was referred to the vthwcvm . muscles and peripheral nerves were collected from this pup for evaluation and a congenital myopathy was diagnosed . dna tests were submitted for the cnm mutation and for the golden retriever dmd mutation , which were not found . according to the owner , the dam of this litter had 2 previous litters , sired by different males , each producing multiple male pups with early onset progressive muscle atrophy and weakness . this dam was presented to the vthwcvm for euthanasia because of behavioral problems a few months after evaluation of pup 1 . a litter of 5 male and 3 female chocolate labrador retriever puppies was born to a clinically normal 3yearold female chocolate labrador retriever bred to a clinically normal male chocolate labrador retriever.5 two male pups from this litter developed signs of progressive muscular weakness and atrophy and were presented for veterinary evaluation between 3 and 4 months of age . muscle specimens and cheek swabs for dna analysis were collected from 1 of the affected male puppies ( pup 2 ) immediately after humane euthanasia by the general veterinarian . the second affected puppy ( pup 3 ) was referred to the vthwcvm . when questioned , the breeder indicated that 3 of the dam 's male littermates had been euthanized at a young age for progressive muscle weakness ( fig . 1 ) . this dam and 1 female puppy from the litter subsequently were examined at the wcvm and determined to be clinically and neurologically normal . litter 3 . a litter of 9 male ( 8 black and 1 chocolate ) and 2 female ( 1 black and 1 chocolate ) puppies was born to a clinically normal 5yearold female black labrador retriever bred to a clinically normal 3.5yearold male black labrador retriever.5 in this litter , 4 black male puppies showed signs of muscle weakness and atrophy at 7 weeks of age ( fig . 1 ) the affected puppies seemed to improve , but then were reevaluated for progressive muscle weakness and atrophy at 1318 weeks of age . limited clinical information was available from 1 of the affected male puppies ( pup 4 ) . the remaining 3 affected male puppies were evaluated at the vthwcvm ( pups 5 , 6 , and 7 ) . one additional male puppy from this litter reportedly died shortly after birth from accidental trauma , and it was uncertain whether that puppy was affected . this dam had 1 previous litter that was the result of an accidental breeding to a nonpurebred male dog , and the breeder reported no problems with any of the puppies . in total , 5 affected male puppies from 3 separate litters , ranging from 14 to 26 weeks of age were evaluated over a 3year period at the vthwcvm . clinical information , tissues , or both also were available from 2 affected puppies not evaluated at the wcvm ( pup 2 from litter 2 and pup 4 from litter 3 ; fig . 1 ) . all affected puppies were considered small for their age and all were thin to emaciated with body condition scores ( bs ) 0.51.5/5 when they were evaluated at the vthwcvm . although most owners and breeders did not notice clinical abnormalities until the puppies were approximately 14 weeks of age ( range , 818 weeks ; median , 14 weeks ) , the veterinarian who examined litter 3 at 7 weeks of age reported that the affected male puppies were abnormal at that time . those 4 puppies ( pups 47 ) were small for their age ( 2.02.7 kg ) and thin ( bcs 2 ) , and they all had pelvic limb weakness , generalized muscle atrophy , and masticatory muscle weakness leading to a dropped jaw . deworming , hand feeding , and physiotherapy reportedly resulted in improvement and the puppies were sent to their new owners . all of these pups were represented to a veterinarian between 13 and 18 weeks of age because of progressive weakness and muscle atrophy . clinical abnormalities in all affected puppies progressed rapidly once they were severe enough to be noted by owners . by the age of 1523 weeks , most puppies were unable to stand and walk . the owners of pup 6 were convinced that muscular weakness became much more pronounced during cold weather when the ambient temperature remained below 30c for several consecutive days . pups 1 and 2 had been treated with corticosteroids ( prednisone 1.52.0 mg / kg bw q24h ) for presumed juvenile cellulitis early in the course of their weakness and pup 7 had been treated with corticosteroids after becoming nonambulatory . pups 1 and 6 had been treated with nonsteroidal antiinflammatory drugs ( meloxicam 0.1 mg / kg bw po q24 h ) . there was no noticeable improvement or slowing of disease progression with either of these treatments . body temperature , pulse , and respiratory rate were normal in all dogs evaluated at the vthwcvm with the exception of the oldest and most severely affected puppy ( pup 7 ; 26 weeks of age ) that exhibited rapid , shallow , abdominal respirations ( 60/min ) , suggesting intercostal muscle weakness . complete ophthalmologic examination including fundoscopic evaluation was normal in the 2 dogs in which this was evaluated . orthopedic examinations were unremarkable in all dogs ; no bone , joint , or spinal pain was identified . only 3 of the puppies ( pups 1 , 3 , and 6 ) could still walk with assistance when they were evaluated at the vthwcvm . they stood with an arched spine and were unable to lift their heads , resulting in low head carriage ( fig . 2 ) . when they walked , they exhibited severe weakness , with a short , choppy stride , and collapsing after only a few steps . within 1 week of presentation , these pups were unable to walk , even with assistance . image of pup 1 ( left ) showing an arched spine and typical low head carriage resulting from neck muscle weakness . proprioception , as assessed by response to knuckling and hopping , was normal in all affected pups that could be evaluated . pup 7 did not have voluntary movement in any limbs , and postural reactions could not be assessed . generalized muscle atrophy was pronounced and muscle tone was decreased in all affected pups , with flaccid muscles reported in the most severely affected pups . although paravertebral skin pinch elicited vocalization in pup 7 , there was no detectable twitch of the cutaneous trunci muscle . skin sensation , tail function , anal tone , and the perineal reflex were normal in all affected pups . mentation was assessed to be normal in all pups and the palpebral reflex was present and was not fatigable . the most severely affected pups ( pups 5 , 6 , and 7 ) had dropped jaws attributed to severe atrophy and weakness of the muscles of mastication . tongue strength and gag response were subjectively decreased in pup 7 . laryngeal , pharyngeal , and esophageal dysfunction were suspected or confirmed in all 5 affected dogs evaluated at the vthwcvm . laryngeal dysfunction was suspected based on a hoarse bark and confirmed by visual observation of failure of the arytenoid cartilages to abduct during inspiration in unsedated pups . fluoroscopic swallowing and esophagram studies in 2 pups ( pups 5 and 6 ) confirmed pharyngeal and esophageal dysfunction . laboratory results were available from the 5 affected pups evaluated at the vthwcvm and from 1 other affected pup from litter 3 ( pup 4 ) . none of the cbc findings were remarkable ; 1 affected pup ( pup 1 ) had a mild neutropenia ( 2.295 10/l ; reference range , 3.010 10/l ) and a few pups had reactive lymphocytes thought to be secondary to recent vaccination . serum biochemical abnormalities included mild increases in alkaline phosphatase activity ( 103172 u / l ; reference range , 990 u / l ) in 3 pups and mild increases in alanine aminotransferase activity ( 136211 u / l ; reference range , 1959 u / l ) in all pups that had been treated previously with glucocorticoids . mild hyperphosphatemia ( range , 2.252.72 mmol / l ; reference range , 0.632.41 mmol / l ) was evident in 4 of the affected pups consistent with agerelated bone turnover . serum creatinine concentration was slightly decreased in the affected pups ( range , 1541 mol / l ; reference range , 6197 mol / l ) with the decrease most marked in the oldest and most severely affected pup ( pup 7 ) suggesting that decreased muscle mass was the cause . serum creatine kinase ( ck ) activity was either normal ( n = 3 ) or only slightly increased ( n = 3 ) in all puppies tested ( range , 196972 u / l ; reference range , 51418 u / l ) . thoracic radiographs were evaluated in 2 pups ( pups 1 and 7 ) and were normal . after pain was identified during deep palpation of the limbs of pup 2 , radiographs of all limbs were performed and findings were normal . fluoroscopic swallowing studies and esophagrams were performed in 2 pups ( pups 5 and 6 ) using liquid barium and then multiple consistencies of barium coated food . pup 5 was unable to form a bolus with liquid barium , and showed impaired bolusforming ability with canned food and dry kibble until a large volume of food had accumulated in the oropharynx , but then esophageal transit was normal . pup 6 exhibited normal swallowing and esophageal transit with canned food , but slightly delayed bolus formation and decreased primary and secondary esophageal contractions with liquid and with dry kibble . echocardiography was performed in pup 7 , the oldest and most severely affected dog , with normal cardiac dimensions and blood flow identified with slightly decreased fractional shortening ( 29.9% ) , suggesting mild myocardial dysfunction . electromyographic studies ( emgs ) performed under general anesthesia showed nonspecific abnormalities suggesting destabilization of the sarcolemmal membrane in the 3 pups tested ( pups 2 , 5 , and 6 ) . positive sharp waves were identified in the neck and shoulder girdle muscles of pup 2 . moderate to severe fibrillation potentials were recorded in the left digastricus , masseter , and temporalis muscles of pup 5 and positive sharp waves were found in the left cranial tibial muscle . positive sharp waves were recorded in the cervical , temporalis , lingual , gastrocnemius , lumbar muscles , and extensor carpi radialis muscles in pup 6 ( fig . 3 ) . electromyographic tracings from pup 6 showing positive sharp waves recorded from the left extensor carpi radialis and lingual muscles . edrophonium chloride3 ( 0.2 mg / kg bw iv ) was administered to pups 1 and 5 with neither showing any improvement in muscle strength . circulating serum antibodies against acetylcholine receptors were measured and were normal ( 0.21 nmol / l ; reference range , < 0.6 nmol / l ) in pup 1 . serological testing for antibodies to toxoplasma gondii and neospora caninum were negative in the 2 pups tested ( pups 1 and 2 ) and cerebrospinal fluid ( csf ) cytology and protein content were normal in these 2 pups as well . dna testing for the ptpla exon 2 insertion associated with autosomal recessive cnm of labrador retrievers was negative for affected and carrier states in all puppies . the dam of pup 1 was tested for cnm and also for the dmd gene mutation causing xlinked dystrophindeficient muscular dystrophy in golden retrievers and was negative for both . comprehensive descriptions of histopathology , histochemistry , immunohistochemistry , and ultrastructural analysis of muscle biopsies from these affected pups and controls have been reported previously.5 pathologic changes were diagnostic of myotubular myopathy . typical pathologic changes included excessive variability in myofiber size , type 1 fiber predominance , centrally placed nuclei in most myofibers , and subsarcolemmal ringed and central dense areas highlighted with mitochondrial specific reactions typical of necklace fibers.11 by immunohistochemical and ultrastructural analysis , centrally located muscle nuclei were confirmed , and muscle triads were disorganized and exhibited an abnormal orientation of t tubules . none of these changes were evident in similarly processed muscle biopsy specimens obtained from the dam of litter 1 and a normal female puppy from litter 2 . peroneal nerve biopsies from pups 1 , 5 , and 7 were evaluated in frozen and resin sections . the nerve biopsies were normal without evidence of axonal degeneration , demyelination , or abnormalities of supporting structures . as previously published , inspection of the pedigrees confirmed that the myopathy in these dogs was inherited as an xlinked recessive trait ( fig . 1).5 all affected dogs evaluated were male and each had multiple affected male siblings and all female siblings were normal . all affected puppies resulted from the mating of a clinically normal dam and sire . breeding a carrier female to multiple normal sires resulted in affected puppies , typical of xlinked inheritance . subsequent genetic analysis confirmed that each of the affected pups described above carried a pathogenic mtm1 gene mutation that was considered responsible for their severe skeletal muscle myopathy.5 here , we provide a complete clinical description of a cohort of puppies with xlinked myotubular myopathy . the initial clinical presentation of these puppies was similar to that expected with cnm , with 3 to 4monthold puppies exhibiting weakness , muscle atrophy , absent patellar reflexes , and relatively normal serum ck activities.12 , 13 , 14 , 15 , 16 , 17 there were , however , several differences between these xlmtm puppies and puppies with cnm , which prompted further evaluation . all affected puppies were male , and pedigree analysis supported an xlinked mode of inheritance rather than the autosomal recessive inheritance typical of cnm in labrador retrievers.12 in contrast to dogs with ptpla mutations , clinical abnormalities in the xlmtm puppies were more severe and rapidly progressive , uniformly resulting in death or euthanasia by 1526 weeks of age . autosomal recessive cnm usually is a less severe , more slowly progressive disorder , with many dogs surviving into adulthood.12 , 13 , 14 , 15 , 16 , 17 all of these puppies were negative for the ptpla mutation responsible for cnm in labrador retrievers . muscular dystrophy has been recognized in many breeds of dogs and initially was considered in the differential diagnosis for these puppies.12 the most common form of muscular dystrophy in dogs , canine xlinked muscular dystrophy ( cxlmd ) , is associated with a mutation in the gene encoding the sarcolemmal protein dystrophin.14 , 15 this disorder usually results in an absence of dystrophin and severe muscular degeneration , but rarely a mutation can lead to a structurally altered and partially functional dystrophin protein resulting in a less severe phenotype.14 , 15 , 18 dystrophindeficient cxlmd has been reported in 1 male labrador retriever puppy1 and has been extensively investigated in golden retrievers.19 most puppies with dystrophindeficient cxlmd show muscular weakness as soon as they begin to ambulate . unlike puppies with cnm and xlmtm , patellar reflexes are maintained until muscle fibrosis and contractures occur.1 , 14 , 20 a reliable feature of dystrophindeficient cxlmd is markedly increased serum ck activity as early as 7 days of age and peaking at 68 weeks of age at 100 times normal.12 , 14 , 20 despite the observation that only males were affected , our clinical findings of absent patellar reflexes and normal ck activities , and normal immunohistochemical staining and immunoblotting for dystrophin and associated proteins , eliminated cxlmd as a diagnosis in the puppies described here . based on the histopathologic diagnosis of myotubular myopathy in these puppies , the xlinked nature of the pedigree analysis , and the absence of myotubularin in the muscles on immunohistochemistry and immunoblotting , a candidate gene approach was used to test for a mutation in the myotubulin gene mtm1.5 all 15 exons of the canine mtm1 gene located on the x chromosome were sequenced . a ctoa transversion in exon 7 was present in all 7 affected males described here.5 affected puppies were hemizygous for the mutation , whereas known carrier females were heterozygous . the mutation was not detected in unaffected male dogs from the 3 litters , in a panel of control dna samples obtained from 237 unrelated and unaffected labrador retrievers from throughout north america , europe , and australia , or in 59 additional control dogs from 25 breeds , supporting it as the pathogenic causative mutation . although litters 1 and 2 initially were thought to be unrelated , further investigation revealed the relationship illustrated in figure 1 . insufficient pedigree details are available for the third litter to prove or disprove a relationship , however , genetic haplotype has shown that a spontaneous mutation occurred in a local bitch and was transmitted to affected puppies in all 3 litters through a founder effect.5 similar to the clinical course in these puppies , xlmtm in humans is a severe disease that , in the absence of intensive medical support , often follows a fatal course over the first few years of life . affected male infants are clinically abnormal at or even before birth , exhibiting decreased muscle tone , decreased movement , and areflexia.5 , 21 , 22 tube feeding usually is required because of impaired ability to suck and swallow . respiratory muscle weakness is severe , often requiring mechanical ventilation and complications related to ventilation cause death in many patients during infancy or early childhood.5 , 21 , 22 diagnosis is based on typical histopathologic findings on muscle biopsy in combination with suggestive clinical features . biopsies disclose uniformly small muscle fibers with centrally placed nuclei resembling fetal myotubes.23 mutations in the myotubularin ( mtm1 ) gene on human chromosome xq28 are responsible for xlmtm . the incidence is estimated at 1/50000 male births.5 , 22 over 200 different spontaneous deletion , insertion , nonsense , missense , and splice mutations have been found in the mtm1 gene , with the majority occurring in exons 12 , 4 , 11 , 8 , and 9.5 the mutation identified in these puppies was a unique exon 7 missense mutation that has not been reported in a human patient.4 identification of the mutation in the affected puppies allowed us to not only identify carrier females and counsel owners regarding future breedings but also allowed us to recruit carrier females to establish a breeding colony . establishment of a breeding colony has contributed to ongoing investigation of the pathophysiology of muscle deterioration and weakness in individuals with xlmtm,24 and to evaluation of potential treatments.25 spontaneous mutation in the mtm1 gene resulting in xlmtm in male offspring is well described in human medicine but only recently reported in dogs.4 , 5 given the frequency with which these mutations occur in humans , it is likely that similar mutations occur occasionally in dogs , but that the resulting myopathies are incorrectly diagnosed as some other congenital myopathy . evaluation of affected labrador retriever puppies in this report highlights the clinical differences between xlmtm and the other reported congenital myopathies in this breed , and may help veterinarians recognize similar clinical presentations in other breeds .

backgroundseven male labrador retriever puppies from 3 different litters , born to clinically normal dams and sires , were evaluated for progressive weakness and muscle atrophy . muscle biopsies identified a congenital myopathy with pathologic features consistent with myotubular myopathy . further investigations identified a pathogenic mutation in the myotubularin gene , confirming that these puppies had xlinked myotubular myopathy ( xlmtm).objectiveto review the clinical phenotype , electrodiagnostic and laboratory features of xlmtm in this cohort of labrador retrievers.resultsmale puppies with xlmtm were small and thin compared with their normal littermates . generalized weakness and muscle atrophy were present by 7 weeks of age in some puppies and evident to most owners by 14 weeks of age . affected puppies stood with an arched spine and low head carriage , and walked with a short , choppy stride . muscle atrophy was severe and progressive . patellar reflexes were absent . laryngeal and esophageal dysfunction , and weakness of the masticatory muscles occurred in puppies surviving beyond 4 months of age . serum creatine kinase activity was normal or only mildly increased . emg findings were nonspecific and included positive sharp waves and fibrillation potentials . clinical signs progressed rapidly , with most affected puppies unable to walk within 34 weeks after clinical signs were first noticed.conclusions and clinical importancealthough initial clinical signs of xlmtm are similar to the phenotypically milder centronuclear myopathy in labrador retrievers , xlmtm is a rapidly progressive and fatal myopathy . clinicians should be aware of these 2 distinct myopathies with similar clinical presentations in the labrador retriever breed .

Materials and Methods Results Familial History for Affected Male Puppies from 3 Different Litters Clinical Findings in Affected Male Puppies Clinical Pathology Diagnostic Imaging Electromyography Miscellaneous Diagnostic Testing Postmortem Findings Muscle and Peripheral Nerve Biopsies Pedigree Analysis Discussion

five young related male labrador retrievers with a rapidly progressive disorder causing muscle atrophy and weakness were evaluated at the veterinary teaching hospital at the western college of veterinary medicine ( vthwcvm ) , university of saskatchewan between august 2006 and march 2009 . all affected puppies were tested for the mutation in the ptpla gene causing autosomal recessive cnm in labrador retrievers2 1 and 1 was tested for the mutation causing dystrophindeficient muscular dystrophy ( dmd ) in golden retrievers.2 complete blood counts and routine serum chemistry profiles were performed on all dogs and toxoplasma gondii and neospora caninum serology were performed on the first two dogs evaluated . seven male and 4 female puppies were born to a clinically normal 4yearold female chocolate labrador retriever bred to a clinically normal male chocolate labrador retriever ( fig . as reported previously,5 , 10 5 of the male puppies developed signs of muscle weakness and atrophy between 12 and 17 weeks of age . muscles and peripheral nerves were collected from this pup for evaluation and a congenital myopathy was diagnosed . according to the owner , the dam of this litter had 2 previous litters , sired by different males , each producing multiple male pups with early onset progressive muscle atrophy and weakness . a litter of 5 male and 3 female chocolate labrador retriever puppies was born to a clinically normal 3yearold female chocolate labrador retriever bred to a clinically normal male chocolate labrador retriever.5 two male pups from this litter developed signs of progressive muscular weakness and atrophy and were presented for veterinary evaluation between 3 and 4 months of age . when questioned , the breeder indicated that 3 of the dam 's male littermates had been euthanized at a young age for progressive muscle weakness ( fig . a litter of 9 male ( 8 black and 1 chocolate ) and 2 female ( 1 black and 1 chocolate ) puppies was born to a clinically normal 5yearold female black labrador retriever bred to a clinically normal 3.5yearold male black labrador retriever.5 in this litter , 4 black male puppies showed signs of muscle weakness and atrophy at 7 weeks of age ( fig . 1 ) the affected puppies seemed to improve , but then were reevaluated for progressive muscle weakness and atrophy at 1318 weeks of age . the remaining 3 affected male puppies were evaluated at the vthwcvm ( pups 5 , 6 , and 7 ) . in total , 5 affected male puppies from 3 separate litters , ranging from 14 to 26 weeks of age were evaluated over a 3year period at the vthwcvm . all affected puppies were considered small for their age and all were thin to emaciated with body condition scores ( bs ) 0.51.5/5 when they were evaluated at the vthwcvm . although most owners and breeders did not notice clinical abnormalities until the puppies were approximately 14 weeks of age ( range , 818 weeks ; median , 14 weeks ) , the veterinarian who examined litter 3 at 7 weeks of age reported that the affected male puppies were abnormal at that time . those 4 puppies ( pups 47 ) were small for their age ( 2.02.7 kg ) and thin ( bcs 2 ) , and they all had pelvic limb weakness , generalized muscle atrophy , and masticatory muscle weakness leading to a dropped jaw . all of these pups were represented to a veterinarian between 13 and 18 weeks of age because of progressive weakness and muscle atrophy . pups 1 and 2 had been treated with corticosteroids ( prednisone 1.52.0 mg / kg bw q24h ) for presumed juvenile cellulitis early in the course of their weakness and pup 7 had been treated with corticosteroids after becoming nonambulatory . body temperature , pulse , and respiratory rate were normal in all dogs evaluated at the vthwcvm with the exception of the oldest and most severely affected puppy ( pup 7 ; 26 weeks of age ) that exhibited rapid , shallow , abdominal respirations ( 60/min ) , suggesting intercostal muscle weakness . complete ophthalmologic examination including fundoscopic evaluation was normal in the 2 dogs in which this was evaluated . only 3 of the puppies ( pups 1 , 3 , and 6 ) could still walk with assistance when they were evaluated at the vthwcvm . they stood with an arched spine and were unable to lift their heads , resulting in low head carriage ( fig . when they walked , they exhibited severe weakness , with a short , choppy stride , and collapsing after only a few steps . within 1 week image of pup 1 ( left ) showing an arched spine and typical low head carriage resulting from neck muscle weakness . generalized muscle atrophy was pronounced and muscle tone was decreased in all affected pups , with flaccid muscles reported in the most severely affected pups . the most severely affected pups ( pups 5 , 6 , and 7 ) had dropped jaws attributed to severe atrophy and weakness of the muscles of mastication . laryngeal , pharyngeal , and esophageal dysfunction were suspected or confirmed in all 5 affected dogs evaluated at the vthwcvm . fluoroscopic swallowing and esophagram studies in 2 pups ( pups 5 and 6 ) confirmed pharyngeal and esophageal dysfunction . mild hyperphosphatemia ( range , 2.252.72 mmol / l ; reference range , 0.632.41 mmol / l ) was evident in 4 of the affected pups consistent with agerelated bone turnover . serum creatine kinase ( ck ) activity was either normal ( n = 3 ) or only slightly increased ( thoracic radiographs were evaluated in 2 pups ( pups 1 and 7 ) and were normal . pup 5 was unable to form a bolus with liquid barium , and showed impaired bolusforming ability with canned food and dry kibble until a large volume of food had accumulated in the oropharynx , but then esophageal transit was normal . electromyographic studies ( emgs ) performed under general anesthesia showed nonspecific abnormalities suggesting destabilization of the sarcolemmal membrane in the 3 pups tested ( pups 2 , 5 , and 6 ) . positive sharp waves were identified in the neck and shoulder girdle muscles of pup 2 . moderate to severe fibrillation potentials were recorded in the left digastricus , masseter , and temporalis muscles of pup 5 and positive sharp waves were found in the left cranial tibial muscle . positive sharp waves were recorded in the cervical , temporalis , lingual , gastrocnemius , lumbar muscles , and extensor carpi radialis muscles in pup 6 ( fig . electromyographic tracings from pup 6 showing positive sharp waves recorded from the left extensor carpi radialis and lingual muscles . dna testing for the ptpla exon 2 insertion associated with autosomal recessive cnm of labrador retrievers was negative for affected and carrier states in all puppies . comprehensive descriptions of histopathology , histochemistry , immunohistochemistry , and ultrastructural analysis of muscle biopsies from these affected pups and controls have been reported previously.5 pathologic changes were diagnostic of myotubular myopathy . typical pathologic changes included excessive variability in myofiber size , type 1 fiber predominance , centrally placed nuclei in most myofibers , and subsarcolemmal ringed and central dense areas highlighted with mitochondrial specific reactions typical of necklace fibers.11 by immunohistochemical and ultrastructural analysis , centrally located muscle nuclei were confirmed , and muscle triads were disorganized and exhibited an abnormal orientation of t tubules . as previously published , inspection of the pedigrees confirmed that the myopathy in these dogs was inherited as an xlinked recessive trait ( fig . seven male and 4 female puppies were born to a clinically normal 4yearold female chocolate labrador retriever bred to a clinically normal male chocolate labrador retriever ( fig . as reported previously,5 , 10 5 of the male puppies developed signs of muscle weakness and atrophy between 12 and 17 weeks of age . muscles and peripheral nerves were collected from this pup for evaluation and a congenital myopathy was diagnosed . according to the owner , the dam of this litter had 2 previous litters , sired by different males , each producing multiple male pups with early onset progressive muscle atrophy and weakness . a litter of 5 male and 3 female chocolate labrador retriever puppies was born to a clinically normal 3yearold female chocolate labrador retriever bred to a clinically normal male chocolate labrador retriever.5 two male pups from this litter developed signs of progressive muscular weakness and atrophy and were presented for veterinary evaluation between 3 and 4 months of age . a litter of 9 male ( 8 black and 1 chocolate ) and 2 female ( 1 black and 1 chocolate ) puppies was born to a clinically normal 5yearold female black labrador retriever bred to a clinically normal 3.5yearold male black labrador retriever.5 in this litter , 4 black male puppies showed signs of muscle weakness and atrophy at 7 weeks of age ( fig . 1 ) the affected puppies seemed to improve , but then were reevaluated for progressive muscle weakness and atrophy at 1318 weeks of age . this dam had 1 previous litter that was the result of an accidental breeding to a nonpurebred male dog , and the breeder reported no problems with any of the puppies . in total , 5 affected male puppies from 3 separate litters , ranging from 14 to 26 weeks of age were evaluated over a 3year period at the vthwcvm . although most owners and breeders did not notice clinical abnormalities until the puppies were approximately 14 weeks of age ( range , 818 weeks ; median , 14 weeks ) , the veterinarian who examined litter 3 at 7 weeks of age reported that the affected male puppies were abnormal at that time . those 4 puppies ( pups 47 ) were small for their age ( 2.02.7 kg ) and thin ( bcs 2 ) , and they all had pelvic limb weakness , generalized muscle atrophy , and masticatory muscle weakness leading to a dropped jaw . all of these pups were represented to a veterinarian between 13 and 18 weeks of age because of progressive weakness and muscle atrophy . clinical abnormalities in all affected puppies progressed rapidly once they were severe enough to be noted by owners . body temperature , pulse , and respiratory rate were normal in all dogs evaluated at the vthwcvm with the exception of the oldest and most severely affected puppy ( pup 7 ; 26 weeks of age ) that exhibited rapid , shallow , abdominal respirations ( 60/min ) , suggesting intercostal muscle weakness . only 3 of the puppies ( pups 1 , 3 , and 6 ) could still walk with assistance when they were evaluated at the vthwcvm . they stood with an arched spine and were unable to lift their heads , resulting in low head carriage ( fig . when they walked , they exhibited severe weakness , with a short , choppy stride , and collapsing after only a few steps . image of pup 1 ( left ) showing an arched spine and typical low head carriage resulting from neck muscle weakness . generalized muscle atrophy was pronounced and muscle tone was decreased in all affected pups , with flaccid muscles reported in the most severely affected pups . the most severely affected pups ( pups 5 , 6 , and 7 ) had dropped jaws attributed to severe atrophy and weakness of the muscles of mastication . laryngeal , pharyngeal , and esophageal dysfunction were suspected or confirmed in all 5 affected dogs evaluated at the vthwcvm . none of the cbc findings were remarkable ; 1 affected pup ( pup 1 ) had a mild neutropenia ( 2.295 10/l ; reference range , 3.010 10/l ) and a few pups had reactive lymphocytes thought to be secondary to recent vaccination . serum creatine kinase ( ck ) activity was either normal ( n = 3 ) or only slightly increased ( n = 3 ) in all puppies tested ( range , 196972 u / l ; reference range , 51418 u / l ) . after pain was identified during deep palpation of the limbs of pup 2 , radiographs of all limbs were performed and findings were normal . pup 5 was unable to form a bolus with liquid barium , and showed impaired bolusforming ability with canned food and dry kibble until a large volume of food had accumulated in the oropharynx , but then esophageal transit was normal . electromyographic studies ( emgs ) performed under general anesthesia showed nonspecific abnormalities suggesting destabilization of the sarcolemmal membrane in the 3 pups tested ( pups 2 , 5 , and 6 ) . positive sharp waves were identified in the neck and shoulder girdle muscles of pup 2 . moderate to severe fibrillation potentials were recorded in the left digastricus , masseter , and temporalis muscles of pup 5 and positive sharp waves were found in the left cranial tibial muscle . positive sharp waves were recorded in the cervical , temporalis , lingual , gastrocnemius , lumbar muscles , and extensor carpi radialis muscles in pup 6 ( fig . electromyographic tracings from pup 6 showing positive sharp waves recorded from the left extensor carpi radialis and lingual muscles . comprehensive descriptions of histopathology , histochemistry , immunohistochemistry , and ultrastructural analysis of muscle biopsies from these affected pups and controls have been reported previously.5 pathologic changes were diagnostic of myotubular myopathy . peroneal nerve biopsies from pups 1 , 5 , and 7 were evaluated in frozen and resin sections . all affected puppies resulted from the mating of a clinically normal dam and sire . subsequent genetic analysis confirmed that each of the affected pups described above carried a pathogenic mtm1 gene mutation that was considered responsible for their severe skeletal muscle myopathy.5 here , we provide a complete clinical description of a cohort of puppies with xlinked myotubular myopathy . the initial clinical presentation of these puppies was similar to that expected with cnm , with 3 to 4monthold puppies exhibiting weakness , muscle atrophy , absent patellar reflexes , and relatively normal serum ck activities.12 , 13 , 14 , 15 , 16 , 17 there were , however , several differences between these xlmtm puppies and puppies with cnm , which prompted further evaluation . all affected puppies were male , and pedigree analysis supported an xlinked mode of inheritance rather than the autosomal recessive inheritance typical of cnm in labrador retrievers.12 in contrast to dogs with ptpla mutations , clinical abnormalities in the xlmtm puppies were more severe and rapidly progressive , uniformly resulting in death or euthanasia by 1526 weeks of age . autosomal recessive cnm usually is a less severe , more slowly progressive disorder , with many dogs surviving into adulthood.12 , 13 , 14 , 15 , 16 , 17 all of these puppies were negative for the ptpla mutation responsible for cnm in labrador retrievers . muscular dystrophy has been recognized in many breeds of dogs and initially was considered in the differential diagnosis for these puppies.12 the most common form of muscular dystrophy in dogs , canine xlinked muscular dystrophy ( cxlmd ) , is associated with a mutation in the gene encoding the sarcolemmal protein dystrophin.14 , 15 this disorder usually results in an absence of dystrophin and severe muscular degeneration , but rarely a mutation can lead to a structurally altered and partially functional dystrophin protein resulting in a less severe phenotype.14 , 15 , 18 dystrophindeficient cxlmd has been reported in 1 male labrador retriever puppy1 and has been extensively investigated in golden retrievers.19 most puppies with dystrophindeficient cxlmd show muscular weakness as soon as they begin to ambulate . unlike puppies with cnm and xlmtm , patellar reflexes are maintained until muscle fibrosis and contractures occur.1 , 14 , 20 a reliable feature of dystrophindeficient cxlmd is markedly increased serum ck activity as early as 7 days of age and peaking at 68 weeks of age at 100 times normal.12 , 14 , 20 despite the observation that only males were affected , our clinical findings of absent patellar reflexes and normal ck activities , and normal immunohistochemical staining and immunoblotting for dystrophin and associated proteins , eliminated cxlmd as a diagnosis in the puppies described here . based on the histopathologic diagnosis of myotubular myopathy in these puppies , the xlinked nature of the pedigree analysis , and the absence of myotubularin in the muscles on immunohistochemistry and immunoblotting , a candidate gene approach was used to test for a mutation in the myotubulin gene mtm1.5 all 15 exons of the canine mtm1 gene located on the x chromosome were sequenced . the mutation was not detected in unaffected male dogs from the 3 litters , in a panel of control dna samples obtained from 237 unrelated and unaffected labrador retrievers from throughout north america , europe , and australia , or in 59 additional control dogs from 25 breeds , supporting it as the pathogenic causative mutation . insufficient pedigree details are available for the third litter to prove or disprove a relationship , however , genetic haplotype has shown that a spontaneous mutation occurred in a local bitch and was transmitted to affected puppies in all 3 litters through a founder effect.5 similar to the clinical course in these puppies , xlmtm in humans is a severe disease that , in the absence of intensive medical support , often follows a fatal course over the first few years of life . biopsies disclose uniformly small muscle fibers with centrally placed nuclei resembling fetal myotubes.23 mutations in the myotubularin ( mtm1 ) gene on human chromosome xq28 are responsible for xlmtm . the incidence is estimated at 1/50000 male births.5 , 22 over 200 different spontaneous deletion , insertion , nonsense , missense , and splice mutations have been found in the mtm1 gene , with the majority occurring in exons 12 , 4 , 11 , 8 , and 9.5 the mutation identified in these puppies was a unique exon 7 missense mutation that has not been reported in a human patient.4 identification of the mutation in the affected puppies allowed us to not only identify carrier females and counsel owners regarding future breedings but also allowed us to recruit carrier females to establish a breeding colony . establishment of a breeding colony has contributed to ongoing investigation of the pathophysiology of muscle deterioration and weakness in individuals with xlmtm,24 and to evaluation of potential treatments.25 spontaneous mutation in the mtm1 gene resulting in xlmtm in male offspring is well described in human medicine but only recently reported in dogs.4 , 5 given the frequency with which these mutations occur in humans , it is likely that similar mutations occur occasionally in dogs , but that the resulting myopathies are incorrectly diagnosed as some other congenital myopathy . evaluation of affected labrador retriever puppies in this report highlights the clinical differences between xlmtm and the other reported congenital myopathies in this breed , and may help veterinarians recognize similar clinical presentations in other breeds .

even relatively small cartilage lesions frequently induce pain and disability for the patient . without repair commonly advocated repair techniques for these smaller lesions are microfracture , autologous chondrocyte implantation ( aci ) , and autologous osteochondral transplantation ( aot ) [ 2 , 28 ] . compared with aci , the advantage of aot is that it is a one - stage procedure and mature hyaline cartilage is present in the defect site directly after transplantation . in literature both very positive reports [ 79 , 13 , 14 ] and concerns [ 1 , 18 ] have been presented for aot in the knee . concerns are related to donor site morbidity [ 6 , 24 , 27 ] , to survival of the transplanted cartilage [ 5 , 11 , 26 ] , and to the reported negative outcome in some patient series [ 3 , 18 ] . particularly mal - indications , treatment of too large defects , the use of too large plugs or co - morbidity within the knee could all negatively influence the clinical outcome of aot [ 1 , 18 ] . adequate positioning , together with intrinsic stability of the transplanted plugs are likely to be important prerequisites to reach clinical success . both are important for adequate osseous integration of the transplanted plug in the subchondral bone and for survival of the hyaline cartilage [ 20 , 23 ] . in addition , osteochondral transplants should be placed flush with the joint surface , since leaving the plugs proud generates stresses at the edges leading to early damage to the graft [ 19 , 23 , 29 ] . subsidence of plugs , on the other hand , leads to stresses at the rim of the host cartilage [ 10 , 15 , 16 ] , with similar damage there . earlier biomechanical studies on an aot cadaver model , using the oats system , reported on relatively high forces needed to displace bottomed plugs beyond flush level as compared to plugs placed with a gap between the cylinder graft and the bottom of the recipient subchondral bone defect ( unbottomed plugs ) . from this biomechanical study , well - seated ( bottomed ) osteochondral plugs thus seemed less susceptible for subsidence [ 16 , 17 ] . the aim of this goat study was to compare bottomed versus unbottomed osteochondral transplants in vivo . it was hypothesized that bottomed plugs would indeed be superior in maintaining their flush level and by that would lead to a better gross morphology of the reconstruction and to a better survival of hyaline cartilage of both donor and host cartilage at a follow - up period of 6 weeks . prior to surgery , each goat had been randomly assigned to receive two bottomed or unbottomed plugs . centrally , in the weight - bearing area of the medial femoral condyle , a standardized oval defect ( length 12 mm , width 6 mm ) of 3 mm depth was created in an ap orientation with the use of a customized drill guide , a circular drill of 6 mm diameter and a small sharp osteotome ( recipient site ) ( fig . in accordance with clinical practice , a cylindrical osteochondral transplantation was performed to restore the articular defect using a 6 mm diameter osteochondral autograft transfer system ( oats ; arthrex , st . 1a standardized 3-mm - deep oval defect on the medial femoral condyle ( length 12 mm , width 6 mm ) . b final result of a two - plug osteochondral transplantation on the medial femoral condyle a standardized 3-mm - deep oval defect on the medial femoral condyle ( length 12 mm , width 6 mm ) . b final result of a two - plug osteochondral transplantation on the medial femoral condyle two donor osteochondral plugs were harvested from the lateral trochlea of the goat knee , which is in accordance with the situation in patients . care was taken to harvest plugs as much perpendicular to the articular surface as possible without penetrating the lateral wall of the trochlear groove . the harvested donor plugs were adjusted to an exact length of 8 mm , measured with a precision slide calliper , by removal of some deep trabecular bone by a surgical bone nibbling plier . depending on the assigned treatment , a recipient hole with an 8 or 10 mm depth was created in the femoral condyle defect for a bottomed or unbottomed situation , respectively . the bottom of the recipient hole was softly tampered , with the provided steel rod , to flatten it in order to ensure an exact matching depth and direct contact of the transplanted plug on the bottom of the recipient hole in case of a bottomed transplantation . subsequently , the available donor osteochondral plug was gently tampered into place until flush level with the adjacent articular cartilage was obtained . after the first plug was in place , the entire procedure was repeated for the second transplant ( fig . plugs were seated exactly on the flattened bottom , and in the unbottomed situation , the plugs were floating 2 mm above the flattened bottom . the trochlear donor site defects were either left empty in six goats or were filled with impacted remainders of the available subchondral bone derived from preparing the recipient holes , leaving small , but shallow defects ( 12 goats ) . in six of these 12 goats , the remaining shallow defects were completely filled up to a flush level with a commercially available hemostatic collagen type i sponge ( spongostan ; ferrosan a / s , soeborg , denmark ) . it was hypothesized that partly restoring the donor defect with the available impacted subchondral bone would lead to a better healing of these defects as compared to empty defects . in addition , filling the remaining defect with a collagen type i sponge would then possibly further enhance the healing process of the donor defect . directly after surgery , the goats were placed in a hammock for 7 days . the first 3 days the goats received standard injections of flunixine ( 1 mg / kg ) ( finadyne ; schering - plough animal health , oss , the netherlands ) and buprenorphine ( 0.018 ml / kg ) ( temgesic ; msd animal health , oss , the netherlands ) as analgesic . antibiotic prophylaxis consisted of ampicillin subcutaneously administered for 5 days postoperatively ( 7.5 ml / day ) ( albupen ; intervet , boxmeer , the netherlands ) . after 7 days , the goats were transported in the hammock to an outdoor farm where they were allowed to move freely and unrestricted . after 2 weeks , tetracycline ( 25 mg / kg ) was administered , after 4 weeks calcein green ( 25 mg / kg ) and finally after 6 weeks , 2 days before killing , alizarin red ( 30 mg / kg ) . after 6 weeks , the goats were killed with an overdose of pentobarbital ( nembutal , ceva sant animale , maassluis , the netherlands ) and the knee joint was completely dissected from femur en tibia . all investigations were conducted in conformity with ethical principles of research and all protocols were approved by the institutional animal ethics committee ( ru - dec2007 - 106 ) . directly after harvesting , the knee joint was opened and photographs of the recipient and donor sites were taken . the photographs of the reconstructions were evaluated by three blinded observers ( nk , gh , and pb ) and scored for gross morphological appearance of the surface congruency of individual plugs ( table 1 ) . subsidence , protrusion , tilting , sclerosis , and fibrous overgrowth were considered to be negative signs . the separate scores of each of both transplanted plugs were added , resulting in a maximum score of 4 per goat.table 1macroscopic scoring systemcharacteristicsscoreclinical acceptable or flush virtually no negative signs2clinical suboptimal minor negative signs1clinical failure obvious negative signs0subsidence , protrusion , tilting , sclerosis , and fibrous overgrowth were considered as negative signs macroscopic scoring system subsidence , protrusion , tilting , sclerosis , and fibrous overgrowth were considered as negative signs the operated condyle was dissected with a diamond - coated and water - cooled sawing blade ( blade thickness 300 m ) exactly in two halves in the ap direction through the center of the two plugs and photographs were taken again . thereafter , two slices were made from each section plane of both halves of the defect and processed for histology . the two slices were used for vitality testing with cell tracker green cmfda ( 5-chloromethylfluorescein diacetate ; molecular probes , invitrogen ltd , uk ) and propidium iodide ( molecular probes , invitrogen ltd . , uk ) . directly following coloring , the specimens were photographed through a fluorescence photomicroscope ( zeiss axioplan 2 ; carl zeiss bv , sliedrecht , the netherlands ) and frozen afterward . in a later stage , they were defrosted and vital cells were colored by nadh - diaphorase ( sigma - aldrich chemie bv , zwijndrecht , the netherlands ) with nitroblue - tetrazolium ( nbt ; sigma - aldrich chemie bv , zwijndrecht , the netherlands ) . after taking photographs , these slices were further processed for routine histology as described below . all specimens were fixated in a 4% buffered formalin solution ( ph 7.4 ) for at least 1 day . the specimens were embedded in polymethylmethacrylate and sectioned ( 7-m - thick sections ) with a microtome ( leica rm 2155 , leica microsystems nederland bv , rijswijk , the netherlands ) , and stained with hematoxylin and eosin , alcian blue and safranin o. serial sections of the trochlear donor defect were prepared in the transverse direction . the reconstructions were graded for graft level with surrounding cartilage , graft cartilage thickness , cartilage integration , degenerative changes at cartilage edges , changes in tidemarks at cartilage edges , integration of adjacent subchondral bone , and subchondral cavity formation ( table 2 ) . this scoring system was adapted from similar scales for osteochondral cartilage repair published by other researchers [ 4 , 22 ] . the same three blinded observers ( nk , gh , and pb ) scored two specimens from the center of the repaired defect independent of each other . in each reconstruction , 3 locations were evaluated , the anterior host plug contact area ( a ) , the central plug plug contact area ( b ) , and the posterior plug host contact area ( c ) . the scores for each location were summed to obtain a total score for each reconstruction.table 2histological scoring systemcharacteristicsscoregraft level with surrounding cartilage level4 < 50% of graft cartilage thickness raised2 > 50% of graft cartilage thickness below1 > 50% of graft cartilage thickness raised0graft cartilage thickness 75100% of adjacent cartilage2 5075%1 < 50%0cartilage integration bonding through cartilage like tissue3 fissure fully filled with fibrous tissue2 fissure partially filled with fibrous tissue1 empty fissure0degenerative changes at cartilage edges normal cellularity , no clusters , normal staining3 mild hypocellularity , some clusters , moderate staining2 moderate hypocellularity , slight staining1 severe hypocellularity , poor or no staining0changes in tidemarks at cartilage edges both normal2 one damaged or abnormal1 both damaged or abnormal0integration of adjacent subchondral bone fully osseous integration4 fully integrated with partial fibrous tissue3 fully integrated only fibrous tissue2 partially integrated1 no integration0subchondral cavity formation no cavities3 small cavities1 large cavity0total maximum score per location21 in each reconstruction , 3 locations were evaluated , the anterior host plug contact area ( a ) , the central plug the scores for each location were summed to obtain a total score for each reconstruction histological scoring system in each reconstruction , 3 locations were evaluated , the anterior host plug contact area ( a ) , the central plug the scores for each location were summed to obtain a total score for each reconstruction datasets from gross morphological scoring and histological grading were evaluated nonparametrically , as normal distribution could not be assumed for all parameters . directly after harvesting , the knee joint was opened and photographs of the recipient and donor sites were taken . the photographs of the reconstructions were evaluated by three blinded observers ( nk , gh , and pb ) and scored for gross morphological appearance of the surface congruency of individual plugs ( table 1 ) . subsidence , protrusion , tilting , sclerosis , and fibrous overgrowth were considered to be negative signs . the separate scores of each of both transplanted plugs were added , resulting in a maximum score of 4 per goat.table 1macroscopic scoring systemcharacteristicsscoreclinical acceptable or flush virtually no negative signs2clinical suboptimal minor negative signs1clinical failure obvious negative signs0subsidence , protrusion , tilting , sclerosis , and fibrous overgrowth were considered as negative signs macroscopic scoring system subsidence , protrusion , tilting , sclerosis , and fibrous overgrowth were considered as negative signs the operated condyle was dissected with a diamond - coated and water - cooled sawing blade ( blade thickness 300 m ) exactly in two halves in the ap direction through the center of the two plugs and photographs were taken again . thereafter , two slices were made from each section plane of both halves of the defect and processed for histology . the two slices were used for vitality testing with cell tracker green cmfda ( 5-chloromethylfluorescein diacetate ; molecular probes , invitrogen ltd , uk ) and propidium iodide ( molecular probes , invitrogen ltd . , uk ) . directly following coloring , the specimens were photographed through a fluorescence photomicroscope ( zeiss axioplan 2 ; carl zeiss bv , sliedrecht , the netherlands ) and frozen afterward . in a later stage , they were defrosted and vital cells were colored by nadh - diaphorase ( sigma - aldrich chemie bv , zwijndrecht , the netherlands ) with nitroblue - tetrazolium ( nbt ; sigma - aldrich chemie bv , zwijndrecht , the netherlands ) . after taking photographs , these slices were further processed for routine histology as described below . all specimens were fixated in a 4% buffered formalin solution ( ph 7.4 ) for at least 1 day . the specimens were embedded in polymethylmethacrylate and sectioned ( 7-m - thick sections ) with a microtome ( leica rm 2155 , leica microsystems nederland bv , rijswijk , the netherlands ) , and stained with hematoxylin and eosin , alcian blue and safranin o. serial sections of the trochlear donor defect were prepared in the transverse direction . the reconstructions were graded for graft level with surrounding cartilage , graft cartilage thickness , cartilage integration , degenerative changes at cartilage edges , changes in tidemarks at cartilage edges , integration of adjacent subchondral bone , and subchondral cavity formation ( table 2 ) . this scoring system was adapted from similar scales for osteochondral cartilage repair published by other researchers [ 4 , 22 ] . the same three blinded observers ( nk , gh , and pb ) scored two specimens from the center of the repaired defect independent of each other . in each reconstruction , 3 locations were evaluated , the anterior host plug contact area ( a ) , the central plug plug contact area ( b ) , and the posterior plug host contact area ( c ) . the scores for each location were summed to obtain a total score for each reconstruction.table 2histological scoring systemcharacteristicsscoregraft level with surrounding cartilage level4 < 50% of graft cartilage thickness below1 > 50% of graft cartilage thickness raised0graft cartilage thickness 75100% of adjacent cartilage2 5075%1 < 50%0cartilage integration bonding through cartilage like tissue3 fissure fully filled with fibrous tissue2 fissure partially filled with fibrous tissue1 empty fissure0degenerative changes at cartilage edges normal cellularity , no clusters , normal staining3 mild hypocellularity , some clusters , moderate staining2 moderate hypocellularity , slight staining1 severe hypocellularity , poor or no staining0changes in tidemarks at cartilage edges both normal2 one damaged or abnormal1 both damaged or abnormal0integration of adjacent subchondral bone fully osseous integration4 fully integrated with partial fibrous tissue3 fully integrated only fibrous tissue2 partially integrated1 no integration0subchondral cavity formation no cavities3 small cavities1 large cavity0total maximum score per location21 in each reconstruction , 3 locations were evaluated , the anterior host plug contact area ( a ) , the central plug plug contact area ( b ) , and the posterior plug the scores for each location were summed to obtain a total score for each reconstruction histological scoring system in each reconstruction , 3 locations were evaluated , the anterior host plug contact area ( a ) , the central plug plug contact area ( b ) , and the posterior plug host contact area ( c ) . datasets from gross morphological scoring and histological grading were evaluated nonparametrically , as normal distribution could not be assumed for all parameters . the gross morphological scoring showed a significantly higher score for surface congruency in bottomed plugs as compared to unbottomed reconstructions ( p = 0.04 ) ( table 3).table 3macroscopic scoresbottomed ( n = 9)unbottomed ( n = 9)p valueanterior plug1.5 0.40.8 0.70.03posterior plug1.2 0.70.7 0.8n.s.total2.7 0.81.5 0.40.04values are mean sdn.s . = p 0.05 no differences between local and total histological grading scores for bottomed and unbottomed plugs were found ( table 4).table 4microscopic scoresbottomed ( n = 9)unbottomed ( n = 9)p valuea11.1 3.612.3 3.0n.s.b8.8 3.09.7 3.3n.s.c12.3 2.511.3 3.7n.s.total32.0 6.032.1 8.5n.s.values are mean sd . ( a ) anterior host plug contact area , ( b ) the central plug plug contact area , and ( c ) the posterior plug ( a ) anterior host plug contact area , ( b ) the central plug plug contact area , and ( c ) the posterior plug host contact area both the nadh - diaphorase staining and the life dead assay showed a similar distribution of vital and necrotic cells ( fig . 2 ) . necrotic chondrocytes were present in equal low numbers in both host and plug articular cartilage . only at the edges toward the interface between plugs , there was a thin acellular zone of cartilage ( fig . clustered chondrocytes were present in almost all plugs and bordering host cartilage ( fig . 2a , b , d ) . occasionally , larger gaps were also present ( figs . 2c , 3b ) . the cartilage interface between plugs was in most cases very thin and plugs seemed to be press - fit ( fig . no integration of cartilage in between plugs or between plugs and host cartilage was found.fig . 2histology of sections stained with nadh - diaphorase with nbt ( nitroblue - tetrazolium ) for vitality ( a , b , c ) and section ( d ) in which living cells are stained with cell tracker green . notice difference in thickness between host ( h ) and plug ( p ) cartilage . in b 3routine histology staining of thin sections with safranin o ( a , b ) and haematoxylin and eosin ( c , d ) . a , c bottomed plugs , b , d unbottomed plugs . a notice cartilage - like tissue formation in the gap between the two inserted plugs ( p ) . in b a larger nonfilled gap is present between host ( h ) and plug ( p ) cartilage . d new bone formation in subchondral bone of transplanted plug histology of sections stained with nadh - diaphorase with nbt ( nitroblue - tetrazolium ) for vitality ( a , b , c ) and section ( d ) in which living cells are stained with cell tracker green . notice difference in thickness between host ( h ) and plug ( p ) cartilage . in b a , b bottomed reconstructions , c , d unbottomed reconstructions routine histology staining of thin sections with safranin o ( a , b ) and haematoxylin and eosin ( c , d ) . a , c bottomed plugs , b , d unbottomed plugs . a notice cartilage - like tissue formation in the gap between the two inserted plugs ( p ) . in b a larger nonfilled gap is present between host ( h ) and plug ( p ) cartilage . d new bone formation in subchondral bone of transplanted plug the tidemarks were generally intact ( fig . particularly , if the subchondral bone was revascularized , small capillaries of the newly formed fibrous subchondral tissue were penetrating the tidemark , and by that , the integrity of the tidemark was reduced ( fig . bv is a blood vessel penetrating to the tidemark subchondral bone of host ( a ) , and of unbottomed plug . bv is a blood vessel penetrating to the tidemark the central part of the subchondral bone of the transplanted plugs was still mainly avascular and necrotic with empty osteocyte lacunae and necrotic medullary tissue . from the deeper parts of the defect and from the interfaces with the host bone , this process was characterized by abundant osteoclastic resorption sites of the transplanted bone ( fig . 3c ) , new bone formation on the remnants of the bone of the plugs and bone remodeling . 3d ) ; however , irrespective of the treatment group , in various locations in the subchondral areas , small areas of enchondral bone formation were present . irrespective to the treatment modality , all donor sites showed rather similar histological appearances . in the defects , the surface of the newly formed tissue was either slightly depressed under the original contour of the defect or in a few cases it was elevated above the original contour ( fig . in only two defects that were filled with the hemostatic collagen type i sponge , centrally a few remnants of the original graft material could be found ( fig . , it was surrounded by some mononuclear lymphocytic cells but without a clear immunological reaction . in all defects that were filled with bone graft , this the edges of the donor site showed in all cases some form of depletion of the matrix , decreased chondrocyte vitality and clustering of chondrocytes ( fig . 5d).fig . 5a , b haematoxylin and eosin - stained sections showing representative micrographs of donor site . a empty defect . d edge of host cartilage ( hc ) with clustered chondrocytes ( arrows ) a , b haematoxylin and eosin - stained sections showing representative micrographs of donor site . a empty defect . the gross morphological scoring showed a significantly higher score for surface congruency in bottomed plugs as compared to unbottomed reconstructions ( p = 0.04 ) ( table 3).table 3macroscopic scoresbottomed ( n = 9)unbottomed ( n = 9)p valueanterior plug1.5 0.40.8 0.70.03posterior plug1.2 0.70.7 0.8n.s.total2.7 0.81.5 0.40.04values are mean sdn.s . no differences between local and total histological grading scores for bottomed and unbottomed plugs were found ( table 4).table 4microscopic scoresbottomed ( n = 9)unbottomed ( n = 9)p valuea11.1 3.612.3 3.0n.s.b8.8 3.09.7 3.3n.s.c12.3 2.511.3 3.7n.s.total32.0 6.032.1 8.5n.s.values are mean sd . ( a ) anterior host plug contact area , ( b ) the central plug ( a ) anterior host plug contact area , ( b ) the central plug both the nadh - diaphorase staining and the life dead assay showed a similar distribution of vital and necrotic cells ( fig . 2 ) . necrotic chondrocytes were present in equal low numbers in both host and plug articular cartilage . only at the edges toward the interface between plugs , there was a thin acellular zone of cartilage ( fig . clustered chondrocytes were present in almost all plugs and bordering host cartilage ( fig . occasionally , larger gaps were also present ( figs . 2c , 3b ) . the cartilage interface between plugs was in most cases very thin and plugs seemed to be press - fit ( fig . no integration of cartilage in between plugs or between plugs and host cartilage was found.fig . 2histology of sections stained with nadh - diaphorase with nbt ( nitroblue - tetrazolium ) for vitality ( a , b , c ) and section ( d ) in which living cells are stained with cell tracker green . notice difference in thickness between host ( h ) and plug ( p ) cartilage . in b 3routine histology staining of thin sections with safranin o ( a , b ) and haematoxylin and eosin ( c , d ) . a , c bottomed plugs , b , d unbottomed plugs . a notice cartilage - like tissue formation in the gap between the two inserted plugs ( p ) . in b a larger nonfilled gap is present between host ( h ) and plug ( p ) cartilage . d new bone formation in subchondral bone of transplanted plug histology of sections stained with nadh - diaphorase with nbt ( nitroblue - tetrazolium ) for vitality ( a , b , c ) and section ( d ) in which living cells are stained with cell tracker green . notice difference in thickness between host ( h ) and plug ( p ) cartilage . in b a , b bottomed reconstructions , c , d unbottomed reconstructions routine histology staining of thin sections with safranin o ( a , b ) and haematoxylin and eosin ( c , d ) . a , c bottomed plugs , b , d unbottomed plugs . a notice cartilage - like tissue formation in the gap between the two inserted plugs ( p ) . in b a larger nonfilled gap is present between host ( h ) and plug ( p ) cartilage . particularly , if the subchondral bone was revascularized , small capillaries of the newly formed fibrous subchondral tissue were penetrating the tidemark , and by that , the integrity of the tidemark was reduced ( fig . bv is a blood vessel penetrating to the tidemark subchondral bone of host ( a ) , and of unbottomed plug . the central part of the subchondral bone of the transplanted plugs was still mainly avascular and necrotic with empty osteocyte lacunae and necrotic medullary tissue . from the deeper parts of the defect and from the interfaces with the host bone , a process of creeping substitution of the necrotic bone was present . this process was characterized by abundant osteoclastic resorption sites of the transplanted bone ( fig . 3c ) , new bone formation on the remnants of the bone of the plugs and bone remodeling . 3d ) ; however , irrespective of the treatment group , in various locations in the subchondral areas , small areas of enchondral bone formation were present . irrespective to the treatment modality , all donor sites showed rather similar histological appearances . in the defects , the surface of the newly formed tissue was either slightly depressed under the original contour of the defect or in a few cases it was elevated above the original contour ( fig . 5b ) , without clear correlation with the filling material in the defect . in only two defects that were filled with the hemostatic collagen type i sponge , centrally a few remnants of the original graft material could be found ( fig . , it was surrounded by some mononuclear lymphocytic cells but without a clear immunological reaction . in all defects that were filled with bone graft , this the edges of the donor site showed in all cases some form of depletion of the matrix , decreased chondrocyte vitality and clustering of chondrocytes ( fig . 5d).fig . 5a , b haematoxylin and eosin - stained sections showing representative micrographs of donor site . a empty defect . d edge of host cartilage ( hc ) with clustered chondrocytes ( arrows ) a , b haematoxylin and eosin - stained sections showing representative micrographs of donor site . a empty defect . the most important finding of the present study was that chondrocyte vitality was maintained after osteochondral transplantation . on gross appearance , bottomed plugs seemed to reveal lesser subsidence tendency than unbottomed plugs ; however , on histological evaluation , no significant differences could be detected between groups . in addition , the repair tendency of the donor defects was not improved by the two different filling techniques evaluated in this animal model . gross morphological scoring showed that the bottomed plugs performed superior in maintaining flush level with the surrounding subchondral bone . the fact that on histological scoring bottomed and unbottomed plugs failed to reveal a significant difference may be induced by the scoring method itself . the histological scoring was performed in the mid - sagittal plane only , whereas for macroscopic evaluation , the entire contour of the transplantation was judged and not just the appearance of the mid - sagittal plane ( see also fig . 6 for further explanation ) . irrespective the fact that histological scoring was performed on the midsagittal plane only , it has to be recognized that no major differences were encountered between bottomed and unbottomed plugs . clearly the extent of subsidence was relatively low in this animal model , which may be related to the compact subchondral bone structure of goats . irrespective of the absence of a significant difference between the two groups , the evaluated different aspects of the histological score are still important parameters for the long - term success of the aot technique . the most important long - term success aspect of the aot technique above the aci techniques or microfracture is the instantaneous restoration of the defect site with mature healthy hyaline cartilage , which was indeed confirmed in our study by an excellent chondrocyte vitality after transplantation.fig . 6schematics for illustrating the possible discrepancy in judging the ( flush ) level of a whole graft through microscopic sections of the specimen . the dotted lines indicate the outer boundaries of the two thick sections , which were made . notice the less than optimal perpendicularity of the plug s cartilage surface in respect to its subchondral bone in this schematic representation . c represents the lateral view of the theoretical sections made at the dotted lines in b. 1 notice the subsidence below flush level of the posterior ( right ) plug . 3 notice the protrusion above flush level of the posterior ( right ) plug schematics for illustrating the possible discrepancy in judging the ( flush ) level of a whole graft through microscopic sections of the specimen . the dotted lines indicate the outer boundaries of the two thick sections , which were made . notice the less than optimal perpendicularity of the plug s cartilage surface in respect to its subchondral bone in this schematic representation . c represents the lateral view of the theoretical sections made at the dotted lines in b. 1 notice the subsidence below flush level of the posterior ( right ) plug . 3 notice the protrusion above flush level of the posterior ( right ) plug chondrocyte survival also appears to be dependent on the harvesting procedure of the osteochondral graft , where sharper cutting devices reduce the thickness of the layer of dead chondrocytes . moreover , harvesting by hand is superior above power trephine harvesting . in this study , only small margins of the plugs were acellular , indicating that indeed also limited chondrocyte death took place during the harvest . besides the harvesting procedure , tampering the plugs into the bone might also induce chondrocyte death at the surface of the plugs . the fact that in both groups the surface layer was populated with vital cells indicated that the tempering probably did not induce a necrosis of the superficial chondrocytes . this indicates that the clinically used forces to insert plugs are most likely within the margin to induce cartilage damage , especially since goat cartilage is thinner and thereby more prone to damage , and higher tempering force is needed in the more compact goat bone , compared with human bone . the potential for donor site morbidity is still a major drawback of the aot technique and is held responsible for inferior results in some studies . for that reason , we evaluated some simple attempts to enhance the healing potential of these defects . however , filling the donor site defects with impacted cancellous bone , with or without a collagen type i layer , did not induce any new subchondral bone restoration as compared to empty defects . although in the deeper parts of the defects some new bone was formed , there was no relation with the graft material . . however , the results and recommendations from this study for patients should be carefully interpreted within the limitations of the model . bone of healthy animals is of good quality and because of high loads more compact than in humans , which may have resulted in a more tightly press - fit placement of the plugs and thus a relative resistance to subsidence tendency . this phenomenon may have played a role in the encountered absence of a significant difference between bottomed and furthermore , it has to be recognized that in this study an osteochondral transplantation using the oats system was evaluated , which system on theoretical grounds may offer less intrinsic stability to the transplanted plugs than the alternatively available mosaicplasty system . in contrast to the oats system , in the mosaicplasty system , primary stability of the transplants is not only dependent on the standard shear forces between plug and host bone , but also on the conically shaped bottom part of the defect created by means of a dilator . the recommendation to bottom osteochondral plugs may thus mainly apply to the currently evaluated oats system . subsidence of the transplanted plugs may be detrimental for the eventual clinical outcome and should be avoided . in this animal model , using the oats system , macroscopic scoring revealed less subsidence tendency when the transplanted plugs were bottomed . in spite of the fact that the encountered benefit of bottoming was only limited in this animal model , bottoming is still recommended for clinical practice , especially when the oats system is used . larger comparative clinical trials will be necessary to truly elucidate the potential clinical benefit of bottoming .

purposethe aim of this study was to evaluate subsidence tendency , surface congruency , chondrocyte survival and plug incorporation after osteochondral transplantation in an animal model . the potential benefit of precise seating of the transplanted osteochondral plug on the recipient subchondral host bone ( bottoming ) on these parameters was assessed in particular.methodsin 18 goats , two osteochondral autografts were harvested from the trochlea of the ipsilateral knee joint and inserted press - fit in a standardized articular cartilage defect in the medial femoral condyle . in half of the goats , the transplanted plugs were matched exactly to the depth of the recipient hole ( bottomed plugs ; n = 9 ) , whereas in the other half of the goats , a gap of 2 mm was left between the plugs and the recipient bottom ( unbottomed plugs ; n = 9 ) . after 6 weeks , all transplants were evaluated on gross morphology , subsidence , histology , and chondrocyte vitality.resultsthe macroscopic morphology scored significantly higher for surface congruency in bottomed plugs as compared to unbottomed reconstructions ( p = 0.04 ) . however , no differences in histological subsidence scoring between bottomed and unbottomed plugs were found . the transplanted articular cartilage of both bottomed and unbottomed plugs was vital . only at the edges some matrix destaining , chondrocyte death and cluster formation was observed . at the subchondral bone level , active remodeling occurred , whereas integration at the cartilaginous surface of the osteochondral plugs failed to occur . subchondral cysts were found in both groups.conclusionsin this animal model , subsidence tendency was significantly lower after bottomed versus unbottomed osteochondral transplants on gross appearance , whereas for histological scoring no significant differences were encountered . since the clinical outcome may be negatively influenced by subsidence , the use of bottomed grafts is recommended for osteochondral transplantation in patients .

Introduction Materials and methods Macroscopic scoring Microscopic preparation Histological scoring Statistical analysis Results Macroscopic scoring Microscopic scoring Histology of the plugs Integrity of the tidemark Subchondral bone remodeling Donor site Discussion Conclusions

concerns are related to donor site morbidity [ 6 , 24 , 27 ] , to survival of the transplanted cartilage [ 5 , 11 , 26 ] , and to the reported negative outcome in some patient series [ 3 , 18 ] . particularly mal - indications , treatment of too large defects , the use of too large plugs or co - morbidity within the knee could all negatively influence the clinical outcome of aot [ 1 , 18 ] . both are important for adequate osseous integration of the transplanted plug in the subchondral bone and for survival of the hyaline cartilage [ 20 , 23 ] . in addition , osteochondral transplants should be placed flush with the joint surface , since leaving the plugs proud generates stresses at the edges leading to early damage to the graft [ 19 , 23 , 29 ] . subsidence of plugs , on the other hand , leads to stresses at the rim of the host cartilage [ 10 , 15 , 16 ] , with similar damage there . earlier biomechanical studies on an aot cadaver model , using the oats system , reported on relatively high forces needed to displace bottomed plugs beyond flush level as compared to plugs placed with a gap between the cylinder graft and the bottom of the recipient subchondral bone defect ( unbottomed plugs ) . the aim of this goat study was to compare bottomed versus unbottomed osteochondral transplants in vivo . it was hypothesized that bottomed plugs would indeed be superior in maintaining their flush level and by that would lead to a better gross morphology of the reconstruction and to a better survival of hyaline cartilage of both donor and host cartilage at a follow - up period of 6 weeks . centrally , in the weight - bearing area of the medial femoral condyle , a standardized oval defect ( length 12 mm , width 6 mm ) of 3 mm depth was created in an ap orientation with the use of a customized drill guide , a circular drill of 6 mm diameter and a small sharp osteotome ( recipient site ) ( fig . 1a standardized 3-mm - deep oval defect on the medial femoral condyle ( length 12 mm , width 6 mm ) . b final result of a two - plug osteochondral transplantation on the medial femoral condyle a standardized 3-mm - deep oval defect on the medial femoral condyle ( length 12 mm , width 6 mm ) . b final result of a two - plug osteochondral transplantation on the medial femoral condyle two donor osteochondral plugs were harvested from the lateral trochlea of the goat knee , which is in accordance with the situation in patients . depending on the assigned treatment , a recipient hole with an 8 or 10 mm depth was created in the femoral condyle defect for a bottomed or unbottomed situation , respectively . the bottom of the recipient hole was softly tampered , with the provided steel rod , to flatten it in order to ensure an exact matching depth and direct contact of the transplanted plug on the bottom of the recipient hole in case of a bottomed transplantation . plugs were seated exactly on the flattened bottom , and in the unbottomed situation , the plugs were floating 2 mm above the flattened bottom . it was hypothesized that partly restoring the donor defect with the available impacted subchondral bone would lead to a better healing of these defects as compared to empty defects . after 6 weeks , the goats were killed with an overdose of pentobarbital ( nembutal , ceva sant animale , maassluis , the netherlands ) and the knee joint was completely dissected from femur en tibia . directly after harvesting , the knee joint was opened and photographs of the recipient and donor sites were taken . the photographs of the reconstructions were evaluated by three blinded observers ( nk , gh , and pb ) and scored for gross morphological appearance of the surface congruency of individual plugs ( table 1 ) . the separate scores of each of both transplanted plugs were added , resulting in a maximum score of 4 per goat.table 1macroscopic scoring systemcharacteristicsscoreclinical acceptable or flush virtually no negative signs2clinical suboptimal minor negative signs1clinical failure obvious negative signs0subsidence , protrusion , tilting , sclerosis , and fibrous overgrowth were considered as negative signs macroscopic scoring system subsidence , protrusion , tilting , sclerosis , and fibrous overgrowth were considered as negative signs the operated condyle was dissected with a diamond - coated and water - cooled sawing blade ( blade thickness 300 m ) exactly in two halves in the ap direction through the center of the two plugs and photographs were taken again . the specimens were embedded in polymethylmethacrylate and sectioned ( 7-m - thick sections ) with a microtome ( leica rm 2155 , leica microsystems nederland bv , rijswijk , the netherlands ) , and stained with hematoxylin and eosin , alcian blue and safranin o. serial sections of the trochlear donor defect were prepared in the transverse direction . in each reconstruction , 3 locations were evaluated , the anterior host plug contact area ( a ) , the central plug plug contact area ( b ) , and the posterior plug host contact area ( c ) . the scores for each location were summed to obtain a total score for each reconstruction.table 2histological scoring systemcharacteristicsscoregraft level with surrounding cartilage level4 < 50% of graft cartilage thickness raised2 > 50% of graft cartilage thickness below1 > 50% of graft cartilage thickness raised0graft cartilage thickness 75100% of adjacent cartilage2 5075%1 < 50%0cartilage integration bonding through cartilage like tissue3 fissure fully filled with fibrous tissue2 fissure partially filled with fibrous tissue1 empty fissure0degenerative changes at cartilage edges normal cellularity , no clusters , normal staining3 mild hypocellularity , some clusters , moderate staining2 moderate hypocellularity , slight staining1 severe hypocellularity , poor or no staining0changes in tidemarks at cartilage edges both normal2 one damaged or abnormal1 both damaged or abnormal0integration of adjacent subchondral bone fully osseous integration4 fully integrated with partial fibrous tissue3 fully integrated only fibrous tissue2 partially integrated1 no integration0subchondral cavity formation no cavities3 small cavities1 large cavity0total maximum score per location21 in each reconstruction , 3 locations were evaluated , the anterior host plug contact area ( a ) , the central plug the scores for each location were summed to obtain a total score for each reconstruction histological scoring system in each reconstruction , 3 locations were evaluated , the anterior host plug contact area ( a ) , the central plug the scores for each location were summed to obtain a total score for each reconstruction datasets from gross morphological scoring and histological grading were evaluated nonparametrically , as normal distribution could not be assumed for all parameters . directly after harvesting , the knee joint was opened and photographs of the recipient and donor sites were taken . the photographs of the reconstructions were evaluated by three blinded observers ( nk , gh , and pb ) and scored for gross morphological appearance of the surface congruency of individual plugs ( table 1 ) . the separate scores of each of both transplanted plugs were added , resulting in a maximum score of 4 per goat.table 1macroscopic scoring systemcharacteristicsscoreclinical acceptable or flush virtually no negative signs2clinical suboptimal minor negative signs1clinical failure obvious negative signs0subsidence , protrusion , tilting , sclerosis , and fibrous overgrowth were considered as negative signs macroscopic scoring system subsidence , protrusion , tilting , sclerosis , and fibrous overgrowth were considered as negative signs the operated condyle was dissected with a diamond - coated and water - cooled sawing blade ( blade thickness 300 m ) exactly in two halves in the ap direction through the center of the two plugs and photographs were taken again . thereafter , two slices were made from each section plane of both halves of the defect and processed for histology . the specimens were embedded in polymethylmethacrylate and sectioned ( 7-m - thick sections ) with a microtome ( leica rm 2155 , leica microsystems nederland bv , rijswijk , the netherlands ) , and stained with hematoxylin and eosin , alcian blue and safranin o. serial sections of the trochlear donor defect were prepared in the transverse direction . in each reconstruction , 3 locations were evaluated , the anterior host plug contact area ( a ) , the central plug plug contact area ( b ) , and the posterior plug host contact area ( c ) . the scores for each location were summed to obtain a total score for each reconstruction.table 2histological scoring systemcharacteristicsscoregraft level with surrounding cartilage level4 < 50% of graft cartilage thickness below1 > 50% of graft cartilage thickness raised0graft cartilage thickness 75100% of adjacent cartilage2 5075%1 < 50%0cartilage integration bonding through cartilage like tissue3 fissure fully filled with fibrous tissue2 fissure partially filled with fibrous tissue1 empty fissure0degenerative changes at cartilage edges normal cellularity , no clusters , normal staining3 mild hypocellularity , some clusters , moderate staining2 moderate hypocellularity , slight staining1 severe hypocellularity , poor or no staining0changes in tidemarks at cartilage edges both normal2 one damaged or abnormal1 both damaged or abnormal0integration of adjacent subchondral bone fully osseous integration4 fully integrated with partial fibrous tissue3 fully integrated only fibrous tissue2 partially integrated1 no integration0subchondral cavity formation no cavities3 small cavities1 large cavity0total maximum score per location21 in each reconstruction , 3 locations were evaluated , the anterior host plug contact area ( a ) , the central plug plug contact area ( b ) , and the posterior plug the scores for each location were summed to obtain a total score for each reconstruction histological scoring system in each reconstruction , 3 locations were evaluated , the anterior host plug contact area ( a ) , the central plug plug contact area ( b ) , and the posterior plug host contact area ( c ) . the gross morphological scoring showed a significantly higher score for surface congruency in bottomed plugs as compared to unbottomed reconstructions ( p = 0.04 ) ( table 3).table 3macroscopic scoresbottomed ( n = 9)unbottomed ( n = 9)p valueanterior plug1.5 0.40.8 0.70.03posterior plug1.2 0.70.7 0.8n.s.total2.7 0.81.5 0.40.04values are mean sdn.s . = p 0.05 no differences between local and total histological grading scores for bottomed and unbottomed plugs were found ( table 4).table 4microscopic scoresbottomed ( n = 9)unbottomed ( n = 9)p valuea11.1 3.612.3 3.0n.s.b8.8 3.09.7 3.3n.s.c12.3 2.511.3 3.7n.s.total32.0 6.032.1 8.5n.s.values are mean sd . particularly , if the subchondral bone was revascularized , small capillaries of the newly formed fibrous subchondral tissue were penetrating the tidemark , and by that , the integrity of the tidemark was reduced ( fig . bv is a blood vessel penetrating to the tidemark subchondral bone of host ( a ) , and of unbottomed plug . bv is a blood vessel penetrating to the tidemark the central part of the subchondral bone of the transplanted plugs was still mainly avascular and necrotic with empty osteocyte lacunae and necrotic medullary tissue . from the deeper parts of the defect and from the interfaces with the host bone , this process was characterized by abundant osteoclastic resorption sites of the transplanted bone ( fig . 3c ) , new bone formation on the remnants of the bone of the plugs and bone remodeling . 3d ) ; however , irrespective of the treatment group , in various locations in the subchondral areas , small areas of enchondral bone formation were present . in the defects , the surface of the newly formed tissue was either slightly depressed under the original contour of the defect or in a few cases it was elevated above the original contour ( fig . the gross morphological scoring showed a significantly higher score for surface congruency in bottomed plugs as compared to unbottomed reconstructions ( p = 0.04 ) ( table 3).table 3macroscopic scoresbottomed ( n = 9)unbottomed ( n = 9)p valueanterior plug1.5 0.40.8 0.70.03posterior plug1.2 0.70.7 0.8n.s.total2.7 0.81.5 0.40.04values are mean sdn.s . no differences between local and total histological grading scores for bottomed and unbottomed plugs were found ( table 4).table 4microscopic scoresbottomed ( n = 9)unbottomed ( n = 9)p valuea11.1 3.612.3 3.0n.s.b8.8 3.09.7 3.3n.s.c12.3 2.511.3 3.7n.s.total32.0 6.032.1 8.5n.s.values are mean sd . the cartilage interface between plugs was in most cases very thin and plugs seemed to be press - fit ( fig . particularly , if the subchondral bone was revascularized , small capillaries of the newly formed fibrous subchondral tissue were penetrating the tidemark , and by that , the integrity of the tidemark was reduced ( fig . bv is a blood vessel penetrating to the tidemark subchondral bone of host ( a ) , and of unbottomed plug . the central part of the subchondral bone of the transplanted plugs was still mainly avascular and necrotic with empty osteocyte lacunae and necrotic medullary tissue . from the deeper parts of the defect and from the interfaces with the host bone , a process of creeping substitution of the necrotic bone was present . 3c ) , new bone formation on the remnants of the bone of the plugs and bone remodeling . 3d ) ; however , irrespective of the treatment group , in various locations in the subchondral areas , small areas of enchondral bone formation were present . in the defects , the surface of the newly formed tissue was either slightly depressed under the original contour of the defect or in a few cases it was elevated above the original contour ( fig . the most important finding of the present study was that chondrocyte vitality was maintained after osteochondral transplantation . on gross appearance , bottomed plugs seemed to reveal lesser subsidence tendency than unbottomed plugs ; however , on histological evaluation , no significant differences could be detected between groups . in addition , the repair tendency of the donor defects was not improved by the two different filling techniques evaluated in this animal model . the fact that on histological scoring bottomed and unbottomed plugs failed to reveal a significant difference may be induced by the scoring method itself . the histological scoring was performed in the mid - sagittal plane only , whereas for macroscopic evaluation , the entire contour of the transplantation was judged and not just the appearance of the mid - sagittal plane ( see also fig . irrespective the fact that histological scoring was performed on the midsagittal plane only , it has to be recognized that no major differences were encountered between bottomed and unbottomed plugs . clearly the extent of subsidence was relatively low in this animal model , which may be related to the compact subchondral bone structure of goats . 3 notice the protrusion above flush level of the posterior ( right ) plug chondrocyte survival also appears to be dependent on the harvesting procedure of the osteochondral graft , where sharper cutting devices reduce the thickness of the layer of dead chondrocytes . in this study , only small margins of the plugs were acellular , indicating that indeed also limited chondrocyte death took place during the harvest . besides the harvesting procedure , tampering the plugs into the bone might also induce chondrocyte death at the surface of the plugs . however , filling the donor site defects with impacted cancellous bone , with or without a collagen type i layer , did not induce any new subchondral bone restoration as compared to empty defects . however , the results and recommendations from this study for patients should be carefully interpreted within the limitations of the model . bone of healthy animals is of good quality and because of high loads more compact than in humans , which may have resulted in a more tightly press - fit placement of the plugs and thus a relative resistance to subsidence tendency . this phenomenon may have played a role in the encountered absence of a significant difference between bottomed and furthermore , it has to be recognized that in this study an osteochondral transplantation using the oats system was evaluated , which system on theoretical grounds may offer less intrinsic stability to the transplanted plugs than the alternatively available mosaicplasty system . in contrast to the oats system , in the mosaicplasty system , primary stability of the transplants is not only dependent on the standard shear forces between plug and host bone , but also on the conically shaped bottom part of the defect created by means of a dilator . subsidence of the transplanted plugs may be detrimental for the eventual clinical outcome and should be avoided . in this animal model , using the oats system , macroscopic scoring revealed less subsidence tendency when the transplanted plugs were bottomed . in spite of the fact that the encountered benefit of bottoming was only limited in this animal model , bottoming is still recommended for clinical practice , especially when the oats system is used .

gene duplication makes an important contribution to the evolution of novel functions and the modifications of existing functions ( reviewed in prince and pickett 2002 ) , and duplicated genes are prevalent throughout metazoans ( holland et al . 1994 ; amores et al . 1998 ; force et al . 1999 ; holland 1999 ; cresko et al . one theory ( the neo - functionalization model ) postulates that one of the duplicated genes evolves a novel function while losing some aspect of the ancestral function . thus , both genes are maintained by natural selection , one for the ancestral function and the other for the new function ( ohno 1970 ) . alternatively , each gene could accumulate complementary degenerative mutations in either coding or regulatory regions , resulting in the loss of a subset of the pre - duplication gene activity . both copies will then be maintained by selection since both are needed to preserve the ancestral function ( the sub - functionalization model ) ( force et al . both models predict that , once duplicated genes occupy different functional niches , they may come under different selective regimes . paralogous gene regions responsible for redundant functions may experience similar selective pressures , if their overall activity affects fitness traits . on the other hand , selection acting on different functions may shape sequence evolution at functionally differentiated regions , and the mode and intensity of this selection may be different for each trait . as a result , duplicated genes would evolve in different modes and at different rates , with different functional elements dominating the evolution of each paralog . these ideas have primarily been tested using recently duplicated genes , but even old duplicates can share some functions . here , we analyze the bric brac ( bab ) locus of drosophila melanogaster , which contains the duplicated paralogs bab1 and bab2 to determine whether we can detect these patterns using intraspecific variation . bab1 and bab2 are located in a 148 kb continuous region of the genome , and their transcripts span ~57 and ~28 kb , respectively ( fig . 1 ) . the large size of the bab locus and the low levels of linkage disequilibrium in the region ( fig . 2 ) suggest that separate regulatory modules and coding regions could evolve independently . both genes function in the proximo - distal patterning of legs and antennae , the development of terminal filaments in the ovary , and the patterning of abdominal sensory organs and abdominal pigmentation ( couderc et al . 2002 ) . although bab1 and bab2 have overlapping and partially redundant roles in development ( kopp et al . 2002 ) , the maintenance of these duplicates in all of the currently sequenced drosophila genomes ( unpublished analysis ) suggests that they may have subtle differences in function.fig . the bab genes are shown in black , cg32334 , cg9205 , and the 5 region of trio in gray . the sites of two transposable element insertions in the d. melanogaster reference genome are marked with trianglesfig . the mean value of r ( a ) and d ( b ) was calculated for all polymorphisms separated by a given distance and combined into 10 bp bins the bab genomic region . the bab genes are shown in black , cg32334 , cg9205 , and the 5 region of trio in gray . the sites of two transposable element insertions in the d. melanogaster reference genome are marked with triangles linkage disequilibrium in the bab region . for each graph , the mean value of r ( a ) and d ( b ) was calculated for all polymorphisms separated by a given distance and combined into 10 bp bins many of the structures patterned by the bab genes are sexually dimorphic , including the gonad ( sahut - barnola et al . 1995 ) , the sex combs on the front legs of males ( godt et al . 1993 ; barmina and kopp 2007 ; randsholt and santamaria 2008 ) , ventral abdominal bristles ( kopp et al . 2000 ) , and the dorsal abdominal pigmentation pattern ( kopp et al . 2000 ; williams et al . 2008 ) , which suggests that sexual selection may have acted on the bab locus . furthermore , it has been demonstrated that sex combs are important for male mating success ( ng and kopp 2008 ) , ovaries are critical for reproduction and fecundity , and abdominal pigmentation plays a role in thermoregulation and desiccation resistance ( gibert et al . 1996 ; brisson et al . 2005 ) , suggesting that the bab1 and bab2 genes may experience selection on a variety of functions . the bab1 and bab2 genes have sequence conservation in two protein domains , btb / poz and babcd ( bric brac conserved domain ) ( couderc et al . 2002 ) . the btb ( broad , tramtrac , bab ) domain is shared by a large number of developmentally regulated genes and is involved in protein protein interactions ( zollman et al . 1994 ) , including bab1 homodimerization in vitro ( chen et al . the babcd is composed of a psq and at - hook domains that are both involved in dna binding , suggesting that the bab genes may act as transcriptional regulators ( reeves and nissen 1990 ; lehmann et al . 1998 ; couderc et al . 2002 ; both bab genes contain a single large intron ( 20 and 50 kb , respectively ) that is present in an evolutionarily conserved position ( couderc et al . 2002 ) . in both genes , this intron separates 5 exons , which contain the protein interaction domain ( btb ) from 3 exons that contain the dna binding region ( babcd ) ( fig . 1 ) . bab1 and bab2 have largely overlapping expression patterns , with bab1 present in a subset of bab2-expressing cells . in the ovary , bab1 is expressed exclusively in the terminal filaments , while bab2 is expressed strongly in the terminal filaments and more weakly in apical cells of the ovary ( couderc et al . flies with bab mutations that affect both paralogs show defects in terminal filament formation , apical cells , and basal stalk primordium , resulting in sterile females and ovaries with only a few rudimentary ovarioles , while mutations that affect a single bab gene result in weaker phenotypes ( godt and laski 1995 ; couderc et al . both duplicate genes also contribute to the patterning of distal antennae and legs during larval and pupal development ( godt et al . again , the strongest phenotypes result from bab mutations that affect both bab1 and bab2 , causing a complete fusion of the second through fifth tarsal segments , while mutations that affect only one of the genes result in intermediate phenotypes . in the abdomen , the bab genes play a central role is specifying sexually dimorphic pigmentation patterns ( kopp et al . bab mutations have a dominant effect resulting in wider pigmentation bands , with the strongest phenotype seen in the most posterior segments ( couderc et al . moreover , genetic variation at the bab locus is associated with intraspecific variation in the pigmentation of posterior abdominal segments in d. melanogaster females ( kopp et al . bab1 and bab2 are expressed in similar spatial patterns in the developing abdominal epidermis ( kopp et al . 2000 ; williams et al . 2008 ) , and artificial over - expression experiments show that both genes are capable of partially rescuing the bab mutant phenotypes ( bardot et al . 2002 ) . in all tissues , despite slight differences in bab expression , bab1 and bab2 mutations have very similar phenotypes . detailed functional analysis of the bab locus has revealed a number of distinct cis - regulatory elements ( cres ) ( williams et al . separate enhancers were identified for pupal abdominal epidermis ( large intron of bab1 ) , legs ( intergenic region between bab1 and bab2 ) , and oenocytes ( large intron of bab2 ) . surprisingly , only a single regulatory element was identified for each tissue that expresses both bab1 and bab2 , raising the possibility that both paralogs may be controlled by the same core cres . this does not rule out the existence of other , paralog - specific regulatory elements that modulate the expression of each gene in a more subtle way . if such modifier elements exist , the expression of each paralog could evolve independently and be subject to different selective regimes . in summary , the two bab genes have largely overlapping expression and developmental roles , yet they show evidence of distinct functional specificities . at the same time , their involvement in a variety of sex - specific processes suggests that these genes could experience many competing selective pressures . in principle , both paralogs could be dominated by similar selective pressures , reflecting their shared functions . alternatively , bab1 and bab2 could show different patterns of selection , suggesting that unique functions of the paralogs are shaping sequence evolution in the region . to distinguish between these modes of evolution we have resequenced the bab genomic region including the bab1 and bab2 genes and the flanking intergenic regions from 94 inbred strains extracted from a single natural population at the wolfskill orchard in winters , ca . the 35 wolfskill-1 ( w1 ) , 56 wolfskill-3 ( w3 ) , and 3 a1 lines were all collected from the same orchard but in separate years . eighty - three of the wolfskill lines were chosen at random , while the remaining lines were chosen for inclusion because of their light abdominal pigmentation pattern . all lines from the wolfskill collections were inbred by full - sib mating for a minimum of 20 generations , while the a1 lines were inbred for at least 10 generations by the same method . overlapping 1 kb amplicons were designed across the region ; successful amplicons were sequenced from both strands . base calls and polymorphisms were initially identified using phred and polyphred 6.11 ( ewing and green 1998 ; ewing et al . using consed , insertion / deletions ( indels ) were identified and polymorphisms were checked for accuracy ( gordon et al . although effort was made to obtain complete coverage , we were unable to sequence any of the strains for two regions that together cover approximately 5 kb . these regions are identified as repetitive by repeatmasker ( smit 19962004 ) , and each region contains a transposable element in the d. melanogaster reference genome sequence ( adams et al . 2000 ) . since transposable elements present in the reference annotation are rarely found in other strains at appreciable frequencies ( petrov et al . in preparation ) , we did not attempt to verify their presence in our lines . on average , we have sequence information from 90% of the lines for any given polymorphism . sliding window analysis was used to calculate population - genetic test statistics in 10 kb windows that were moved by 2 kb steps across the length of the bab region . theta values ( , w , and h ) , tajima s d , fu and li s d and fu s f were calculated using the compute implementation of libsequence library ( thornton 2003 ) and custom scripts , using the d. simulans genome sequence as an outgroup when appropriate ( tajima 1989 ; fu and li 1993 ; fay and wu 2000 ; thornton 2003 ; zeng et al . 2006 ) . kreitman ( mk ) tests ( mcdonald and kreitman 1991 ) were performed as described by begun et al . we sequenced the 148 kb bab region from 94 inbred strains of d. melanogaster collected from winters , ca . this region includes bab1 and bab2 in their entirety as well as two additional open reading frames , cg9205 and cg32334 , for which no information about expression or function is available ( fig . 1 ) . in this region , we identified 5566 single nucleotide polymorphisms ( snps ) , 5405 of which contained two alleles and 161 contained three alleles ( table 1 ) . we also identified 1211 short insertion / deletion ( indel ) polymorphisms , ranging in size from 1 to 526 bp . this is likely to be an underestimate of the number of indels because we have no information from the repeat regions ( fig . 1 ) and longer repeat variants from other regions were likely to result in a failed sequencing reaction . the mean indel length was 9.37 bp , and the most common length 4 bp . although indels are less frequent than snps , we find that indels tend to have a higher tajima s d statistic than snps , indicating that they are more likely to be present at intermediate frequencies ( table 1 ) . this result is unlikely to be caused by sequencing errors , since the removal of singletons ( snps present in a single line ) results in the same pattern ( data not shown).table 1summary statistics of sequence variation in the bab regionbpssingletonswdtajima s dentire region147959665513820.900.870.13snps553511890.750.710.18indels11201930.150.160.15coding8692292370.670.710.23utrs4572156290.680.640.225-utrs8572650.610.580.173-utrs3715130240.700.650.23introns7587033667460.890.840.19intergenic5882528595730.970.950.07number of base pairs represented in the samplenumber of polymorphisms identifiedthe number of polymorphisms that contain a single observation of the alternative allelewaterson s theta per 100 bpthe average pairwise diversity per 100 bp summary statistics of sequence variation in the bab region number of base pairs represented in the sample number of polymorphisms identified the number of polymorphisms that contain a single observation of the alternative allele waterson s theta per 100 bp the average pairwise diversity per 100 bp nucleotide diversity ( ) and estimates of the population mutation rate ( w ) were generally higher in non - coding than coding dna , suggesting that non - coding sequences are under less functional constraint ( table 1 ) . intronic , intergenic , and utr regions have similar values of tajima s d , which is consistent with genome - wide studies in d. melanogaster ( andolfatto 2005 ) . the bab region shows little linkage disequilibrium ( ld ) , with average correlation between polymorphisms ( r ) dropping off rapidly within 300 bps ( fig . d , a quantitative measure of ld normalized for allele frequency ( lewontin 1964 ) , has a slower decline and is constant after 1 kb ( fig . the short range of ld suggests that different regions of the bab locus can , in principle , evolve independently of one another . to investigate and compare the evolutionary forces acting on the bab paralogs , we used several tests to examine the allele frequency spectrum across the bab locus . tajima s d statistic compares two estimators of the population mutation parameter : , a measure of average pairwise differences between sequences that is strongly influenced by common alleles , and w , which weighs all polymorphisms equally and is thus more strongly influenced by rare alleles ( watterson 1975 ; tajima 1989 ) . sliding window analysis shows variable values of tajima s d statistic across the bab locus . a region centered on the non - coding 3 utr of bab2 transcript ( near the 110,000 bp mark ) has negative d values ( fig . 3b ) , indicating an excess of low frequency alleles that may be due to recent selection . the remainder of the bab locus has tajima s d values near zero , indicating that this region is evolving neutrally.fig . 3sliding window analysis of sequence variation in the bab region . a three estimates of including ( solid line ) , w ( dashed line ) , and h ( dotted line ) . d fu and li s d ( dashed line ) and f ( solid line ) statistics sliding window analysis of sequence variation in the bab region . a three estimates of including ( solid line ) , w ( dashed line ) , and h ( dotted line ) . d fu and li s d ( dashed line ) and f ( solid line ) statistics we also compared high - frequency derived and intermediate - frequency alleles using fay and wu s h statistic ( fay and wu 2000 ; zeng et al . 2006 ) . a low value of h indicates a higher than expected number of derived alleles , making it a powerful test for detecting positive selection and the initial stages of balancing selection ( zeng et al . we used d. simulans genome sequence as an outgroup to polarize snp alleles in d. melanogaster . similar to tajima s d , the strongest negative values of fay and wu s h are found near the 3 end of the bab2 transcript , with no comparable signature in the paralogous bab1 region ( fig . this pattern provides additional evidence for directional selection acting on the region near the 3 end of bab2 . in addition , the h statistic shows a region in the large intron of bab1 with strongly negative values , suggesting an additional region under selection , which was not detected with the d statistic . fu and li s d and fu s f statistics compare the frequencies of derived and ancestral alleles to detect deviations from the neutral expectation ( fu and li 1993 ; fu 1997 ) . negative values of d and f indicate an excess of derived mutations ( an excess of external branches in the gene tree ) , while positive values show a deficiency of derived alleles ( excess of internal branches ) . a reduction of diversity at a neutral locus due to selection against linked deleterious mutations ( charlesworth et al . we find negative values of d and f in the large introns of both bab1 and bab2 , with peak values in the 10 kb windows centered near the 28,000 and 122,000 bp marks ( fig . these regions overlap with the locations of repetitive sequences and transposable element insertions in the reference genome sequence ( fig . 1 ) . repetitive sequences are often a source of frequently occurring deleterious mutations , and the low values of d and f may arise when these mutations are removed by background selection . our sequencing sample is drawn primarily from two collections , wolfskill 1 ( w1 ) and wolfskill 3 ( w3 ) , which were collected from the same location but in separate years . in general , sequence variation in the w1 and w3 samples does not differ significantly across the bab region ( table 2 ) . however , the strong negative values of tajima s d near the 3 end of bab2 are caused primarily by the w3 sample ( fig . sliding window analysis of population differentiation ( fst ) between w1 and w3 reveals elevated levels of differentiation in the same region ( fig . further collections would be necessary to understand if these differences are the result of seasonal or yearly differences in selection for the abiotic environment or can be explained by some other process.table 2summary statistics comparing the w1 and w3 sequence samples across the bab regionbpssingletonswdtajima s dw1147959532313470.900.890.04w3147959584513840.880.870.06number of base pairs represented in the samplenumber of polymorphisms identifiedthe number of polymorphisms that contain a single observation of the alternative allelewaterson s theta per 100 bpthe average pairwise diversity per 100 bpfig . all analyses are in 10 kb windows offset by 2 kb . a tajima s d statistic showing pooled collections ( solid line ) , w1 alone ( dashed line ) and w3 alone ( dotted line ) . b population differentiation ( fst ) between the w1 and w3 samples summary statistics comparing the w1 and w3 sequence samples across the bab region number of base pairs represented in the sample number of polymorphisms identified the number of polymorphisms that contain a single observation of the alternative allele waterson s theta per 100 bp the average pairwise diversity per 100 bp comparison of the w1 and w3 collections . all analyses are in 10 kb windows offset by 2 kb . a tajima s d statistic showing pooled collections ( solid line ) , w1 alone ( dashed line ) and w3 alone ( dotted line ) . b population differentiation ( fst ) between the w1 and w3 samples to investigate the role of selection in the evolution of bab1 and bab2 coding sequences , we used the mcdonald kreitman ( mk ) test , which compares the ratio of synonymous ( s ) and non - synonymous ( ns ) nucleotide substitutions between and within species ( mcdonald and kreitman 1991 ) . to identify nucleotide substitutions that occurred specifically in the d. melanogaster lineage , we used the genome sequences of d. simulans and d. yakuba to polarize the direction of change . we then compared the fixed substitutions that occurred on the d. melanogaster evolutionary lineage to polymorphisms segregating within the d. melanogaster population ( table 3 ) . we found a significant skew in bab1 , such that it has a lack of fixed ns changes and/or an excess of polymorphic ns substitutions ( two tailed fisher s exact test ; p < 0.05 ) . no such pattern is seen in the bab2 gene or in the other predicted genes in the bab region ( table 3 ) . the ns changes in bab1 are distributed throughout the transcript , although none of these changes are found in the btb ( protein interaction ) or babcd ( dna binding ) functional domains . this pattern could be due to functional constraint on the bab1 coding region or balancing selection maintaining multiple alleles of bab1 . to differentiate between these possibilities , we looked at how many of the polymorphic sites were represented by a single individual ( singletons ) , which are more likely to be deleterious variants ( table 3 ) . a large number of the ns polymorphisms in bab1 are singletons ( seven of 15 ) suggesting that bab1 polymorphisms are under strong purifying selection preventing their fixation.table 3mcdonald kreitman test for genes in the bab regiongenecodonsreplacement ( ns)silent ( s)p - valuepolymorphicfixedpolymorphicfixedcg9205199001071bab179115 ( 7)156 ( 3)280.034*cg3233413551601bab282813 ( 4)679 ( 9)300.784p values are the results of a two - tailed fisher s exact test kreitman test for genes in the bab region p values are the results of a two - tailed fisher s exact test . values below p = 0.05 are indicated if the bab genes are functionally redundant , it is possible that deleterious alleles in one gene are compensated by a functional allele of the other gene . we found no correlation between the number of low frequency ( likely deleterious ) alleles in the bab1 and bab2 coding regions ( p > 0.05 for synonymous , non - synonymous , and total changes ) , nor do we find long - range ld between polymorphisms in the bab1 and bab2 transcripts . duplicate genes persist in the genome due to the acquisition of new functions or the subdivision of the ancestral role . over time , paralogous proteins may acquire subtle functional changes or gain entirely different biological activities ( hirth et al . alternatively , the proteins may share similar specificity while gene expression patterns diverge due to cis - regulatory changes , leading to the acquisition of different functional roles ( greer et al . the two mechanisms are not mutually exclusive , and both can operate on the same pair of paralogs . at the bab locus , the two duplicated genes have similar but non - identical expression patterns ( couderc et al . 2002 ) despite sharing at least some cres ( williams et al . this suggests that some functions of these paralogs may experience shared constraints , while others may evolve independently . numerous studies have shown that duplicated genes diverge rapidly in expression ( gu et al . 2002 ; makova and li 2003 ; gu et al . 2005 ) , and that the rate of expression divergence is highest immediately after gene duplication and slows down over time ( jordan et al . this pattern is consistent with either directional selection ( neo - functionalization model ) or the relaxation of purifying selection ( sub - functionalization model ) acting during the early stages of gene divergence , and the relative contributions of these forces continue to be debated ( yu et al . 2003 ; castillo - davis et al . generally , paralogous genes are more likely to lose ancestral expression domains than to acquire new ones , indicating that sub - functionalization is probably more common than neo - functionalization ( oakley et al . both models predict that , once duplicate genes acquire non - identical functions , they may come under different selective regimes . in this study , we used a population genetic approach to assess the evolutionary forces acting on the bab paralogs . first , a region near the 3 end of bab2 ( which includes bab2 3 exons , introns , and intergenic region ) appears to experience directional selection ( fig . furthermore , selection in this region may vary over time , as indicated by the difference between population samples collected in different years . surprisingly , no cres have been found in this region ( williams et al . 2008 ) , although it remains possible that it contains regulatory elements that modulate transcriptional activity but can not function independently in transgenic assays . future analysis is required to determine whether the coding or non - coding dna is driving this signature of selection . the second region that appears to be under selection is located in the large intron of bab1 ( fig . this region contains the cre that controls female specific expression of bab in the abdominal epidermis ( williams et al . 2008 ) , suggesting that selection may be acting on the sexually dimorphic pigmentation of d. melanogaster . furthermore , a recent study found that this same region was differentiated between northern and southern d. melanogaster populations in north america and australia ( turner et al . one possible explanation is that the two proteins have somewhat different functional activities despite being expressed in largely overlapping patterns . given our data , it seems that the bab homolgs are most likely maintained due to sub - functionalization . previous work on the bab locus has shown that both bab genes are expressed in the same tissues during development ( couderc et al . we have found that the coding and non - coding dna show differences in sequence evolution . thus , within the ancestral functions it is likely that the bab genes have divided their roles such that both are indispensable and thus maintained . several recent studies have used comparative genomic approaches to examine the role of selection in the evolution of duplicate genes . such analyses are based on variation in the rate of expression divergence over time ( jordan et al . 2004 ; gu et al . 2005 ) , across phylogenetic lineages ( shiu et al . 2006 ) , or on the correlation between the rate of expression and sequence divergence ( yu et al . 2003 ; castillo - davis et al . however , these long - term evolutionary patterns are consistent with either selective or neutral explanations ( castillo - davis et al . 2004 ; kondrashov and kondrashov 2006 ) , and are best suited for detecting selection at the genome - wide level rather than individual loci . a population - genetic approach brings an alternative perspective to this question , since it is explicitly designed to test for selection acting on specific dna sequences . as genome - wide analyses of intraspecific variation become possible ( begun et al . 2007 ) , an integration of population - genetic and comparative - genomic approaches will shed new light on the relative importance of positive selection and neutral changes in the maintenance and evolution of paralogous genes .

genes with overlapping expression and function may gradually diverge despite retaining some common functions . to test whether such genes show distinct patterns of molecular evolution within species , we examined sequence variation at the bric brac ( bab ) locus of drosophila melanogaster . this locus is composed of two anciently duplicated paralogs , bab1 and bab2 , which are involved in patterning the adult abdomen , legs , and ovaries . we have sequenced the 148 kb genomic region spanning the bab1 and bab2 genes from 94 inbred lines of d. melanogaster sampled from a single location . two non - coding regions , one in each paralog , appear to be under selection . the strongest evidence of directional selection is found in a region of bab2 that has no known functional role . the other region is located in the bab1 paralog and is known to contain a cis - regulatory element that controls sex - specific abdominal pigmentation . the coding region of bab1 appears to be under stronger functional constraint than the bab2 coding sequences . thus , the two paralogs are evolving under different selective regimes in the same natural population , illuminating the different evolutionary trajectories of partially redundant duplicate genes .

Introduction Materials and Methods Results Discussion

thus , both genes are maintained by natural selection , one for the ancestral function and the other for the new function ( ohno 1970 ) . alternatively , each gene could accumulate complementary degenerative mutations in either coding or regulatory regions , resulting in the loss of a subset of the pre - duplication gene activity . both models predict that , once duplicated genes occupy different functional niches , they may come under different selective regimes . on the other hand , selection acting on different functions may shape sequence evolution at functionally differentiated regions , and the mode and intensity of this selection may be different for each trait . here , we analyze the bric brac ( bab ) locus of drosophila melanogaster , which contains the duplicated paralogs bab1 and bab2 to determine whether we can detect these patterns using intraspecific variation . bab1 and bab2 are located in a 148 kb continuous region of the genome , and their transcripts span ~57 and ~28 kb , respectively ( fig . the large size of the bab locus and the low levels of linkage disequilibrium in the region ( fig . both genes function in the proximo - distal patterning of legs and antennae , the development of terminal filaments in the ovary , and the patterning of abdominal sensory organs and abdominal pigmentation ( couderc et al . although bab1 and bab2 have overlapping and partially redundant roles in development ( kopp et al . the bab genes are shown in black , cg32334 , cg9205 , and the 5 region of trio in gray . the sites of two transposable element insertions in the d. melanogaster reference genome are marked with trianglesfig . the bab genes are shown in black , cg32334 , cg9205 , and the 5 region of trio in gray . the sites of two transposable element insertions in the d. melanogaster reference genome are marked with triangles linkage disequilibrium in the bab region . 2000 ) , and the dorsal abdominal pigmentation pattern ( kopp et al . furthermore , it has been demonstrated that sex combs are important for male mating success ( ng and kopp 2008 ) , ovaries are critical for reproduction and fecundity , and abdominal pigmentation plays a role in thermoregulation and desiccation resistance ( gibert et al . 2005 ) , suggesting that the bab1 and bab2 genes may experience selection on a variety of functions . the bab1 and bab2 genes have sequence conservation in two protein domains , btb / poz and babcd ( bric brac conserved domain ) ( couderc et al . the btb ( broad , tramtrac , bab ) domain is shared by a large number of developmentally regulated genes and is involved in protein protein interactions ( zollman et al . the babcd is composed of a psq and at - hook domains that are both involved in dna binding , suggesting that the bab genes may act as transcriptional regulators ( reeves and nissen 1990 ; lehmann et al . 2002 ; both bab genes contain a single large intron ( 20 and 50 kb , respectively ) that is present in an evolutionarily conserved position ( couderc et al . bab1 and bab2 have largely overlapping expression patterns , with bab1 present in a subset of bab2-expressing cells . in the ovary , bab1 is expressed exclusively in the terminal filaments , while bab2 is expressed strongly in the terminal filaments and more weakly in apical cells of the ovary ( couderc et al . flies with bab mutations that affect both paralogs show defects in terminal filament formation , apical cells , and basal stalk primordium , resulting in sterile females and ovaries with only a few rudimentary ovarioles , while mutations that affect a single bab gene result in weaker phenotypes ( godt and laski 1995 ; couderc et al . again , the strongest phenotypes result from bab mutations that affect both bab1 and bab2 , causing a complete fusion of the second through fifth tarsal segments , while mutations that affect only one of the genes result in intermediate phenotypes . in the abdomen , the bab genes play a central role is specifying sexually dimorphic pigmentation patterns ( kopp et al . bab mutations have a dominant effect resulting in wider pigmentation bands , with the strongest phenotype seen in the most posterior segments ( couderc et al . moreover , genetic variation at the bab locus is associated with intraspecific variation in the pigmentation of posterior abdominal segments in d. melanogaster females ( kopp et al . bab1 and bab2 are expressed in similar spatial patterns in the developing abdominal epidermis ( kopp et al . 2008 ) , and artificial over - expression experiments show that both genes are capable of partially rescuing the bab mutant phenotypes ( bardot et al . in all tissues , despite slight differences in bab expression , bab1 and bab2 mutations have very similar phenotypes . detailed functional analysis of the bab locus has revealed a number of distinct cis - regulatory elements ( cres ) ( williams et al . separate enhancers were identified for pupal abdominal epidermis ( large intron of bab1 ) , legs ( intergenic region between bab1 and bab2 ) , and oenocytes ( large intron of bab2 ) . surprisingly , only a single regulatory element was identified for each tissue that expresses both bab1 and bab2 , raising the possibility that both paralogs may be controlled by the same core cres . this does not rule out the existence of other , paralog - specific regulatory elements that modulate the expression of each gene in a more subtle way . if such modifier elements exist , the expression of each paralog could evolve independently and be subject to different selective regimes . in summary , the two bab genes have largely overlapping expression and developmental roles , yet they show evidence of distinct functional specificities . at the same time , their involvement in a variety of sex - specific processes suggests that these genes could experience many competing selective pressures . alternatively , bab1 and bab2 could show different patterns of selection , suggesting that unique functions of the paralogs are shaping sequence evolution in the region . to distinguish between these modes of evolution we have resequenced the bab genomic region including the bab1 and bab2 genes and the flanking intergenic regions from 94 inbred strains extracted from a single natural population at the wolfskill orchard in winters , ca . the 35 wolfskill-1 ( w1 ) , 56 wolfskill-3 ( w3 ) , and 3 a1 lines were all collected from the same orchard but in separate years . all lines from the wolfskill collections were inbred by full - sib mating for a minimum of 20 generations , while the a1 lines were inbred for at least 10 generations by the same method . although effort was made to obtain complete coverage , we were unable to sequence any of the strains for two regions that together cover approximately 5 kb . these regions are identified as repetitive by repeatmasker ( smit 19962004 ) , and each region contains a transposable element in the d. melanogaster reference genome sequence ( adams et al . since transposable elements present in the reference annotation are rarely found in other strains at appreciable frequencies ( petrov et al . on average , we have sequence information from 90% of the lines for any given polymorphism . theta values ( , w , and h ) , tajima s d , fu and li s d and fu s f were calculated using the compute implementation of libsequence library ( thornton 2003 ) and custom scripts , using the d. simulans genome sequence as an outgroup when appropriate ( tajima 1989 ; fu and li 1993 ; fay and wu 2000 ; thornton 2003 ; zeng et al . we sequenced the 148 kb bab region from 94 inbred strains of d. melanogaster collected from winters , ca . this region includes bab1 and bab2 in their entirety as well as two additional open reading frames , cg9205 and cg32334 , for which no information about expression or function is available ( fig . in this region , we identified 5566 single nucleotide polymorphisms ( snps ) , 5405 of which contained two alleles and 161 contained three alleles ( table 1 ) . this is likely to be an underestimate of the number of indels because we have no information from the repeat regions ( fig . 1 ) and longer repeat variants from other regions were likely to result in a failed sequencing reaction . although indels are less frequent than snps , we find that indels tend to have a higher tajima s d statistic than snps , indicating that they are more likely to be present at intermediate frequencies ( table 1 ) . this result is unlikely to be caused by sequencing errors , since the removal of singletons ( snps present in a single line ) results in the same pattern ( data not shown).table 1summary statistics of sequence variation in the bab regionbpssingletonswdtajima s dentire region147959665513820.900.870.13snps553511890.750.710.18indels11201930.150.160.15coding8692292370.670.710.23utrs4572156290.680.640.225-utrs8572650.610.580.173-utrs3715130240.700.650.23introns7587033667460.890.840.19intergenic5882528595730.970.950.07number of base pairs represented in the samplenumber of polymorphisms identifiedthe number of polymorphisms that contain a single observation of the alternative allelewaterson s theta per 100 bpthe average pairwise diversity per 100 bp summary statistics of sequence variation in the bab region number of base pairs represented in the sample number of polymorphisms identified the number of polymorphisms that contain a single observation of the alternative allele waterson s theta per 100 bp the average pairwise diversity per 100 bp nucleotide diversity ( ) and estimates of the population mutation rate ( w ) were generally higher in non - coding than coding dna , suggesting that non - coding sequences are under less functional constraint ( table 1 ) . intronic , intergenic , and utr regions have similar values of tajima s d , which is consistent with genome - wide studies in d. melanogaster ( andolfatto 2005 ) . to investigate and compare the evolutionary forces acting on the bab paralogs , we used several tests to examine the allele frequency spectrum across the bab locus . tajima s d statistic compares two estimators of the population mutation parameter : , a measure of average pairwise differences between sequences that is strongly influenced by common alleles , and w , which weighs all polymorphisms equally and is thus more strongly influenced by rare alleles ( watterson 1975 ; tajima 1989 ) . a region centered on the non - coding 3 utr of bab2 transcript ( near the 110,000 bp mark ) has negative d values ( fig . 3sliding window analysis of sequence variation in the bab region . d fu and li s d ( dashed line ) and f ( solid line ) statistics sliding window analysis of sequence variation in the bab region . a three estimates of including ( solid line ) , w ( dashed line ) , and h ( dotted line ) . we used d. simulans genome sequence as an outgroup to polarize snp alleles in d. melanogaster . similar to tajima s d , the strongest negative values of fay and wu s h are found near the 3 end of the bab2 transcript , with no comparable signature in the paralogous bab1 region ( fig . this pattern provides additional evidence for directional selection acting on the region near the 3 end of bab2 . in addition , the h statistic shows a region in the large intron of bab1 with strongly negative values , suggesting an additional region under selection , which was not detected with the d statistic . negative values of d and f indicate an excess of derived mutations ( an excess of external branches in the gene tree ) , while positive values show a deficiency of derived alleles ( excess of internal branches ) . we find negative values of d and f in the large introns of both bab1 and bab2 , with peak values in the 10 kb windows centered near the 28,000 and 122,000 bp marks ( fig . repetitive sequences are often a source of frequently occurring deleterious mutations , and the low values of d and f may arise when these mutations are removed by background selection . our sequencing sample is drawn primarily from two collections , wolfskill 1 ( w1 ) and wolfskill 3 ( w3 ) , which were collected from the same location but in separate years . in general , sequence variation in the w1 and w3 samples does not differ significantly across the bab region ( table 2 ) . however , the strong negative values of tajima s d near the 3 end of bab2 are caused primarily by the w3 sample ( fig . sliding window analysis of population differentiation ( fst ) between w1 and w3 reveals elevated levels of differentiation in the same region ( fig . further collections would be necessary to understand if these differences are the result of seasonal or yearly differences in selection for the abiotic environment or can be explained by some other process.table 2summary statistics comparing the w1 and w3 sequence samples across the bab regionbpssingletonswdtajima s dw1147959532313470.900.890.04w3147959584513840.880.870.06number of base pairs represented in the samplenumber of polymorphisms identifiedthe number of polymorphisms that contain a single observation of the alternative allelewaterson s theta per 100 bpthe average pairwise diversity per 100 bpfig . b population differentiation ( fst ) between the w1 and w3 samples summary statistics comparing the w1 and w3 sequence samples across the bab region number of base pairs represented in the sample number of polymorphisms identified the number of polymorphisms that contain a single observation of the alternative allele waterson s theta per 100 bp the average pairwise diversity per 100 bp comparison of the w1 and w3 collections . b population differentiation ( fst ) between the w1 and w3 samples to investigate the role of selection in the evolution of bab1 and bab2 coding sequences , we used the mcdonald kreitman ( mk ) test , which compares the ratio of synonymous ( s ) and non - synonymous ( ns ) nucleotide substitutions between and within species ( mcdonald and kreitman 1991 ) . to identify nucleotide substitutions that occurred specifically in the d. melanogaster lineage , we used the genome sequences of d. simulans and d. yakuba to polarize the direction of change . we then compared the fixed substitutions that occurred on the d. melanogaster evolutionary lineage to polymorphisms segregating within the d. melanogaster population ( table 3 ) . no such pattern is seen in the bab2 gene or in the other predicted genes in the bab region ( table 3 ) . the ns changes in bab1 are distributed throughout the transcript , although none of these changes are found in the btb ( protein interaction ) or babcd ( dna binding ) functional domains . this pattern could be due to functional constraint on the bab1 coding region or balancing selection maintaining multiple alleles of bab1 . to differentiate between these possibilities , we looked at how many of the polymorphic sites were represented by a single individual ( singletons ) , which are more likely to be deleterious variants ( table 3 ) . a large number of the ns polymorphisms in bab1 are singletons ( seven of 15 ) suggesting that bab1 polymorphisms are under strong purifying selection preventing their fixation.table 3mcdonald kreitman test for genes in the bab regiongenecodonsreplacement ( ns)silent ( s)p - valuepolymorphicfixedpolymorphicfixedcg9205199001071bab179115 ( 7)156 ( 3)280.034*cg3233413551601bab282813 ( 4)679 ( 9)300.784p values are the results of a two - tailed fisher s exact test kreitman test for genes in the bab region p values are the results of a two - tailed fisher s exact test . we found no correlation between the number of low frequency ( likely deleterious ) alleles in the bab1 and bab2 coding regions ( p > 0.05 for synonymous , non - synonymous , and total changes ) , nor do we find long - range ld between polymorphisms in the bab1 and bab2 transcripts . duplicate genes persist in the genome due to the acquisition of new functions or the subdivision of the ancestral role . alternatively , the proteins may share similar specificity while gene expression patterns diverge due to cis - regulatory changes , leading to the acquisition of different functional roles ( greer et al . the two mechanisms are not mutually exclusive , and both can operate on the same pair of paralogs . at the bab locus , the two duplicated genes have similar but non - identical expression patterns ( couderc et al . 2005 ) , and that the rate of expression divergence is highest immediately after gene duplication and slows down over time ( jordan et al . this pattern is consistent with either directional selection ( neo - functionalization model ) or the relaxation of purifying selection ( sub - functionalization model ) acting during the early stages of gene divergence , and the relative contributions of these forces continue to be debated ( yu et al . both models predict that , once duplicate genes acquire non - identical functions , they may come under different selective regimes . in this study , we used a population genetic approach to assess the evolutionary forces acting on the bab paralogs . first , a region near the 3 end of bab2 ( which includes bab2 3 exons , introns , and intergenic region ) appears to experience directional selection ( fig . future analysis is required to determine whether the coding or non - coding dna is driving this signature of selection . the second region that appears to be under selection is located in the large intron of bab1 ( fig . this region contains the cre that controls female specific expression of bab in the abdominal epidermis ( williams et al . 2008 ) , suggesting that selection may be acting on the sexually dimorphic pigmentation of d. melanogaster . one possible explanation is that the two proteins have somewhat different functional activities despite being expressed in largely overlapping patterns . previous work on the bab locus has shown that both bab genes are expressed in the same tissues during development ( couderc et al . we have found that the coding and non - coding dna show differences in sequence evolution . thus , within the ancestral functions it is likely that the bab genes have divided their roles such that both are indispensable and thus maintained . several recent studies have used comparative genomic approaches to examine the role of selection in the evolution of duplicate genes . 2006 ) , or on the correlation between the rate of expression and sequence divergence ( yu et al . 2004 ; kondrashov and kondrashov 2006 ) , and are best suited for detecting selection at the genome - wide level rather than individual loci . a population - genetic approach brings an alternative perspective to this question , since it is explicitly designed to test for selection acting on specific dna sequences .

preeclampsia is defined as a new - onset hypertensive disorder with elevated urine protein , which occurs in pregnant women after 20 weeks gestation . severe preeclampsia , a liver disease that is unique to pregnancy , is often complicated by severe liver damage . dani et al . detected microvesicular fat droplets in the liver of patients with preeclampsia . this indicates that preeclampsia and acute fatty liver in pregnancy , which is another liver disease that is unique to pregnancy , belong in the same disease spectrum . dysregulation of fatty acid oxidative metabolism may be common to all these liver diseases that are unique to pregnancy . plasma free fatty acid ( ffa ) and triglyceride ( tg ) levels increase in patients with preeclampsia . this may be a result of a decrease in the ffa oxidation - related enzyme long - chain 3-hydroxyacyl - coa dehydrogenase ( lchad ) mrna expression and a reduced capacity for ffa oxidation in preeclampsia patients . other dysregulated lipid metabolism may be involved in preeclampsia , such as the increased serum apolipoprotein c - iii ( apoc3 ) lipid transport protein . in addition , derzsy et al . found that complement activation and c - reactive protein levels in patients with preeclampsia were higher than those in normal pregnancy , suggesting that activation of the inflammatory response is associated with preeclampsia . antiphospholipid syndrome ( aps ) is also associated with early - onset preeclampsia , and abnormal lipid metabolism and inflammatory activation also exist in aps patients . therefore , maternal underlying diseases that are presented in prepregnancy , such as hyperlipidemia and aps , and adverse environmental factors during pregnancy , such as inflammation , are all related to preeclampsia . poon et al . detected different placental hemodynamics , pregnancy - associated plasma protein - a and abnormal placental growth factors between patients with early - onset and late - onset preeclampsia as well as in healthy pregnant women in the first trimester . our previous study found that enzyme protein expression related to placental ffa oxidation in severe preeclampsia patients with an onset time before 32 weeks gestation , especially in patients with liver injury , was dramatically decreased compared with normal pregnancy , while there were no significant differences in severe preeclampsia patients with an onset time after 32 weeks gestation . in early- and mid - onset preeclampsia - like mouse models , lchad protein expression was significantly reduced in the placenta , but the late - onset model showed no significant difference from controls , suggesting that different onset times of preeclampsia may result in different lipid metabolism . according to different pathogenesis hypotheses , various animal models of preeclampsia have been established , such as the reduced uterine perfusion pressure model , tumor necrosis factor--injected model , nitric oxide synthase inhibitor - injected model , endotoxin - injected model and inflammatory factor - injected model . our previous study found that serum ffa increased in wild type or apoc3 knock - in transgenic preeclampsia - like mouse models induced by nw - nitro - l - arginine - methyl ester ( l - name ) or 2 glycoprotein i ( gpi ) , and hepatic and placental fatty infiltration were observed in these models . however , serum ffa , hepatic and placental fatty infiltration in the lipopolysaccharide ( lps ) group were not significantly different from control . carnitine palmitoyltransferase 1 , carnitine palmitoyltransferase 2 and lchad protein expression were abnormal in l - name or gpi preeclampsia - like models , and all these enzymes are localized in mitochondria . trophoblastic mitochondria are an important place for long - chain fatty acid oxidation during pregnancy , and abnormal long - chain fatty acid oxidation may induce mitochondrial damage in trophoblast cells . in addition , a previous study reported that lps can inhibit bcl-2 expression and promote bax and caspase-8 expression to induce apoptosis in trophoblast cells via the mitochondrial pathway . the liver and placenta are important organs for ffa -oxidation during pregnancy , and complex links may exist between them in different preeclampsia - like models . the present study used l - name and lps injections , knock - in mice with the human apoc3 gene as an adverse genetic background , and a basic maternal disease model with aps to create different pathogenic preeclampsia - like mouse models . all models were created at the preimplantation ( pi ) , early- , mid- and late - gestation ( eg , mg , lg ) stages to study the relationship between lipid metabolism and liver injury in different models that are created at different times . in addition , trophoblastic mitochondrial damage as a possible common pathway of different preeclampsia - like mouse models was investigated . animal experiments were approved by the animal care committee and medical ethics committee of peking university , and all procedures were conducted in strict accordance with the guidelines of the principles of laboratory animal care , published by the nih . randomly - selected 810-week - old virgin female and 1014-week - old male spf c57bl/6j wild type mice and apoc3 knock - in transgenic mice were purchased from the department of laboratory animal science of peking university health science center and mated . the c57bl/6j mice were randomly assigned to one of six groups : the l - name ( ln ) group ( injected with l - name ) , lps group , gpi group , and their normal saline - injected control groups ( the ln - c , lps - c , and gpi - c groups , respectively ) . the apoc3 knock - in mice were randomly divided into the apo - ln group and a normal saline - injected control group , the apo - c group . mice in the ln and apo - ln groups received daily subcutaneous injections of l - name at a dose of 50 mgkgd . mice in the lps group received a single intraperitoneal injection of ultra - low - dose lps ( 1 g / kg ) . the ln , lps and apo - ln groups and their control groups were further subdivided depending on when the first injection was administered : pi subgroup , day 3 of gestation ; eg subgroup , day 7 of gestation ; mg subgroup , day 11 of gestation ; and lg subgroup , day 16 of gestation . two and 3 weeks before mating , we divided another 20 c57bl/6j mice into two groups , and subcutaneously injected them with human gpi dissolved in incomplete freund 's adjuvant ( sigma , usa ; 0.1 ml ) or physiological saline ( 0.1 ml ) to create the gpi group and its control group , respectively . the coda noninvasive tail - cuff acquisition system ( kent scientific corporation , usa ) was used to measure the mean arterial pressure ( map ) every 2 days , starting from day 2 after mating . mice were placed in standard metabolic cages on the 17 day of gestation , and 24-h urine samples were collected from them for the measurement of the urine protein level , using a protein assay kit ( bio - rad protein assay kit i , usa ) . on the 18 day of pregnancy , the pregnant mice were sacrificed using an injection of 10% chloral hydrate ( 3 ml / kg ) . blood samples were immediately collected from the retro - orbital plexus and centrifuged ; the supernatant was stored at 80c . serum tg concentration and total cholesterol ( tc ) were measured using a blood lipid chemical assay kit ( wako chemicals , japan ) . the maternal liver was embedded in paraffin sliced into 5-m sections , which underwent routine hematoxylin and eosin staining , and observed and photographed under an optical microscope ( nikon , japan ) . the placenta was sliced into 1-mm thick sections and fixed with 3.0% glutaraldehyde at 4c . after dehydration using a series of graded alcohol concentrations , the placenta was embedded in epon812 epoxy , and sliced ultrathin into 50100 nm sections , double - stained with uranyl acetate and lead citrate , and observed and photographed using transmission electron microscopy ( jem1200ex , japan ) . a student 's t - test was used for comparing two groups , and one - way anova followed by the least - significant difference post - hoc test was used for differences between multiple groups . animal experiments were approved by the animal care committee and medical ethics committee of peking university , and all procedures were conducted in strict accordance with the guidelines of the principles of laboratory animal care , published by the nih . randomly - selected 810-week - old virgin female and 1014-week - old male spf c57bl/6j wild type mice and apoc3 knock - in transgenic mice were purchased from the department of laboratory animal science of peking university health science center and mated . the c57bl/6j mice were randomly assigned to one of six groups : the l - name ( ln ) group ( injected with l - name ) , lps group , gpi group , and their normal saline - injected control groups ( the ln - c , lps - c , and gpi - c groups , respectively ) . the apoc3 knock - in mice were randomly divided into the apo - ln group and a normal saline - injected control group , the apo - c group . mice in the ln and apo - ln groups received daily subcutaneous injections of l - name at a dose of 50 mgkgd . mice in the lps group received a single intraperitoneal injection of ultra - low - dose lps ( 1 g / kg ) . the ln , lps and apo - ln groups and their control groups were further subdivided depending on when the first injection was administered : pi subgroup , day 3 of gestation ; eg subgroup , day 7 of gestation ; mg subgroup , day 11 of gestation ; and lg subgroup , day 16 of gestation . two and 3 weeks before mating , we divided another 20 c57bl/6j mice into two groups , and subcutaneously injected them with human gpi dissolved in incomplete freund 's adjuvant ( sigma , usa ; 0.1 ml ) or physiological saline ( 0.1 ml ) to create the gpi group and its control group , respectively . the coda noninvasive tail - cuff acquisition system ( kent scientific corporation , usa ) was used to measure the mean arterial pressure ( map ) every 2 days , starting from day 2 after mating . mice were placed in standard metabolic cages on the 17 day of gestation , and 24-h urine samples were collected from them for the measurement of the urine protein level , using a protein assay kit ( bio - rad protein assay kit i , usa ) . on the 18 day of pregnancy , the pregnant mice were sacrificed using an injection of 10% chloral hydrate ( 3 ml / kg ) . blood samples were immediately collected from the retro - orbital plexus and centrifuged ; the supernatant was stored at 80c . serum tg concentration and total cholesterol ( tc ) were measured using a blood lipid chemical assay kit ( wako chemicals , japan ) . the maternal liver was embedded in paraffin sliced into 5-m sections , which underwent routine hematoxylin and eosin staining , and observed and photographed under an optical microscope ( nikon , japan ) . the placenta was sliced into 1-mm thick sections and fixed with 3.0% glutaraldehyde at 4c . after dehydration using a series of graded alcohol concentrations , the placenta was embedded in epon812 epoxy , and sliced ultrathin into 50100 nm sections , double - stained with uranyl acetate and lead citrate , and observed and photographed using transmission electron microscopy ( jem1200ex , japan ) . a student 's t - test was used for comparing two groups , and one - way anova followed by the least - significant difference post - hoc test was used for differences between multiple groups . within each experimental group , map was significantly higher in every experimental subgroup than in the corresponding control subgroup ( p < 0.05 ) . moreover , the earlier the injection began , the higher was the map ( p < 0.05 ) . among the experimental groups , map in the apo - ln subgroups was higher than that of the corresponding subgroups in the other three experimental groups , but only the map in the apo - ln - mp and apo - ln - lp subgroups was significantly different ( p < 0.05 ) . map was also significantly higher in the apo - c group than in other control groups that were established using wild type mice ( p < 0.05 ) . the map level in the gpi group was similar to that in the ln - pi and lps - pi subgroups . there was no significant difference in map between the gpi - c , ln - c and lps - c groups [ figure 1a ] . * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , compared with the corresponding l - name subgroup ; p < 0.05 , compared with the corresponding lipopolysaccharide subgroup . pi : preimplantation ; ep : early pregnancy ; mp : mid - pregnancy ; lp : late pregnancy . every experimental subgroup showed significantly higher 24-h urine protein concentrations than their corresponding control subgroups at 18 day of pregnancy ( p < 0.05 ) . moreover , the earlier the injection began , the more the urine protein increased ( p < 0.05 ) . in the ln , lps and apo - ln experimental groups , the pi , ep and mp subgroups showed significantly higher urine protein levels than those in lp subgroups ( p < 0.05 ) . the urine protein level in the apo - ln group was higher than those in the other experimental groups , but only that in the apo - ln - lp subgroup showed significance ( p < 0.05 ) . the urine protein level in the apo - c group was not significantly different from that in the control groups of wild type mice . the urine protein level in the gpi group was similar to that in the ln - pi and lps - pi subgroups , and the difference between the gpi - c , ln - c and lps - c groups was not significant [ figure 1b ] . within each experimental group , pi , ep and mp subgroups placental and fetal weight was less than that of the corresponding control subgroup for ln , lps and apo - ln ( p < 0.05 ) . ln - lp placental and fetal weight were not significantly different within the control subgroups . lps - lp placental and fetal weight were significantly lower than that of the control subgroup ( p < 0.05 ) . apo - ln - lp fetal weight was significantly less than the control subgroup ( p < 0.05 ) , but placental weight was not significantly different from the control subgroup . gpi placental and fetal weight were significantly lower than the control group ( p < 0.05 ) , and there was no significant difference between ln - pi and lps - pi . within each experimental group , the earlier the preeclampsia onset , the more severe the placental and fetal damage ( p < 0.05 ) . lipopolysaccharide - lp placental weight was significantly lower than that of the other late subgroups . fetal weight in all the lps and apo - ln subgroups was significantly less than the corresponding ln subgroups ( p < 0.05 ) . fetal weight in the apo - ln - pi , ep and mp groups was significantly lower than in the corresponding lps subgroups ( p < 0.05 ) , but lps - lp was significantly lower than apo - ln - lp ( p < 0.05 ) . there were no significant differences between any other corresponding subgroups [ tables 1 and 2 ] . placental weight on day 18 of pregnancy in each group ( mg ) * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , the lp subgroups were significantly different from the other subgroups in the same group ; p < 0.05 , the lps - lp was significantly different than ln - lp and apo - ln - lp . pi : preimplantation ; ep : early pregnancy ; mp : mid - pregnancy ; lp : late pregnancy ; lps : lipopolysaccharide ; gpi : 2 glycoprotein i ; ln : l - name ; apo - ln : apolipoprotein l - name . fetal weight on day 18 of pregnancy in each group ( g ) * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , the lp subgroups were significantly different from the other subgroups in the same group ; p < 0.05 , subgroups in lps and apo - ln were significantly lower than the corresponding subgroup in ln and gpi ; p < 0.05 , compared with the corresponding lps subgroup . pi : preimplantation ; ep : early pregnancy ; mp : mid - pregnancy ; lp : late pregnancy ; lps : lipopolysaccharide ; gpi : 2 glycoprotein i ; ln : l - name ; apo - ln : apolipoprotein l - name . within each experimental group , the serum tg concentration was significantly higher in all ln subgroups , except for the ln - lg subgroup , compared with those in the corresponding ln - c subgroups ( p < 0.05 ) , and the earlier the injection began , the higher the serum tg concentration . the serum tg concentration in the lps subgroups did not significantly differ from those in the lps - c subgroups . except for the apo - ln - lg subgroup , all apo - ln subgroups showed a significantly higher tg concentration than those in the corresponding apo - c subgroups ( p < 0.05 ) . tc concentrations did not significantly differ among all the groups and subgroups in this study . among the experimental groups , the tg concentrations in all the apo - ln and apo - c subgroups were significantly higher than those in any of the other subgroups ( p < 0.05 ) . the tg concentration in the gpi group was similar to that in the ln - pi subgroup [ figure 2 ] . ( a ) triglyceride and ( b ) total cholesterol concentrations . * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , compared with the corresponding l - name ( ln ) or ln - c subgroup ; p < 0.05 , compared with the corresponding lipopolysaccharide ( lps ) or lps - c subgroup . pi : preimplantation ; eg : early - gestation ; mg : mid - gestation ; lg : late - gestation . spotty necrosis was observed in pi , eg and mg for ln , apo - ln and gpi , but no obvious morphologic changes were observed in the lg subgroups for ln and apo - ln . in the ln group , the earlier the injections began , the more severe were the changes in liver morphology . spotty necrosis was observed in pi , eg , mg of l - name ( ln ) , apo - ln and 2 glycoprotein i ( gpi ) ( black arrow ) . in the ln group , ( a - d ) ln ; ( e ) ln - c ; ( f - i ) lps ; ( j ) lps - c ; ( k - n ) apo - ln ; ( o ) apo - c ; ( p ) gpi ; ( q ) gpi - c . pi : preimplantation ; eg : early - gestation ; mg : mid - gestation ; lg : late - gestation ( h and e ; original magnification , 200 ) . a decrease in microvilli , endoplasmic reticulum expansion , trophoblastic mitochondrial swelling and cristae disappearance were observed in all the experimental groups . there was no obvious difference in cell ultrastructure between the different models or between different onset times [ figure 4 ] . trophoblastic mitochondria swelling and cristae disappearance were observed in all the experimental groups ( black arrow ) . ( a - d ) l - name ( ln ) ; ( e ) ln - c ; ( f - i ) lipopolysaccharide ( lps ) ; ( j ) lps - c ; ( k - n ) apo - ln ; ( o ) apo - c ; ( p ) 2 glycoprotein i ( gpi ) ; ( q ) gpi - c . pi : preimplantation ; eg : early - gestation ; mg : mid - gestation ; lg : late - gestation ( double stained with uranyl acetate and lead citrate , magnification 8000 ) . within each experimental group , map was significantly higher in every experimental subgroup than in the corresponding control subgroup ( p < 0.05 ) . moreover , the earlier the injection began , the higher was the map ( p < 0.05 ) . among the experimental groups , map in the apo - ln subgroups was higher than that of the corresponding subgroups in the other three experimental groups , but only the map in the apo - ln - mp and apo - ln - lp subgroups was significantly different ( p < 0.05 ) . map was also significantly higher in the apo - c group than in other control groups that were established using wild type mice ( p < 0.05 ) . the map level in the gpi group was similar to that in the ln - pi and lps - pi subgroups . there was no significant difference in map between the gpi - c , ln - c and lps - c groups [ figure 1a ] . * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , compared with the corresponding l - name subgroup ; p < 0.05 , compared with the corresponding lipopolysaccharide subgroup . pi : preimplantation ; ep : early pregnancy ; mp : mid - pregnancy ; lp : late pregnancy . every experimental subgroup showed significantly higher 24-h urine protein concentrations than their corresponding control subgroups at 18 day of pregnancy ( p < 0.05 ) . moreover , the earlier the injection began , the more the urine protein increased ( p < 0.05 ) . in the ln , lps and apo - ln experimental groups , the pi , ep and mp subgroups showed significantly higher urine protein levels than those in lp subgroups ( p < 0.05 ) . the urine protein level in the apo - ln group was higher than those in the other experimental groups , but only that in the apo - ln - lp subgroup showed significance ( p < 0.05 ) . the urine protein level in the apo - c group was not significantly different from that in the control groups of wild type mice . the urine protein level in the gpi group was similar to that in the ln - pi and lps - pi subgroups , and the difference between the gpi - c , ln - c and lps - c groups was not significant [ figure 1b ] . within each experimental group , pi , ep and mp subgroups placental and fetal weight was less than that of the corresponding control subgroup for ln , lps and apo - ln ( p < 0.05 ) . ln - lp placental and fetal weight were not significantly different within the control subgroups . lps - lp placental and fetal weight were significantly lower than that of the control subgroup ( p < 0.05 ) . apo - ln - lp fetal weight was significantly less than the control subgroup ( p < 0.05 ) , but placental weight was not significantly different from the control subgroup . gpi placental and fetal weight were significantly lower than the control group ( p < 0.05 ) , and there was no significant difference between ln - pi and lps - pi . within each experimental group , the earlier the preeclampsia onset , the more severe the placental and fetal damage ( p < 0.05 ) . lipopolysaccharide - lp placental weight was significantly lower than that of the other late subgroups . fetal weight in all the lps and apo - ln subgroups was significantly less than the corresponding ln subgroups ( p < 0.05 ) . fetal weight in the apo - ln - pi , ep and mp groups was significantly lower than in the corresponding lps subgroups ( p < 0.05 ) , but lps - lp was significantly lower than apo - ln - lp ( p < 0.05 ) . there were no significant differences between any other corresponding subgroups [ tables 1 and 2 ] . placental weight on day 18 of pregnancy in each group ( mg ) * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , the lp subgroups were significantly different from the other subgroups in the same group ; p < 0.05 , the lps - lp was significantly different than ln - lp and apo - ln - lp . pi : preimplantation ; ep : early pregnancy ; mp : mid - pregnancy ; lp : late pregnancy ; lps : lipopolysaccharide ; gpi : 2 glycoprotein i ; ln : l - name ; apo - ln : apolipoprotein l - name . fetal weight on day 18 of pregnancy in each group ( g ) * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , the lp subgroups were significantly different from the other subgroups in the same group ; p < 0.05 , subgroups in lps and apo - ln were significantly lower than the corresponding subgroup in ln and gpi ; p < 0.05 , compared with the corresponding lps subgroup . pi : preimplantation ; ep : early pregnancy ; mp : mid - pregnancy ; lp : late pregnancy ; lps : lipopolysaccharide ; gpi : 2 glycoprotein i ; ln : l - name ; apo - ln : apolipoprotein l - name . within each experimental group , the serum tg concentration was significantly higher in all ln subgroups , except for the ln - lg subgroup , compared with those in the corresponding ln - c subgroups ( p < 0.05 ) , and the earlier the injection began , the higher the serum tg concentration . the serum tg concentration in the lps subgroups did not significantly differ from those in the lps - c subgroups . except for the apo - ln - lg subgroup , all apo - ln subgroups showed a significantly higher tg concentration than those in the corresponding apo - c subgroups ( p < 0.05 ) . tc concentrations did not significantly differ among all the groups and subgroups in this study . among the experimental groups , the tg concentrations in all the apo - ln and apo - c subgroups were significantly higher than those in any of the other subgroups ( p < 0.05 ) . the tg concentration in the gpi group was similar to that in the ln - pi subgroup [ figure 2 ] . ( a ) triglyceride and ( b ) total cholesterol concentrations . * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , compared with the corresponding l - name ( ln ) or ln - c subgroup ; p < 0.05 , compared with the corresponding lipopolysaccharide ( lps ) or lps - c subgroup . pi : preimplantation ; eg : early - gestation ; mg : mid - gestation ; lg : late - gestation . spotty necrosis was observed in pi , eg and mg for ln , apo - ln and gpi , but no obvious morphologic changes were observed in the lg subgroups for ln and apo - ln . in the ln group , the earlier the injections began , the more severe were the changes in liver morphology . spotty necrosis was observed in pi , eg , mg of l - name ( ln ) , apo - ln and 2 glycoprotein i ( gpi ) ( black arrow ) . in the ln group , ( a - d ) ln ; ( e ) ln - c ; ( f - i ) lps ; ( j ) lps - c ; ( k - n ) apo - ln ; ( o ) apo - c ; ( p ) gpi ; ( q ) gpi - c . pi : preimplantation ; eg : early - gestation ; mg : mid - gestation ; lg : late - gestation ( h and e ; original magnification , 200 ) . a decrease in microvilli , endoplasmic reticulum expansion , trophoblastic mitochondrial swelling and cristae disappearance were observed in all the experimental groups . there was no obvious difference in cell ultrastructure between the different models or between different onset times [ figure 4 ] trophoblastic mitochondria swelling and cristae disappearance were observed in all the experimental groups ( black arrow ) . ( a - d ) l - name ( ln ) ; ( e ) ln - c ; ( f - i ) lipopolysaccharide ( lps ) ; ( j ) lps - c ; ( k - n ) apo - ln ; ( o ) apo - c ; ( p ) 2 glycoprotein i ( gpi ) ; ( q ) gpi - c . pi : preimplantation ; eg : early - gestation ; mg : mid - gestation ; lg : late - gestation ( double stained with uranyl acetate and lead citrate , magnification 8000 ) . the present study used two typical models of preeclampsia : an endothelial injury model generated using l - name and an inflammation model generated using lps . in addition , we used a transgenic model that overexpresses the human apoc3 gene and has an abnormal lipid metabolism background , and the aps model that is generated by gpi , which is injected before mating . map and 24-h urine protein were significantly elevated in pregnant mice after injection , and experimental groups showed preeclampsia - like symptoms . four different models were used , and preeclampsia - like symptoms were induced at different onset times . the earlier the preeclampsia onset , the higher were the map and urine protein levels , indicating that successful preeclampsia - like model generation . in the ln ( except for the late subgroup ) , gpi , apo - ln and apo - c groups , but not in the lps group , serum ffas significantly increased . all experimental groups , except the lps group , showed significant hepatic and placental lipid deposition . roberts et al . proposed that maternal constitutional factors , including genetic , behavioral and environmental factors , affect the clinical manifestations of preeclampsia . different pregestational maternal situations and different pathogenic factors may induce different pathophysiological changes in lipid metabolism , resulting in different clinical outcomes for patients with preeclampsia . in this study , the earlier the preeclampsia onset , the higher were the map and urine protein levels , and the lower were the placental and fetal weights , indicating that early pregnancy is an important time for placental development ; the earlier the adverse factors impact upon the mother , the more severely affected are placental and fetal development . compared with other subgroups , serum tg concentration in ln - lg subgroup liver injury in the apo - ln group was also affected by apoc3 knock - in , and there was no obvious tendency in this group , but tgs and liver injury in the apo - ln - lg subgroup were significantly different from other subgroups . this is consistent with ffa oxidative metabolism and liver injury that often occurred in patients with early - onset preeclampsia , suggesting that the interaction between adverse factors and late - onset preeclampsia patients may be different from early - onset preeclampsia patients . in early - onset the adverse factors act on the mother 's body and affect the developing placenta , and maternal - placental interactions may produce a vicious cycle by influencing the clinical manifestations of preeclampsia . in late - onset preeclampsia , the adverse factors affect the developed placenta , and this may lead to clinical manifestations that are mainly caused by the maternal stress reaction . the classic preeclampsia - like model , the ln group , showed increased serum tgs , indicating that the l - name - generated model had dysregulated lipid metabolism . because of a decrease in placental lchad protein expression , accumulation of long - chain fatty acids may be the reason for the increase in tgs , and increased levels of long - chain fatty acids in the blood , which could lead to more absorption of long - chain fatty acids by the liver . when the anabolism of tgs exceeds the capacity of fatty acid oxidation , lipids in liver and other organs increase , resulting in maternal ectopic deposition of lipids and systemic metabolic disorder . another classic preeclampsia - like model , the lps group , showed a similar map and urine protein level as that in the ln group , but serum tg levels in the lps group differed from those in the other experimental groups . this finding might be a result of inflammatory cytokines stimulated by lps that act on endothelial cells to activate nf-b and other inflammatory pathways , resulting in endothelial cell damage and dysfunction , and leading to preeclampsia - like changes . this indicates that the lps - generated preeclampsia - like model may not have a long - chain fatty acid oxidative metabolism dysfunction . serum apoc3 can cause an increase in hepatic tg anabolism , leading to elevation of serum tg levels . apo - c and apoc3 transgenic mice without l - name injection had an elevated map , but their urine protein was not significantly different from the wild type control groups . after injection of l - name in the apo - ln group , map and urine protein increased significantly , and placental and fetal weight was significantly reduced . this suggests that the apo - ln group showed a preeclampsia - like syndrome , and lchad mrna and protein expression in the liver and placenta were significantly higher for the apo - c , apo - ln and gpi groups and lower for ln group compared with controls . however , this did not differ between the lps group and controls , indicating that different maternal constitutional factors play different roles in pathogenesis of preeclampsia . although some negative factors can not induce preeclampsia - like syndrome alone , they can exacerbate the burden of pregnancy so that preeclampsia - like symptoms will be more serious with other conditions . in the ln group , unoxidized palmitoyl - coa , a ffa metabolite , is the main cause of lipid - generated tissue injury , suggesting that ffas are more harmful than tgs . this difference in the source of serum tgs may account for the absence of liver injury in the apo - c group . this phenomenon indicates that preeclampsia is the result of the interaction between genetic and environmental factors and that a lipid metabolism disorder acting on a different genetic background may result in different pathophysiological changes . in the present study , gpi - generated a preeclampsia - like mouse model that had significantly increased serum tg levels , which were similar to that in the ln - pi subgroup . our previous study showed increased placental lchad mrna and protein expression in the gpi - generated preeclampsia - like model , and decreased placental lchad mrna and protein expression in the l - name - generated preeclampsia - like mouse model , but both models had increased serum ffa . this suggests that dysregulated long - chain fatty acid metabolism exists in the gpi - generated preeclampsia - like mouse model , but fatty acid -oxidation may be differently regulated in these two models . in this study , spotty liver necrosis was observed in the ln and apo - ln groups , which were generated using l - name , and in the gpi group , which was generated using gpi , but the lps - generated model had no obvious hepatic necrosis . fat mobilization increases during pregnancy , and adverse maternal constitutional factors may decrease placental long - chain fatty acid oxidation , resulting in increased serum ffa . the accumulation of long - chain fatty acids in the liver may be the reason for liver injury in preeclampsia . lchad is an important enzyme for long - chain fatty acid oxidation , and the preeclampsia - like ln , apo - ln and gpi groups with abnormal serum tg showed abnormal lchad expression . the lps group had no significant dyslipidemia , and there was also no obvious abnormal lchad expression , confirming that long - chain fatty acid metabolism may be a reason for liver injury in preeclampsia . in addition , apo - ln and apo - c groups showed significant hepatic cytoplasmic vacuoles , which may be caused by congenital hyperlipidemia . the liver and placental protein lchad expression in apo - c were significantly higher than those in the wild type saline control group , but placental lchad expression in the apo - c group was significantly lower than that in apo - ln . this suggests that highly expressed apoc3 plays a role in the regulation of lchad in preeclampsia , and the difference in placental lchad expression may be the reason for different liver injuries between apo - ln and apo - c . these phenomena suggest an association between placental long - chain fatty acid oxidative disorders and liver injury in preeclampsia . previous studies suggested that trophoblast cell dysfunction causes preeclampsia . in this study , no matter what kind of model or induction time , preeclampsia - like symptoms always emerged and they were accompanied by abnormal mitochondrial structure in trophoblast cells . it is unlikely that cholesterol , which is mainly metabolized in the cytosol , is involved , but mitochondria are important places for ffa metabolism in trophoblast cells . experimental groups with abnormal tg levels have abnormal lchad protein expression , suggesting that the trophoblastic mitochondrial damage caused by long - chain fatty acid oxidative metabolic disorders is associated with some preeclampsia , which is also corroborated in the clinic by increased oxidative stress and mitochondrial damage in placental trophoblast cells from preeclampsia patients . in addition , a previous study reported that lps can promote caspase-8 expression in trophoblast cells , leading to apoptosis via the mitochondrial pathway . thus , there is also mitochondrial damage in the lps group without a lipid metabolic disorder . all the preeclampsia models had trophoblast cell mitochondrial damage , suggesting that trophoblastic mitochondrial damage may be the common terminal pathway in different preeclampsia - like models . our results show that the earlier the preeclampsia onset , the higher were the map and urine protein levels , and the lower was the placental and fetal weight . different preeclampsia - like mouse models may have different lipid metabolism , and a long - chain fatty acid oxidation metabolic disorder may be one of the reasons for liver injury in preeclampsia . preeclampsia may be a similar clinical manifestation in a group of diseases , and the trophoblastic mitochondrial damage may be their common terminal pathway .

background : preeclampsia is a multifactorial disease during pregnancy . dysregulated lipid metabolism may be related to some preeclampsia . we investigated the relationship between triglycerides ( tgs ) and liver injury in different preeclampsia - like mouse models and their potential common pathways.methods:preeclampsia-like models ( nw - nitro - l - arginine - methyl ester [ l - name ] , lipopolysaccharide [ lps ] , apolipoprotein c - iii [ apo ] transgnic mice + l - name , 2 glycoprotein i [ gpi ] ) were used in four experimental groups : l - name ( ln ) , lps , apo - ln and gpi , respectively , and controls received saline ( ln - c , lps - c , apo - c , gpi - c ) . the first three models were established in preimplantation ( pi ) , early- , mid- and late - gestation ( eg , mg and lg ) . gpi and controls were injected before implantation . mean arterial pressure ( map ) , 24-hour urine protein , placental and fetal weight , serum tgs , total cholesterol ( tc ) and pathologic liver and trophocyte changes were assessed.results:map and proteinuria were significantly increased in the experimental groups . placenta and fetal weight in pi , ep and mp subgroups were significantly lower than lp . serum tgs significantly increased in most groups but controls . tc was not different between experimental and control groups . spotty hepatic cell necrosis was observed in pi , eg , mg in ln , apo - ln and gpi , but no morphologic changes were observed in the lps group . similar trophoblastic mitochondrial damage was observed in every experimental group.conclusions:earlier preeclampsia onset causes a higher map and urine protein level , and more severe placental and fetal damage . preeclampsia - like models generated by varied means lead to different changes in lipid metabolism and associated with liver injury . trophoblastic mitochondrial damage may be the common terminal pathway in different preeclampsia - like models .

I M Animals Sample collection and tests Statistical analysis R Identification of models Placental and fetal weight Serum triglyceride and total cholesterol concentrations Liver morphologic changes Trophoblast cell ultrastructure D

other dysregulated lipid metabolism may be involved in preeclampsia , such as the increased serum apolipoprotein c - iii ( apoc3 ) lipid transport protein . in early- and mid - onset preeclampsia - like mouse models , lchad protein expression was significantly reduced in the placenta , but the late - onset model showed no significant difference from controls , suggesting that different onset times of preeclampsia may result in different lipid metabolism . our previous study found that serum ffa increased in wild type or apoc3 knock - in transgenic preeclampsia - like mouse models induced by nw - nitro - l - arginine - methyl ester ( l - name ) or 2 glycoprotein i ( gpi ) , and hepatic and placental fatty infiltration were observed in these models . the liver and placenta are important organs for ffa -oxidation during pregnancy , and complex links may exist between them in different preeclampsia - like models . the present study used l - name and lps injections , knock - in mice with the human apoc3 gene as an adverse genetic background , and a basic maternal disease model with aps to create different pathogenic preeclampsia - like mouse models . all models were created at the preimplantation ( pi ) , early- , mid- and late - gestation ( eg , mg , lg ) stages to study the relationship between lipid metabolism and liver injury in different models that are created at different times . in addition , trophoblastic mitochondrial damage as a possible common pathway of different preeclampsia - like mouse models was investigated . the c57bl/6j mice were randomly assigned to one of six groups : the l - name ( ln ) group ( injected with l - name ) , lps group , gpi group , and their normal saline - injected control groups ( the ln - c , lps - c , and gpi - c groups , respectively ) . mice in the ln and apo - ln groups received daily subcutaneous injections of l - name at a dose of 50 mgkgd . the ln , lps and apo - ln groups and their control groups were further subdivided depending on when the first injection was administered : pi subgroup , day 3 of gestation ; eg subgroup , day 7 of gestation ; mg subgroup , day 11 of gestation ; and lg subgroup , day 16 of gestation . the c57bl/6j mice were randomly assigned to one of six groups : the l - name ( ln ) group ( injected with l - name ) , lps group , gpi group , and their normal saline - injected control groups ( the ln - c , lps - c , and gpi - c groups , respectively ) . the ln , lps and apo - ln groups and their control groups were further subdivided depending on when the first injection was administered : pi subgroup , day 3 of gestation ; eg subgroup , day 7 of gestation ; mg subgroup , day 11 of gestation ; and lg subgroup , day 16 of gestation . among the experimental groups , map in the apo - ln subgroups was higher than that of the corresponding subgroups in the other three experimental groups , but only the map in the apo - ln - mp and apo - ln - lp subgroups was significantly different ( p < 0.05 ) . in the ln , lps and apo - ln experimental groups , the pi , ep and mp subgroups showed significantly higher urine protein levels than those in lp subgroups ( p < 0.05 ) . the urine protein level in the apo - ln group was higher than those in the other experimental groups , but only that in the apo - ln - lp subgroup showed significance ( p < 0.05 ) . the urine protein level in the apo - c group was not significantly different from that in the control groups of wild type mice . the urine protein level in the gpi group was similar to that in the ln - pi and lps - pi subgroups , and the difference between the gpi - c , ln - c and lps - c groups was not significant [ figure 1b ] . within each experimental group , pi , ep and mp subgroups placental and fetal weight was less than that of the corresponding control subgroup for ln , lps and apo - ln ( p < 0.05 ) . lps - lp placental and fetal weight were significantly lower than that of the control subgroup ( p < 0.05 ) . apo - ln - lp fetal weight was significantly less than the control subgroup ( p < 0.05 ) , but placental weight was not significantly different from the control subgroup . gpi placental and fetal weight were significantly lower than the control group ( p < 0.05 ) , and there was no significant difference between ln - pi and lps - pi . fetal weight in the apo - ln - pi , ep and mp groups was significantly lower than in the corresponding lps subgroups ( p < 0.05 ) , but lps - lp was significantly lower than apo - ln - lp ( p < 0.05 ) . placental weight on day 18 of pregnancy in each group ( mg ) * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , the lp subgroups were significantly different from the other subgroups in the same group ; p < 0.05 , the lps - lp was significantly different than ln - lp and apo - ln - lp . pi : preimplantation ; ep : early pregnancy ; mp : mid - pregnancy ; lp : late pregnancy ; lps : lipopolysaccharide ; gpi : 2 glycoprotein i ; ln : l - name ; apo - ln : apolipoprotein l - name . fetal weight on day 18 of pregnancy in each group ( g ) * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , the lp subgroups were significantly different from the other subgroups in the same group ; p < 0.05 , subgroups in lps and apo - ln were significantly lower than the corresponding subgroup in ln and gpi ; p < 0.05 , compared with the corresponding lps subgroup . pi : preimplantation ; ep : early pregnancy ; mp : mid - pregnancy ; lp : late pregnancy ; lps : lipopolysaccharide ; gpi : 2 glycoprotein i ; ln : l - name ; apo - ln : apolipoprotein l - name . among the experimental groups , the tg concentrations in all the apo - ln and apo - c subgroups were significantly higher than those in any of the other subgroups ( p < 0.05 ) . * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , compared with the corresponding l - name ( ln ) or ln - c subgroup ; p < 0.05 , compared with the corresponding lipopolysaccharide ( lps ) or lps - c subgroup . spotty necrosis was observed in pi , eg and mg for ln , apo - ln and gpi , but no obvious morphologic changes were observed in the lg subgroups for ln and apo - ln . spotty necrosis was observed in pi , eg , mg of l - name ( ln ) , apo - ln and 2 glycoprotein i ( gpi ) ( black arrow ) . in the ln group , ( a - d ) ln ; ( e ) ln - c ; ( f - i ) lps ; ( j ) lps - c ; ( k - n ) apo - ln ; ( o ) apo - c ; ( p ) gpi ; ( q ) gpi - c . ( a - d ) l - name ( ln ) ; ( e ) ln - c ; ( f - i ) lipopolysaccharide ( lps ) ; ( j ) lps - c ; ( k - n ) apo - ln ; ( o ) apo - c ; ( p ) 2 glycoprotein i ( gpi ) ; ( q ) gpi - c . among the experimental groups , map in the apo - ln subgroups was higher than that of the corresponding subgroups in the other three experimental groups , but only the map in the apo - ln - mp and apo - ln - lp subgroups was significantly different ( p < 0.05 ) . in the ln , lps and apo - ln experimental groups , the pi , ep and mp subgroups showed significantly higher urine protein levels than those in lp subgroups ( p < 0.05 ) . the urine protein level in the apo - ln group was higher than those in the other experimental groups , but only that in the apo - ln - lp subgroup showed significance ( p < 0.05 ) . the urine protein level in the apo - c group was not significantly different from that in the control groups of wild type mice . the urine protein level in the gpi group was similar to that in the ln - pi and lps - pi subgroups , and the difference between the gpi - c , ln - c and lps - c groups was not significant [ figure 1b ] . within each experimental group , pi , ep and mp subgroups placental and fetal weight was less than that of the corresponding control subgroup for ln , lps and apo - ln ( p < 0.05 ) . lps - lp placental and fetal weight were significantly lower than that of the control subgroup ( p < 0.05 ) . apo - ln - lp fetal weight was significantly less than the control subgroup ( p < 0.05 ) , but placental weight was not significantly different from the control subgroup . gpi placental and fetal weight were significantly lower than the control group ( p < 0.05 ) , and there was no significant difference between ln - pi and lps - pi . fetal weight in the apo - ln - pi , ep and mp groups was significantly lower than in the corresponding lps subgroups ( p < 0.05 ) , but lps - lp was significantly lower than apo - ln - lp ( p < 0.05 ) . placental weight on day 18 of pregnancy in each group ( mg ) * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , the lp subgroups were significantly different from the other subgroups in the same group ; p < 0.05 , the lps - lp was significantly different than ln - lp and apo - ln - lp . pi : preimplantation ; ep : early pregnancy ; mp : mid - pregnancy ; lp : late pregnancy ; lps : lipopolysaccharide ; gpi : 2 glycoprotein i ; ln : l - name ; apo - ln : apolipoprotein l - name . fetal weight on day 18 of pregnancy in each group ( g ) * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , the lp subgroups were significantly different from the other subgroups in the same group ; p < 0.05 , subgroups in lps and apo - ln were significantly lower than the corresponding subgroup in ln and gpi ; p < 0.05 , compared with the corresponding lps subgroup . pi : preimplantation ; ep : early pregnancy ; mp : mid - pregnancy ; lp : late pregnancy ; lps : lipopolysaccharide ; gpi : 2 glycoprotein i ; ln : l - name ; apo - ln : apolipoprotein l - name . within each experimental group , the serum tg concentration was significantly higher in all ln subgroups , except for the ln - lg subgroup , compared with those in the corresponding ln - c subgroups ( p < 0.05 ) , and the earlier the injection began , the higher the serum tg concentration . among the experimental groups , the tg concentrations in all the apo - ln and apo - c subgroups were significantly higher than those in any of the other subgroups ( p < 0.05 ) . * p < 0.05 , compared with the corresponding control subgroup ; p < 0.05 , compared with the corresponding l - name ( ln ) or ln - c subgroup ; p < 0.05 , compared with the corresponding lipopolysaccharide ( lps ) or lps - c subgroup . spotty necrosis was observed in pi , eg and mg for ln , apo - ln and gpi , but no obvious morphologic changes were observed in the lg subgroups for ln and apo - ln . spotty necrosis was observed in pi , eg , mg of l - name ( ln ) , apo - ln and 2 glycoprotein i ( gpi ) ( black arrow ) . in the ln group , ( a - d ) ln ; ( e ) ln - c ; ( f - i ) lps ; ( j ) lps - c ; ( k - n ) apo - ln ; ( o ) apo - c ; ( p ) gpi ; ( q ) gpi - c . ( a - d ) l - name ( ln ) ; ( e ) ln - c ; ( f - i ) lipopolysaccharide ( lps ) ; ( j ) lps - c ; ( k - n ) apo - ln ; ( o ) apo - c ; ( p ) 2 glycoprotein i ( gpi ) ; ( q ) gpi - c . map and 24-h urine protein were significantly elevated in pregnant mice after injection , and experimental groups showed preeclampsia - like symptoms . the earlier the preeclampsia onset , the higher were the map and urine protein levels , indicating that successful preeclampsia - like model generation . in the ln ( except for the late subgroup ) , gpi , apo - ln and apo - c groups , but not in the lps group , serum ffas significantly increased . in this study , the earlier the preeclampsia onset , the higher were the map and urine protein levels , and the lower were the placental and fetal weights , indicating that early pregnancy is an important time for placental development ; the earlier the adverse factors impact upon the mother , the more severely affected are placental and fetal development . compared with other subgroups , serum tg concentration in ln - lg subgroup liver injury in the apo - ln group was also affected by apoc3 knock - in , and there was no obvious tendency in this group , but tgs and liver injury in the apo - ln - lg subgroup were significantly different from other subgroups . the classic preeclampsia - like model , the ln group , showed increased serum tgs , indicating that the l - name - generated model had dysregulated lipid metabolism . another classic preeclampsia - like model , the lps group , showed a similar map and urine protein level as that in the ln group , but serum tg levels in the lps group differed from those in the other experimental groups . apo - c and apoc3 transgenic mice without l - name injection had an elevated map , but their urine protein was not significantly different from the wild type control groups . after injection of l - name in the apo - ln group , map and urine protein increased significantly , and placental and fetal weight was significantly reduced . this suggests that the apo - ln group showed a preeclampsia - like syndrome , and lchad mrna and protein expression in the liver and placenta were significantly higher for the apo - c , apo - ln and gpi groups and lower for ln group compared with controls . in the present study , gpi - generated a preeclampsia - like mouse model that had significantly increased serum tg levels , which were similar to that in the ln - pi subgroup . our previous study showed increased placental lchad mrna and protein expression in the gpi - generated preeclampsia - like model , and decreased placental lchad mrna and protein expression in the l - name - generated preeclampsia - like mouse model , but both models had increased serum ffa . this suggests that dysregulated long - chain fatty acid metabolism exists in the gpi - generated preeclampsia - like mouse model , but fatty acid -oxidation may be differently regulated in these two models . in this study , spotty liver necrosis was observed in the ln and apo - ln groups , which were generated using l - name , and in the gpi group , which was generated using gpi , but the lps - generated model had no obvious hepatic necrosis . lchad is an important enzyme for long - chain fatty acid oxidation , and the preeclampsia - like ln , apo - ln and gpi groups with abnormal serum tg showed abnormal lchad expression . the lps group had no significant dyslipidemia , and there was also no obvious abnormal lchad expression , confirming that long - chain fatty acid metabolism may be a reason for liver injury in preeclampsia . in addition , apo - ln and apo - c groups showed significant hepatic cytoplasmic vacuoles , which may be caused by congenital hyperlipidemia . the liver and placental protein lchad expression in apo - c were significantly higher than those in the wild type saline control group , but placental lchad expression in the apo - c group was significantly lower than that in apo - ln . this suggests that highly expressed apoc3 plays a role in the regulation of lchad in preeclampsia , and the difference in placental lchad expression may be the reason for different liver injuries between apo - ln and apo - c . experimental groups with abnormal tg levels have abnormal lchad protein expression , suggesting that the trophoblastic mitochondrial damage caused by long - chain fatty acid oxidative metabolic disorders is associated with some preeclampsia , which is also corroborated in the clinic by increased oxidative stress and mitochondrial damage in placental trophoblast cells from preeclampsia patients . all the preeclampsia models had trophoblast cell mitochondrial damage , suggesting that trophoblastic mitochondrial damage may be the common terminal pathway in different preeclampsia - like models . our results show that the earlier the preeclampsia onset , the higher were the map and urine protein levels , and the lower was the placental and fetal weight . different preeclampsia - like mouse models may have different lipid metabolism , and a long - chain fatty acid oxidation metabolic disorder may be one of the reasons for liver injury in preeclampsia . preeclampsia may be a similar clinical manifestation in a group of diseases , and the trophoblastic mitochondrial damage may be their common terminal pathway .

the evolving scientific and regulatory activities in europe and north america emphasize the need for the development of tools that refine , replace , or reduce the use of animals and human volunteers in pharmacokinetic and toxicity tests [ 13 ] . the ability to base the toxic responses on the target tissue dose or internal concentration of the toxic moiety of the chemicals is key to the predictive tools reflective of the current state of science . therefore , physiologically based pharmacokinetic ( pbpk ) models that are capable of providing a priori prediction of the time course of chemicals in blood and tissues is of tremendous interest . pbpk models are mechanistically based mathematical descriptions of the absorption , distribution , metabolism , and excretion of chemicals or pharmaceutical compounds . in pbpk models , the organism is represented as a set of several tissue compartments interconnected by blood flows . in these models , the internal dose measures ( e.g. , blood or tissue concentrations , amount metabolized ) of a chemical are described on the basis of mass - balance differential equations requiring species - specific properties ( e.g. , alveolar ventilation rate , cardiac output , regional blood flows , and tissue volumes ) and chemical - specific input parameters ( e.g. , partition coefficients and metabolic constants ) . although the species - specific values of several physiological parameters are available in the literature [ 46 ] , the partition coefficients ( pcs ) and metabolic constants need to be determined experimentally or calculated by using animal - replacement methods for each chemical individually . the values of tissue : blood or tissue : plasma partition coefficients essential for developing pbpk models have been estimated for a wide range of chemicals and chemical classes , including drugs , with the use of tissue composition - based algorithms or qsar methods ( e.g. , [ 819 ] ) . regarding the metabolism parameters ( i.e. , hepatic clearance , intrinsic clearance , vmax , km , kcat , free energy of binding , energy of activation , or activation enthalpy ) , some studies have developed 2-d and 3-d qsars but with a specific focus on either a single isozyme , a single reaction or a single class of substances [ 8 , 2038 ] . none of these past efforts succeeded in predicting both vmax and km ( or clint ) of environmental chemicals for direct incorporation within animal or human pbpk models . alternatively , few studies utilized the group contribution method of gao [ 3943 ] , to predict metabolic rates for pbpk models . in this method , the chemical is decomposed into different structural fragments or groups , the contributions of which are obtained by regression analysis . accordingly , these publications demonstrated the feasibility of developing structure - property relationships for the metabolism rates . the group contribution method was successfully used to develop quantitative structure - property relationships ( qsprs ) for the tissue : air partition coefficients as well as intrinsic ( clint ) and hepatic clearance ( clh for a group of low - molecular - weight volatile organic chemicals ( vocs ) in rats [ 41 , 42 ] . these qspr models , in turn , were incorporated within pbpk models to predict reasonably well the blood kinetics of inhaled vocs in rats . as these qsprs are species specific , they could not be used to conduct interspecies extrapolations . to overcome this limitation , bliveau et al . developed biologically based algorithms for pcs and clh to conduct rat to human extrapolations of the inhalation toxicokinetics of vocs . in this study , qsprs based on the group contribution method were developed for the chemical - specific input parameters of the biological algorithms for pcs ( i.e. , oil : air , water : air , and blood protein : air ) and clint ( intrinsic clearance normalized for cytochrome p450 2e1 content ) . more recently , qsprs were developed for the metabolic constants vmax ( maximum velocity of reaction ) and km ( michaelis constant ) and were further incorporated within a rat pbpk model to predict the toxicokinetics of mixtures of vocs . despite the successful use of the group contribution method in qspr modeling of metabolism rates , their principal limitation relates to the fact that the chemical space they cover is extremely limited ( low - molecular - weight vocs containing one or more of the following fragments : ch3 , ch2 , ch , c , c = c , h , br , cl , f , benzene ring , and h on benzene ring ) . more experimental data on diverse chemicals would be needed to determine the contributions of other molecular fragments , as has been done with pow ( e.g. , estimation of the contribution of 130 fragments ( i.e. , groups ) required 1200 measurements of pow ) . to extend the currently available qspr for clint to cover more diverse fragments and at the same time respect a reasonable ratio of the number of parameters to the number of observations , since the critical limitation in the construction of pbpk models for new substances continues to be the metabolism rate , a pragmatic approach particularly for inhaled vocs is to evaluate the maximum and minimum possible blood concentration profiles in exposed individuals . thus , using a hepatic extraction ratio ( e ) of 0 and 1 in the pbpk models , poulin and krishnan obtained simulations of the physiological limits ( i.e. , maximal and minimal blood concentration profiles ) for inhaled vocs in humans . assuming the conceptual pbpk model and the values of its physiological parameters are reliable , the real answer , that is , the actual concentrations and kinetic curve , would be somewhere in between the theoretical limits simulated with these pbpk models . the uncertainty associated with these theoretical bounds can be reduced by developing better estimates of the metabolism constants . this could be done , at a practical level , by developing in silico tools that provide a range of plausible values , in lieu of a single accurate point estimate . such a tool might be of use for the toxicokinetic screening of substances , until the time when the chemical - specific measurements are obtained in vivo , in vitro , or with a highly precise mechanistic in silico method . since human exposures to environmental contaminants in most cases do not attain levels that approach or exceed saturation , it is not crucial to predict vmax and km separately , particularly for simulating kinetics in humans exposed to low atmospheric concentrations of vocs . therefore , the availability of in silico approaches based on easily available parameters to predict plausible range of clint would be desirable as a screening - level tool . the objective of this study was therefore to develop a quantitative property - property relationship ( qppr ) model of animal data to generate initial estimates ( or bounds ) of intrinsic clearance of vocs , for eventual incorporation within a human pbpk model to simulate blood concentration profiles associated with inhalation exposures . in this regard , we focused on evaluating the impact of the uncertainty associated with qppr predictions of clint on the blood kinetics of vocs in humans , relative to that of the uncertainty associated with the total lack of knowledge of the metabolic rate in humans . furthermore , the reliability of applying the qppr to predict the area under the blood concentration versus time curve ( auc ) of parent chemicals was evaluated , as a function of the sensitivity of the metabolism parameter in the pbpk model and the prediction uncertainty of qppr model . the qppr predictions were then compared with experimental data for several vocs and the pharmacokinetics in humans were simulated using integrated qppr - pbpk models for these 26 vocs . the predictions of qppr were evaluated further with an external data set of clint for 11 vocs . the development of a global qppr model for metabolism was initially undertaken using experimental data on the in vivo intrinsic clearance of 26 vocs in rats , collated and evaluated in previous studies by bliveau et al . [ 40 , 41 ] ( 1,1,1,2-tetrachloroethane ; 1,1,2,2-tetrachloroethane ; 1,1,2-trichloroethane ; 1,1-dichloroethane ; 1,1-dichloroethylene ; 1,2-dichloroethane ; benzene ; bromochloromethane ; bromodichloromethane ; carbon tetrachloride ; chloroethane ; chloroform ; cis-1,2-dichloroethylene ; dibromomethane ; dichloromethane ; ethylbenzene ; hexachloroethane ; isoprene ; methyl chloride ; m - xylene ; n - hexane ; pentachloroethane ; styrene ; toluene ; trichloroethylene ; vinyl chloride ) [ 24 , 4653 ] . subsequently , the resulting qppr model was evaluated with experimental in vivo data on clint for 11 additional vocs in rats ( 1,1,1-trichloroethane ; 1,2,4-trimethylbenzene ; bromoform ; dibromochloromethane ; furan ; halothane ; o - xylene ; trans-1,2-dichloroethylene ; tetrachloroethylene ; propylene ; ethylene ) [ 46 , 48 , 5461 ] . these 11 chemicals outside the calibration set were also lipophilic , low - molecular - weight vocs and likely substrates of cytochrome p450 2e1 [ 32 , 62 ] . moreover except for halothane and 1,2,4-trimethylbenzene , the chemicals of the evaluation dataset possess values of pow , ionization potential , and blood : water pc within the range of values for the chemicals in the qppr calibration set . for qppr modeling , clint ( expressed in units of l blood , clintblood , or l phospholipids , clintpl ) initially , clintblood ( l blood / h / kg ) for all the studied chemicals was computed as allometrically scaled vmax ( mol / h / kg)/km ( mol / l blood ) . since cyps are located in the endoplasmic reticulum embedded in the phospholipidic bilayer , the clintpl values reflecting chemical affinity for the phospholipids ( pl ) were subsequently computed . the values of clintpl ( l phospholipid / h / kg ) were obtained by dividing vmax ( mol / h / kg ) with km expressed as mol / l pl . the km values in m of pl were obtained by multiplying the values of km expressed as mol / l blood with the chemical - specific phospholipid : blood partition coefficients ( pplb ) calculated as follows : ( 1)pplb=0.3poa+0.7pwapba , where poa is the n - octanol : air pc , pwa the water : air pc , and pba the blood : air pc . the above equation computes pplb as the ratio of phospholipid : air to blood : air pcs of the vocs , based on poulin and krishnan [ 10 , 12 ] . the input parameters required for converting the clint obtained from the literature were poa , pwa , and pba . ( 1 ) poa and pwathe n - octanol : air pc ( poa ) , was calculated as the product of the n - octanol : water pc ( pow ) and pwa ( inverse of henry 's law constant at 37.5c ) . the n - octanol : air pc ( poa ) , was calculated as the product of the n - octanol : water pc ( pow ) and pwa ( inverse of henry 's law constant at 37.5c ) . ( 2 ) pbaexperimental values were used for rat blood : air [ 54 , 56 , 59 , 6468 ] . the calculated values of pplb for the chemicals used for the development and for the evaluation of the qppr are reported in table 1 . experimental values were used for rat blood : air [ 54 , 56 , 59 , 6468 ] . the calculated values of pplb for the chemicals used for the development and for the evaluation of the qppr are reported in table 1 . the rate and affinity for p450-mediated metabolism would appear to be related to the size , shape , charge , and energy of the substrate ; therefore variables that reflect these properties were chosen for the qppr analysis [ 21 , 23 , 27 , 28 , 32 , 6971 ] . the descriptors of the size and shape of the molecule were the molecular length , width , depth , volume , surface , and the kappa 2 index , as well as two descriptors used in the work of lewis et al . , namely , the ratio of the molecular length to the molecular width ( l / w ) and the ratio of the area of the molecule ( i.e. , length times width ) to the square of the depth ( a / d ) . the dipole moment and ionization potential ( ip ) were used as measure of the charge disposition and the energy in the molecule , respectively . the values of all the previously cited descriptors were calculated using commercially available software ( molecular modeling pro , chem sw , fairfield , ca ) . before calculating the molecular descriptors with molecular modeling pro , the 3d molecules were drawn and minimized using the full mm2 ( molecular mechanics program ) method provided in the software . the dipole moment and the ionization potential hydrophobic descriptors such as log pow ( log of the n - octanol : water pc ) that reflect hydrogen bonding and - stacking have already been correlated to the values of metabolic constants [ 6971 ] . in this study , the following physicochemical parameters were chosen to describe the relative solubility and partitioning into diverse biological media : log pow , log phospholipid : water pc ( log pplw ) ; log blood : water pc ( log pbw ) , and log water : air pc ( log pwa ) . the blood : water and phospholipid : water pcs were obtained by dividing the blood : air and phospholipid : air pcs values by the water : air pc values . the values of pow , pwa , blood : air , and phospholipid : air pcs were obtained as described for the calculation of pplb ( 1 ) . multilinear regression analysis approach was chosen for the qppr analysis of clint because linear regression models are simple , transparent , and easy to reproduce . stepwise regression analysis was performed to select the qpprs based on the most statistically significant independent variable(s ) from an a priori list ( see section 2.1.4 ) . the coefficient of determination r , the adjusted r ( radj ; adjusted for number of variables ) , the standard error of the estimate s , and the value and significance of the f statistic were calculated . the normality of the residuals was checked visually on normal probability plots of the standardized residuals ( i.e. , expected normal cumulative probability versus observed cumulative probability ) . leave - one - out cross - validation was conducted and the results were expressed in terms of q , a measure of precision error of the model . the q was computed as follows : ( 2)q2=1pressssy , where press is that predicted residual sum of squares and ssy the sum of squares of the response values . the statistical significance ( p < 0.05 ) of the regression coefficients was estimated by a t statistic test . multicollinearity refers to the occurrence of correlation between two independent variables in the multiple linear regression model . multicollinearity of the variables in the model was assessed by calculating the variance inflation factor ( vif ) for all independent variables . the value of vif was calculated as follows : ( 3)vifi=11ri2 , where vifi is the variance inflation factor of the independent variable i in the multilinear regression model and ri the coefficient of determination of the regression between the independent variable i and the other independent variables in the multilinear regression model . for each model , the application domain was documented by reporting the ranges of values of the descriptors , the modeled response , and the endpoint . a qppr model was considered adequate when : the values of r and radj were 0.6 , the value of q was 0.6 , and the independent variables were not highly correlated ( i.e. , vif < 4 ) . the predictions of the qppr model were obtained in terms of lower and upper bounds of the 95% mean confidence intervals ( lmci and umci , resp . ) in order to represent the uncertainty associated with the mean predicted value . the lmci and umci for the 11 vocs , not in the qspr calibration dataset , were obtained by adding them in the spss file containing the data used for the qppr , along with the values of their independent variables only . in the pbpk model , the value of intrinsic clearance was calculated as the product of the qppr value of clintpl ( l of pl / h / kg ) and the phospholipid : blood pc ( values of pplb in table 1 ) . the intrinsic clearance ( l blood / h / kg ) was used within the human pbpk models to compute the hepatic clearance . the rate of metabolism was calculated on the basis of hepatic clearance ( i.e. , hepatic clearance times the arterial concentration ) [ 4 , 40 , 41 , 45 ] . for chloroethane , dichloromethane , vinyl chloride , and dibromomethane a first - order constant ( 1 , 2 , 1 , and 0.7 h , resp . ) was included in the calculation of the hepatic clearance , clh ( l / h ) : ( 4)clh = qle , where e = ( clint + kf vl)/((clint + kf vl ) + ql ) , ql is the blood flow through the liver ( l / h ) , clint the intrinsic clearance ( l blood / h ) , kf the first order metabolic constant ( h ) , and vl the liver volume ( l ) . the qppr values of clint were included in a human pbpk model for inhaled vocs . briefly , the pbpk model consisted in four tissue compartments ( i.e. , liver , fat , richly , and poorly perfused tissues ) and a gas exchange lung , which were interconnected by blood flows . the distribution of vocs into tissue compartments was described as perfusion limited , and the metabolism was limited to liver . to evaluate the impact of uncertainty on the metabolic rate , for all the chemicals , pbpk simulations were also conducted by setting the value of e to 0.999 ( emax ) and then to 0.001 ( emin ) , respectively . the human physiological parameters of the pbpk model ( i.e. , body weight = 70 kg ; cardiac output = 18 l / h / kg ; alveolar ventilation = 18 l / h / kg ; tissue compartment volumes , fraction of body weight : liver = 0.026 ; richly perfused tissues = 0.05 ; poorly perfused tissues = 0.62 ; fat = 0.19 ; perfusion of the tissue compartments , fraction of cardiac output : liver = 0.26 ; richly perfused tissues = 0.44 ; poorly perfused tissues = 0.25 ; fat = 0.05 ) were obtained from tardif et al . . table 1 presents the value of the partition coefficients used in the pbpk model ( i.e. , blood : air , tissue : blood , and phospholipid : blood pcs ) . the phospholipid : blood pc was calculated using ( 1 ) , whereas the blood : air pc and tissue : blood pcs were gathered from the literature [ 48 , 50 , 52 , 54 , 5659 , 61 , 66 , 67 , 7780 ] . the pbpk model ( differential and algebraic mass - balance equations , physiological parameters , qspr equations for metabolic constants , and pcs ) was written in acsl ( acslx , version 2.5 , aegis technologies group , inc , huntsville , al ) . the model code is included in the supplementary data available online at doi:10.1155/2012/286079 . to compare the impact of different ( uncertain ) scenarios of rate of metabolism on the pharmacokinetics in human , simulations were carried out by setting ( i ) the value of clint equal to the lower and upper bound of the qppr predicted mean 95% confidence interval , or ( ii ) the liver extraction ratio to 0.001 ( no metabolism ) and 0.999 ( maximum extraction ) . the 24 h venous blood kinetics corresponding to the four scenarios of metabolism were simulated for an 8 h exposure to 1 ppm of each voc . the 24 h area under the curve ( auc24 ) of the venous blood kinetics was also calculated to compare the four scenarios of metabolism simulated with pbpk models . additionally , the venous blood kinetics of m - xylene , toluene , ethylbenzene , dichloromethane , styrene , 1,2,4-trimethylbenzene , and 1,1,1-trichloroethane were compared to experimental data [ 61 , 67 , 8183 ] . the applicability of the qppr model was evaluated on the basis of the level of uncertainty in the qppr estimate and the impact ( sensitivity ) of metabolism on the auc24 . figure 1 illustrates the role of uncertainty and sensitivity in the reliability of the qppr - pbpk modeling framework , based on reference . the sensitivity of the metabolism to the auc was estimated by the ratio of the auc24 obtained with no metabolism ( emin ) to that obtained with the maximum theoretical metabolism ( emax ) . the sensitivity of auc24 to metabolism was considered to be low , medium , or high if the ratio ( aucemin/aucemax ) was within a factor of 2 , within an order of magnitude , or greater . the uncertainty in the qppr prediction was evaluated by comparing it to the experimental data . the prediction uncertainty was considered to be low , medium or high if the prediction was within a factor of two , within an order of magnitude and above 10-fold of the experimental data , respectively . this approach was applied to evaluate the reliability of applying the qppr within the pbpk model for two situations : ( i ) for the calibration set of chemicals , for which the uncertainty of the qppr was evaluated by comparing the predictions of clintpl with the experimentally derived clintpl values and ( ii ) for chemicals in the evaluation dataset , for which the uncertainty in the qppr prediction was considered to be high , to replicate the data poor situations with new or tested chemicals with unknown experimental clint values . the initial effort to develop a qppr model for metabolism rate ( expressed as clintblood , in units of l blood / hr ) , based on a stepwise analysis of its relationship to various molecular descriptors and physicochemical properties , was not successful ( not shown ) . same analysis , repeated for clint expressed in units of l pl / h ( clintpl ) , yielded a qppr that consisted of log pplw , log pbw , and ip ( ionization potential , ev ) as input parameters . this model satisfied the criteria for an acceptable model in terms of coefficient of determination ( r = 0.802 ; radj = 0.775 ) , leave - one - out cross validation ( q = 0.755 ) , and multicollinearity ( vifs : log pplw = 2.42 ; log pbw = 2.38 ; ip = 1.04 ) . the values of the regression coefficients were significant ( p value < 0.001 for the constant , log pplw and log pbw , and 0.007 for ip ) . however , as the value of log pow can be obtained more readily than log pplw , the regression analysis was repeated by using log pow , log pbw , and calculated ip , and it yielded the following qppr : ( 5)log clintpl=5.63(1.187)1.287(0.149)log pow + 1.08(0.233)log pbw 0.328(0.111)ip . this qppr model satisfied the criteria for an acceptable model in terms of coefficient of determination ( r = 0.796 ; radj = 0.768 ) , leave - one - out cross validation ( q = 0.748 ) , and multicollinearity ( vifs : logpow = 2.42 ; log pbw = 2.38 ; ip = 1.04 ) . the application domain of the model can be described with [ min ; max ] as follows : log pow = [ 1.09 ; 4.03 ] ; logpbw [ 0.16 ; 2.49 ] ; calculated ionization potential [ 9.13;11.28 ] . the qppr ( 5 ) was subsequently applied to calculate the clintpl of the vocs in the calibration set . table 2 presents the values of the input parameters , along with the experimental data for the 26 vocs used in qppr development . figure 2 illustrates the comparison of the predicted values of clintpl ( lmci and umci ) and the experimental data . the uncertainty in the predicted log clintpl can be characterized by the difference between the umci and the lmci ; this value ranged from 0.37 ( 1,1-dichloroethane ) to 1.23 ( n - hexane ) with a mean of 0.54 and a standard deviation of 0.18 . the nearest confidence bounds of the predicted log clintpl were higher than 5-fold of the experimental value ( exp . ) for three substances ( cis-1,2-dichloroethylene , lmci = 0.55 versus exp. = 0.09 ; styrene , lmci = 0.45 versus exp. = 0.09 ; and 1,1,2-trichloroethane , umci = 0.46 versus exp. = 0.02 ) . the impact of the imprecision of these qppr predictions of the metabolic constants on the pharmacokinetics in humans was then evaluated by pbpk modeling . figure 3 presents the predictions of the 24 h blood pharmacokinetics following 8 h exposure to 1 ppm of each of the 26 vocs used in the qppr analysis . the bold lines represent the simulations obtained using 0 and 1 as the hepatic extraction ratio , whereas the grey area encompassed by thin lines represents the simulation obtained using lmci and umci of predicted clint in pbpk models . overall , the envelope of the concentrations predicted using the qppr predictions reduced the region of uncertainty associated with the complete lack of knowledge of hepatic extraction ratio in humans ( i.e. , ranging from 0 to 1 ) . the average ratio ( standard deviation ) of the pbpk model simulated values of the end - of - exposure blood concentrations ( i.e. , cmax ) obtained with emin and emax was 4.19 1.81 . the lowest and highest ratios , based on the theoretical bounds of hepatic extraction ( i.e. , emin and emax ) , were observed for isoprene ( 1.63 ) and 1,1,2,2-tetrachloroethane ( 8.05 ) , respectively . however , the average ratio ( standard deviation ) of the pbpk model simulated values of the end - of - exposure blood concentrations , based on qppr - generated bounds ( lmci , umci ) , was 1.29 0.27 . this ratio was the highest for hexachloroethane ( 2.39 ) and the lowest for 1,1-dichloroethylene ( 1.06 ) . for the 26 vocs used in the development of the qppr , the values of auc24s for a 1 ppm continuous exposure the ratio of the highest to the lowest auc predicted with emin and emax was 4.3 1.94 ranging from 1.63 ( isoprene ) to 8.7 ( 1,1,2,2-tetrachloroethane ) . the ratio of the maximum to minimum concentrations predicted using the qppr metabolism rate was 1.36 0.4 ranging from 1.06 ( 1,1-dichloroethylene ) to 2.8 ( isoprene ) . figure 4 illustrates the range of predictions of venous blood pharmacokinetics compared to experimental data [ 67 , 81 , 82 ] . overall , the predicted envelope of concentrations approximated reasonably the experimental data for dichloromethane , ethylbenzene , styrene , toluene , and m - xylene . the reliability of applying the qppr within the pbpk model was assessed for the 26 vocs in the calibration dataset ( table 4 ) . the uncertainty of the qppr prediction was estimated as the ratio of predicted clintpl to experimental clintpl . for 3 vocs ( isoprene , 1,1-dichloroethylene , and vinyl chloride ) the sensitivity of auc to clint was low ( ratio of aucs < 2 ) whereas uncertainty of the clint qppr was low for isoprene and vinyl chloride and medium for 1,1-dichloroethylene . for the other 23 vocs , the ratio of aucs was between 2 and 5 . for 16 of the later 23 vocs ( benzene ; bromochloromethane ; bromodichloromethane ; chloroform ; dibromomethane ; 1,2-dichloroethane ; hexachloroethane ; n - hexane ; pentachloroethane ; styrene ; 1,1,1,2-tetrachloroethane ; 1,1,2,2-tetrachloroethane ; toluene ; 1,1,2-trichloroethane ; trichloroethylene ; m - xylene ) the prediction uncertainty was low , thus the confidence in using the qppr in the pbpk model is high for these compounds . the uncertainty was medium for the prediction of clintpl for 7 vocs ( carbon tetrachloride ; chloroethane ; 1,1-dichloroethane ; cis-1,2-dichloroethylene ; dichloromethane ; ethylbenzene ; methyl chloride ) . therefore , for these chemicals , the confidence in using the qppr in an inhalation pbpk model to evaluate the auc is medium . the qppr model was applied to predict the clintpl of 11 vocs that were not in the calibration dataset . table 5 presents the values of the input parameters along with the experimental data for the 11 vocs used in qppr evaluation . figure 5 illustrates the comparison of the predicted values of clintpl ( lmci and umci ) and the experimental data . the average difference ( standard deviation ) between the umci and the lmci was 0.57 0.11 ranging from 0.46 ( bromoform ) to 0.84 ( 1,2,4-trimethylbenzene ) . the highest umci - lmci ranges were obtained for furan ( 0.62 ) , tetrachloroethylene ( 0.63 ) , and 1,2,4-trimethylbenzene ( 0.84 ) . the nearest predicted values of umci and lmci on log clintpl were greater than 5-fold of the experimental data for tetrachloroethylene ( lmci = 0.02 versus exp = 1.8 ) . as in the qppr development section , the impact of the imprecision on these log clint predictions on the pharmacokinetics in humans was evaluated by pbpk modeling . figure 6 presents the predictions of the 24 h blood pharmacokinetics following 8 h exposure to 1 ppm of each of the 11 vocs used in the qppr evaluation . the bold lines represent the simulations obtained using 0 and 1 as the hepatic extraction ratio , whereas the grey area encompassed by thin lines represents the simulation obtained using lmci and umci of predicted clint in pbpk models . the reduction of the region of uncertainty associated with the complete lack of knowledge of hepatic extraction ratio in humans ( i.e. , ranging from 0 to 1 ) by the envelope of the concentrations predicted using the qppr predictions was observed for the 11 vocs . the mean ratio ( standard deviation ) of the pbpk model simulated values of the end - of - exposure blood concentrations obtained with emin and emax was 3.92 2.13 ranging from 1.42 ( ethylene ) to 7.45 ( bromoform ) . however , the same average ratio ( standard deviation ) of pbpk simulated blood concentrations , based on qppr - generated bounds ( lmci and umci ) was 1.2 0.1 , ranging from 1.07 ( ethylene ) to 1.33 ( bromoform ) . table 6 presents the values of the auc24s ( mg / l - h ) for the 11 vocs used in the evaluation of the qppr . the average ratio of the highest to lowest auc predicted using emin and emax was 4.08 2.31 ( mean sd ) . the lowest and highest ratios , based on the theoretical bounds of hepatic extraction ( i.e. , e = 0.001 or 0.999 ) , the ratio of the maximum to minimum concentrations predicted using the qppr metabolism rate was 1.2 0.1 , ranging from 1.07 ( propylene ) to 1.33 ( dibromochloromethane ) . figure 7 illustrates the range of predictions for two of the chemicals in the external dataset ( 1,2,4-trimethylbenzene and 1,1,1-trichloroethane ) venous blood pharmacokinetics compared to experimental data [ 61 , 85 ] . envelope of concentrations simulated reasonably the experimental data for 1,2,4-trimethylbenzene whereas the blood concentrations of 1,1,1-trichloroethane were underestimated by about 30% . the reliability of applying the qppr within the pbpk model was assessed for the 11 vocs in the evaluation dataset , using the framework shown in figure 1 . considering that the experimental data of clintpl for new or untested chemicals will be essentially unknown , it is realistic to consider the uncertainty of the qppr prediction of clintpl to be high for all chemicals in the evaluation dataset . the results of the analysis of applicability for the chemicals in the evaluation dataset are reported in table 7 . for 3 vocs ( ethylene ; propylene ; 1,1,1-trichloroethane ) the sensitivity was low ( ratio of aucs < 2 ) thus the reliability of using their clint qppr in the pbpk was considered high . for the other 8 vocs ( bromoform ; dibromochloromethane ; trans-1,2-dichloroethylene ; furan ; halothane ; tetrachloroethylene ; 1,2,4-trimethylbenzene ; o - xylene ) , the ratio of the maximum to the minimum possible aucs was between 2 and 5 , such that the confidence in using the qppr in an inhalation pbpk model to evaluate the aucs is medium for these chemicals . sars , qsar , qsprs , and qpprs have been developed for various toxicological and chemical properties but only very few studies have focused on developing such models to parameterize pbpk models [ 8 , 86 ] . a limitation in developing pbpk models relates to the availability of the metabolic constants ( clint , vmax , and km ) . quantitative relationships between structure and metabolism rates have been investigated for a limited number of closely related compounds , even though their applicability to pbpk modeling has not been demonstrated ( e.g. , qspr models for kcat and 1/km ) . other works in this area relate to the development of quantum chemical or quantum dynamic methods for prediction of activation energy or enthalpy of activation of p450 mediated reactions [ 20 , 25 , 26 , 31 , 36 , 38 , 8891 ] , which have not been used to derive metabolism constants for direct incorporation within rodent or human pbpk models . the use of the group contribution method to develop qsprs for integration within pbpk models has been successfully demonstrated , particularly for the inhalation toxicokinetics of vocs [ 4043 ] . this approach however is limited to vocs containing one or more of the molecular groups or fragments for which the contribution has been evaluated ( i.e. , ch3 , ch2 , ch , c , c = c , benzene ring , h on benzene ring , and halogens ) . in order to extend the applicability domain then , it is important to investigate the feasibility of developing qsprs based on more global , physicochemical properties . in this regard , the present study investigated the development of a qppr , that used chemical properties rather than chemical structure as input , and it was calibrated to predict clint expressed in terms of chemical affinity to phospholipids in the endoplasmic reticulum in which cyp enzymes are embedded . this logical transformation of clint data , reported here for the first time in literature , facilitated the development of more adequate qppr than the conventional clint based on blood concentrations . all efforts to develop qpprs for predicting clint based on blood concentrations were unsuccessful . the qppr based chemical affinity to phospholipids obtained in this study should be regarded as a screening level tool to provide plausible range of metabolism rates in order to facilitate a first - cut evaluation of the blood concentration of inhaled vocs in humans . the uncertainty associated with this qppr tool should be evaluated along with the sensitivity of clint on the dose metrics of the chemical of interest , in the perspective of intended precision . accordingly , if the dose metric is highly sensitive to clint and the qppr predictions of clint are highly uncertain , then the present tool is of limited use even for screening purposes . in such cases , then in vivo or in vitro studies can be undertaken to get chemical - specific estimates of clint. the qppr predictions were reasonably in accordance with experimental values for most but not all chemicals in the calibration and evaluation datasets . for some chemicals , the predicted values of log clint for 1,1,1-trichloroethane ( figure 5(a ) ) and tetrachloroethylene ( figure 5(k ) ) exceeded the experimental values by two orders of magnitude . the qspr for rat hepatic clearance developed by bliveau and colleagues also overestimated the metabolic rate of these two vocs . however the pbpk model for 1,1,1-trichlorethane indicated that the auc of parent chemical in venous blood is not sensitive to vmax and km [ 92 , 93 ] . this was demonstrated in figure 6(a ) , showing that qppr - overestimation of clintpl of 1,1,1-trichloroethane led only to a minimal impact , in terms of the underestimation of the venous blood concentration . in the case of tetrachloroethylene , a poorly metabolized halogenated voc , the overestimation of the clintpl led to a 3-fold underestimation of the cmax ( figure 6(k ) ) or a 4 - 5-fold underestimation of the auc24 ( table 7 ) . if this magnitude of error is not acceptable for screening - level evaluation , then the metabolic rate should be experimentally determined . the combined assessment of the uncertainty / sensitivity of metabolic constants in pbpk models would facilitate the determination of the applicability of the qppr model , given the level of precision need for an application ( figure 1 ) the qppr developed in this study is a generic tool to provide initial estimates of clint of vocs metabolized by hepatic cyp . it does not take into account stereochemistry or other pathway - specific rates and processes , which may be important for some chemicals ( e.g. , predicted values of clint are almost identical for 1,1-dichloroethylene and cis-1,2-dichloroethylene but experimental values vary by log units of 1.06 ) . therefore , predictions of clint based on generic considerations are likely to be inaccurate for specific chemicals but are of limited use in that the estimates ( along with the bounds , representing the level of uncertainty ) can be integrated with human physiology to provide a first - cut view of the plausible kinetic profiles . the utility of the qppr models depends , in part , on the ability to reproducibly calculate the descriptors . hence , in this study , the descriptors that could be easily calculated and interpreted were chosen and obtained using episuite ( for log pow and pwa ) and mmpro ( for the ionization potential ) . however , the blood solubility parameter ( i.e. , blood : air pc ) is additionally required and this can either be obtained experimentally in vitro or using other qsars that account for protein ( i.e. , haemoglobin and plasma protein ) binding in addition to solubility considerations . there are some algorithms and qsars available in this regard , but further development is necessary to adequately account for the protein binding phenomena in human blood for various classes of chemicals . the qppr developed in this study computes clintpl , which can then be converted to clintblood for use in pbpk modeling . in an effort to evaluate whether the same input parameters can be used to relate to clintblood these yielded the following equation ( significant terms only ) : ( 6)log clintblood=5.1170.305logpow0.324ip . even though ( 5 ) and ( 6 ) give almost identical results ( one for clintpl and the other for clintblood ) despite the differing r values ( 0.796 versus 0.402 ) , it should be noted that ( 5 ) was obtained based on statistical analysis of calibration dataset ( i.e. , modeling ) whereas ( 6 ) was derived simply by fitting clintblood to the specific input parameters . further rearrangements and simplifications of the qppr , as well as the loss of accuracy associated with such attempts , were not performed in the current study . the output of the qppr developed in the present study is log clint , which is useful for simulating pharmacokinetics in humans of chemicals at low levels of exposure . clint is applicable to first - order situations ( i.e. , when blood levels in humans are much lower than the km for metabolizing enzyme ) and is derived by dividing the vmax ( i.e. , the enzyme turn - over ) with km ( representing the affinity of the substrate for the enzyme ) . the input parameters of the qppr , namely , log pow and log pbw , are estimates of the relative solubility in octanol , water , and blood . then , an interpretation of the model for clint could be that the binding to the p450 enzyme is a result of hydrophobic interactions which , in turn , can be estimated with parameters reflective of the solubility in n - octanol and blood . the solubility in blood is the sum of the solubility in its components ( water , phospholipids , neutral lipids , and proteins ) . most of the studied vocs are likely to bind to hemoglobin because of their lipophilicity ( log pow value above 1 ) and low molecular volume . the pbw , thus , is likely an indicator of the binding to proteins , whereas the log pow reflects more the affinity for biotic lipids in the metabolism microenvironment . similar to log pow , the ionization potential has already been correlated with metabolic rates , namely , the vmax and vmax/km , as this latter parameter could be correlated with the energy needed to break a covalent bond for the oxidation of the substrate . the qspr model for clint developed in this study has a defined theoretical endpoint , is nonambiguous , has a defined domain of application , was analyzed using appropriate goodness - of - fit ( r ) and robustness ( q ) , and has an attempt of mechanistic interpretation . the in vivo dataset on 26 vocs used for the qppr calibration was chosen because it was previously collated and used in qspr analyses [ 40 , 41 ] . the qspr analysis was also attempted with the entire dataset of 37 vocs ( calibration + external dataset ) but it did not improve the goodness - of - fit statistics ( not shown ) . the predicted bounds of the 95% confidence interval of intrinsic clearance were incorporated within a pbpk model to predict the blood toxicokinetics of vocs . the simulations of blood kinetics were comparable to experimental data for 6 vocs ( toluene , m - xylene , ethylbenzene , styrene , dichloromethane,1,2,4-trimethylbenzene , and 1,1,1-trichloroethane , figures 4 and 7 ) . the simulations obtained in the present study , using lower and upper confidence intervals on the mean predicted clint , reduced clearly the uncertainty bounds associated with the total lack of knowledge ( i.e. , e ranging anywhere between 0 and 1 ) . furthermore , the present study incorporated the qppr predictions of clint along with physiological parameters , such that impact on in vivo kinetics could be simulated . in effect , in some cases where the uncertainty on clint predictions was high , it did not translate into a proportionate error on the predictions of kinetics , due to the additional consideration of physiological constraints , and such observations are critical in data - poor situations for designing focused studies to generate chemical - specific data in vitro or in vivo . the qppr developed in this study approximated the experimental rat metabolic constants for the various low - molecular - weight vocs ; and it was used along with the human physiology to generate initial or screening level values of clint to construct human pbpk models that could be of potential use to interpret data such as measured biomarker levels or for designing kinetic studies to reduce database uncertainty . as shown with some vocs ( e.g. , figure 3 : 1,1,1,2-tetrachloroethane , hexachloroethane , and n - hexane ) , the blood concentration profile is extremely influenced by clint , such that metabolism can not be neglected in simulating or interpreting human exposure data . and in such cases , the ability to generate at least a range of plausible values of clint , as done in the present study , would facilitate first in - human simulations of pharmacokinetics of parent chemicals . integrating information on the impact of metabolism on dose metrics ( i.e. , auc ) along with prediction uncertainty of the qppr facilitates the determination of the level of confidence in using this screening level tool . depending upon the overall confidence in the qppr application for predicting dose metrics ( low , medium , and high ) relative to the use purposes , overall , the qppr developed in the present study allows to predict the clint of vocs on the basis of generic molecular descriptors rather than with fragment constants as done previously . the chemical concentration in phospholipids , for the first time , was found to be a dose metric amenable to qppr analysis . the qppr was then used to generate range of values of clint ; the level of confidence in these estimates was assessed by considering the impact of clint on the simulated dose metrics ( i.e. , auc of parent chemical in venous blood ) . for other dose metrics and situations , a more robust qppr needs to be developed , and such efforts can be based on the methodological developments accomplished in this study . the qppr - based simulation of pharmacokinetics reduced the range of uncertainty for few substances relative to complete lack of knowledge of the clint , but it needs to be evaluated / refined with much larger dataset should this screening - level approach be adopted for providing more precise estimates of metabolism rates . overall , the integrated qppr - pbpk model developed in this study is a potentially useful tool for characterizing and reducing the uncertainty associated with the complete lack of knowledge of clint in predicting human pharmacokinetics of inhaled vocs .

the objectives of this study were ( i ) to develop a screening - level quantitative property - property relationship ( qppr ) for intrinsic clearance ( clint ) obtained from in vivo animal studies and ( ii ) to incorporate it with human physiology in a pbpk model for predicting the inhalation pharmacokinetics of vocs . clint , calculated as the ratio of the in vivo vmax ( mol / h / kg bw rat ) to the km ( m ) , was obtained for 26 vocs from the literature . the qppr model resulting from stepwise linear regression analysis passed the validation step ( r2 = 0.8 ; leave - one - out cross - validation q2 = 0.75 ) for clint normalized to the phospholipid ( pl ) affinity of the vocs . the qppr facilitated the calculation of clint ( l pl / h / kg bw rat ) from the input data on log pow , log blood : water pc and ionization potential . the predictions of the qppr as lower and upper bounds of the 95% mean confidence intervals ( lmci and umci , resp . ) were then integrated within a human pbpk model . the ratio of the maximum ( using lmci for clint ) to minimum ( using umci for clint ) auc predicted by the qppr - pbpk model was 1.36 0.4 and ranged from 1.06 ( 1,1-dichloroethylene ) to 2.8 ( isoprene ) . overall , the integrated qppr - pbpk modeling method developed in this study is a pragmatic way of characterizing the impact of the lack of knowledge of clint in predicting human pharmacokinetics of vocs , as well as the impact of prediction uncertainty of clint on human pharmacokinetics of vocs .

1. Introduction 2. Methods 3. Results 4. Discussion

the group contribution method was successfully used to develop quantitative structure - property relationships ( qsprs ) for the tissue : air partition coefficients as well as intrinsic ( clint ) and hepatic clearance ( clh for a group of low - molecular - weight volatile organic chemicals ( vocs ) in rats [ 41 , 42 ] . more recently , qsprs were developed for the metabolic constants vmax ( maximum velocity of reaction ) and km ( michaelis constant ) and were further incorporated within a rat pbpk model to predict the toxicokinetics of mixtures of vocs . to extend the currently available qspr for clint to cover more diverse fragments and at the same time respect a reasonable ratio of the number of parameters to the number of observations , since the critical limitation in the construction of pbpk models for new substances continues to be the metabolism rate , a pragmatic approach particularly for inhaled vocs is to evaluate the maximum and minimum possible blood concentration profiles in exposed individuals . therefore , the availability of in silico approaches based on easily available parameters to predict plausible range of clint would be desirable as a screening - level tool . the objective of this study was therefore to develop a quantitative property - property relationship ( qppr ) model of animal data to generate initial estimates ( or bounds ) of intrinsic clearance of vocs , for eventual incorporation within a human pbpk model to simulate blood concentration profiles associated with inhalation exposures . in this regard , we focused on evaluating the impact of the uncertainty associated with qppr predictions of clint on the blood kinetics of vocs in humans , relative to that of the uncertainty associated with the total lack of knowledge of the metabolic rate in humans . furthermore , the reliability of applying the qppr to predict the area under the blood concentration versus time curve ( auc ) of parent chemicals was evaluated , as a function of the sensitivity of the metabolism parameter in the pbpk model and the prediction uncertainty of qppr model . the qppr predictions were then compared with experimental data for several vocs and the pharmacokinetics in humans were simulated using integrated qppr - pbpk models for these 26 vocs . the development of a global qppr model for metabolism was initially undertaken using experimental data on the in vivo intrinsic clearance of 26 vocs in rats , collated and evaluated in previous studies by bliveau et al . subsequently , the resulting qppr model was evaluated with experimental in vivo data on clint for 11 additional vocs in rats ( 1,1,1-trichloroethane ; 1,2,4-trimethylbenzene ; bromoform ; dibromochloromethane ; furan ; halothane ; o - xylene ; trans-1,2-dichloroethylene ; tetrachloroethylene ; propylene ; ethylene ) [ 46 , 48 , 5461 ] . moreover except for halothane and 1,2,4-trimethylbenzene , the chemicals of the evaluation dataset possess values of pow , ionization potential , and blood : water pc within the range of values for the chemicals in the qppr calibration set . for qppr modeling , clint ( expressed in units of l blood , clintblood , or l phospholipids , clintpl ) initially , clintblood ( l blood / h / kg ) for all the studied chemicals was computed as allometrically scaled vmax ( mol / h / kg)/km ( mol / l blood ) . the values of clintpl ( l phospholipid / h / kg ) were obtained by dividing vmax ( mol / h / kg ) with km expressed as mol / l pl . the above equation computes pplb as the ratio of phospholipid : air to blood : air pcs of the vocs , based on poulin and krishnan [ 10 , 12 ] . ( 1 ) poa and pwathe n - octanol : air pc ( poa ) , was calculated as the product of the n - octanol : water pc ( pow ) and pwa ( inverse of henry 's law constant at 37.5c ) . the n - octanol : air pc ( poa ) , was calculated as the product of the n - octanol : water pc ( pow ) and pwa ( inverse of henry 's law constant at 37.5c ) . the descriptors of the size and shape of the molecule were the molecular length , width , depth , volume , surface , and the kappa 2 index , as well as two descriptors used in the work of lewis et al . , namely , the ratio of the molecular length to the molecular width ( l / w ) and the ratio of the area of the molecule ( i.e. the dipole moment and the ionization potential hydrophobic descriptors such as log pow ( log of the n - octanol : water pc ) that reflect hydrogen bonding and - stacking have already been correlated to the values of metabolic constants [ 6971 ] . in this study , the following physicochemical parameters were chosen to describe the relative solubility and partitioning into diverse biological media : log pow , log phospholipid : water pc ( log pplw ) ; log blood : water pc ( log pbw ) , and log water : air pc ( log pwa ) . leave - one - out cross - validation was conducted and the results were expressed in terms of q , a measure of precision error of the model . the predictions of the qppr model were obtained in terms of lower and upper bounds of the 95% mean confidence intervals ( lmci and umci , resp . ) the lmci and umci for the 11 vocs , not in the qspr calibration dataset , were obtained by adding them in the spss file containing the data used for the qppr , along with the values of their independent variables only . in the pbpk model , the value of intrinsic clearance was calculated as the product of the qppr value of clintpl ( l of pl / h / kg ) and the phospholipid : blood pc ( values of pplb in table 1 ) . the intrinsic clearance ( l blood / h / kg ) was used within the human pbpk models to compute the hepatic clearance . was included in the calculation of the hepatic clearance , clh ( l / h ) : ( 4)clh = qle , where e = ( clint + kf vl)/((clint + kf vl ) + ql ) , ql is the blood flow through the liver ( l / h ) , clint the intrinsic clearance ( l blood / h ) , kf the first order metabolic constant ( h ) , and vl the liver volume ( l ) . the qppr values of clint were included in a human pbpk model for inhaled vocs . , body weight = 70 kg ; cardiac output = 18 l / h / kg ; alveolar ventilation = 18 l / h / kg ; tissue compartment volumes , fraction of body weight : liver = 0.026 ; richly perfused tissues = 0.05 ; poorly perfused tissues = 0.62 ; fat = 0.19 ; perfusion of the tissue compartments , fraction of cardiac output : liver = 0.26 ; richly perfused tissues = 0.44 ; poorly perfused tissues = 0.25 ; fat = 0.05 ) were obtained from tardif et al . the phospholipid : blood pc was calculated using ( 1 ) , whereas the blood : air pc and tissue : blood pcs were gathered from the literature [ 48 , 50 , 52 , 54 , 5659 , 61 , 66 , 67 , 7780 ] . to compare the impact of different ( uncertain ) scenarios of rate of metabolism on the pharmacokinetics in human , simulations were carried out by setting ( i ) the value of clint equal to the lower and upper bound of the qppr predicted mean 95% confidence interval , or ( ii ) the liver extraction ratio to 0.001 ( no metabolism ) and 0.999 ( maximum extraction ) . the applicability of the qppr model was evaluated on the basis of the level of uncertainty in the qppr estimate and the impact ( sensitivity ) of metabolism on the auc24 . figure 1 illustrates the role of uncertainty and sensitivity in the reliability of the qppr - pbpk modeling framework , based on reference . the sensitivity of the metabolism to the auc was estimated by the ratio of the auc24 obtained with no metabolism ( emin ) to that obtained with the maximum theoretical metabolism ( emax ) . this approach was applied to evaluate the reliability of applying the qppr within the pbpk model for two situations : ( i ) for the calibration set of chemicals , for which the uncertainty of the qppr was evaluated by comparing the predictions of clintpl with the experimentally derived clintpl values and ( ii ) for chemicals in the evaluation dataset , for which the uncertainty in the qppr prediction was considered to be high , to replicate the data poor situations with new or tested chemicals with unknown experimental clint values . the initial effort to develop a qppr model for metabolism rate ( expressed as clintblood , in units of l blood / hr ) , based on a stepwise analysis of its relationship to various molecular descriptors and physicochemical properties , was not successful ( not shown ) . same analysis , repeated for clint expressed in units of l pl / h ( clintpl ) , yielded a qppr that consisted of log pplw , log pbw , and ip ( ionization potential , ev ) as input parameters . this model satisfied the criteria for an acceptable model in terms of coefficient of determination ( r = 0.802 ; radj = 0.775 ) , leave - one - out cross validation ( q = 0.755 ) , and multicollinearity ( vifs : log pplw = 2.42 ; log pbw = 2.38 ; ip = 1.04 ) . however , as the value of log pow can be obtained more readily than log pplw , the regression analysis was repeated by using log pow , log pbw , and calculated ip , and it yielded the following qppr : ( 5)log clintpl=5.63(1.187)1.287(0.149)log pow + 1.08(0.233)log pbw 0.328(0.111)ip . this qppr model satisfied the criteria for an acceptable model in terms of coefficient of determination ( r = 0.796 ; radj = 0.768 ) , leave - one - out cross validation ( q = 0.748 ) , and multicollinearity ( vifs : logpow = 2.42 ; log pbw = 2.38 ; ip = 1.04 ) . the impact of the imprecision of these qppr predictions of the metabolic constants on the pharmacokinetics in humans was then evaluated by pbpk modeling . figure 3 presents the predictions of the 24 h blood pharmacokinetics following 8 h exposure to 1 ppm of each of the 26 vocs used in the qppr analysis . the bold lines represent the simulations obtained using 0 and 1 as the hepatic extraction ratio , whereas the grey area encompassed by thin lines represents the simulation obtained using lmci and umci of predicted clint in pbpk models . overall , the envelope of the concentrations predicted using the qppr predictions reduced the region of uncertainty associated with the complete lack of knowledge of hepatic extraction ratio in humans ( i.e. however , the average ratio ( standard deviation ) of the pbpk model simulated values of the end - of - exposure blood concentrations , based on qppr - generated bounds ( lmci , umci ) , was 1.29 0.27 . for the 26 vocs used in the development of the qppr , the values of auc24s for a 1 ppm continuous exposure the ratio of the highest to the lowest auc predicted with emin and emax was 4.3 1.94 ranging from 1.63 ( isoprene ) to 8.7 ( 1,1,2,2-tetrachloroethane ) . the ratio of the maximum to minimum concentrations predicted using the qppr metabolism rate was 1.36 0.4 ranging from 1.06 ( 1,1-dichloroethylene ) to 2.8 ( isoprene ) . the uncertainty of the qppr prediction was estimated as the ratio of predicted clintpl to experimental clintpl . as in the qppr development section , the impact of the imprecision on these log clint predictions on the pharmacokinetics in humans was evaluated by pbpk modeling . the bold lines represent the simulations obtained using 0 and 1 as the hepatic extraction ratio , whereas the grey area encompassed by thin lines represents the simulation obtained using lmci and umci of predicted clint in pbpk models . the reduction of the region of uncertainty associated with the complete lack of knowledge of hepatic extraction ratio in humans ( i.e. however , the same average ratio ( standard deviation ) of pbpk simulated blood concentrations , based on qppr - generated bounds ( lmci and umci ) was 1.2 0.1 , ranging from 1.07 ( ethylene ) to 1.33 ( bromoform ) . , e = 0.001 or 0.999 ) , the ratio of the maximum to minimum concentrations predicted using the qppr metabolism rate was 1.2 0.1 , ranging from 1.07 ( propylene ) to 1.33 ( dibromochloromethane ) . for the other 8 vocs ( bromoform ; dibromochloromethane ; trans-1,2-dichloroethylene ; furan ; halothane ; tetrachloroethylene ; 1,2,4-trimethylbenzene ; o - xylene ) , the ratio of the maximum to the minimum possible aucs was between 2 and 5 , such that the confidence in using the qppr in an inhalation pbpk model to evaluate the aucs is medium for these chemicals . the qppr based chemical affinity to phospholipids obtained in this study should be regarded as a screening level tool to provide plausible range of metabolism rates in order to facilitate a first - cut evaluation of the blood concentration of inhaled vocs in humans . the combined assessment of the uncertainty / sensitivity of metabolic constants in pbpk models would facilitate the determination of the applicability of the qppr model , given the level of precision need for an application ( figure 1 ) the qppr developed in this study is a generic tool to provide initial estimates of clint of vocs metabolized by hepatic cyp . therefore , predictions of clint based on generic considerations are likely to be inaccurate for specific chemicals but are of limited use in that the estimates ( along with the bounds , representing the level of uncertainty ) can be integrated with human physiology to provide a first - cut view of the plausible kinetic profiles . hence , in this study , the descriptors that could be easily calculated and interpreted were chosen and obtained using episuite ( for log pow and pwa ) and mmpro ( for the ionization potential ) . the qppr developed in this study computes clintpl , which can then be converted to clintblood for use in pbpk modeling . further rearrangements and simplifications of the qppr , as well as the loss of accuracy associated with such attempts , were not performed in the current study . the output of the qppr developed in the present study is log clint , which is useful for simulating pharmacokinetics in humans of chemicals at low levels of exposure . the input parameters of the qppr , namely , log pow and log pbw , are estimates of the relative solubility in octanol , water , and blood . then , an interpretation of the model for clint could be that the binding to the p450 enzyme is a result of hydrophobic interactions which , in turn , can be estimated with parameters reflective of the solubility in n - octanol and blood . similar to log pow , the ionization potential has already been correlated with metabolic rates , namely , the vmax and vmax/km , as this latter parameter could be correlated with the energy needed to break a covalent bond for the oxidation of the substrate . the qspr model for clint developed in this study has a defined theoretical endpoint , is nonambiguous , has a defined domain of application , was analyzed using appropriate goodness - of - fit ( r ) and robustness ( q ) , and has an attempt of mechanistic interpretation . the predicted bounds of the 95% confidence interval of intrinsic clearance were incorporated within a pbpk model to predict the blood toxicokinetics of vocs . the simulations obtained in the present study , using lower and upper confidence intervals on the mean predicted clint , reduced clearly the uncertainty bounds associated with the total lack of knowledge ( i.e. furthermore , the present study incorporated the qppr predictions of clint along with physiological parameters , such that impact on in vivo kinetics could be simulated . the qppr developed in this study approximated the experimental rat metabolic constants for the various low - molecular - weight vocs ; and it was used along with the human physiology to generate initial or screening level values of clint to construct human pbpk models that could be of potential use to interpret data such as measured biomarker levels or for designing kinetic studies to reduce database uncertainty . and in such cases , the ability to generate at least a range of plausible values of clint , as done in the present study , would facilitate first in - human simulations of pharmacokinetics of parent chemicals . depending upon the overall confidence in the qppr application for predicting dose metrics ( low , medium , and high ) relative to the use purposes , overall , the qppr developed in the present study allows to predict the clint of vocs on the basis of generic molecular descriptors rather than with fragment constants as done previously . the qppr was then used to generate range of values of clint ; the level of confidence in these estimates was assessed by considering the impact of clint on the simulated dose metrics ( i.e. the qppr - based simulation of pharmacokinetics reduced the range of uncertainty for few substances relative to complete lack of knowledge of the clint , but it needs to be evaluated / refined with much larger dataset should this screening - level approach be adopted for providing more precise estimates of metabolism rates . overall , the integrated qppr - pbpk model developed in this study is a potentially useful tool for characterizing and reducing the uncertainty associated with the complete lack of knowledge of clint in predicting human pharmacokinetics of inhaled vocs .

the online version of this article ( doi:10.1007/s40291 - 013 - 0059-y ) contains supplementary material , which is available to authorized users . ovarian cancer has the highest mortality among gynecological malignancies in europe and the usa [ 1 , 2 ] . several factors , including genetic background , are known to increase the risk of ovarian cancer development . to the well - recognized hereditary factors of ovarian cancer belong some of high - penetrance genes : brca1 ( 36 % ) , brca2 ( 13 % ) , and hnpcc dna mismatch repair genes ( 12 % ) [ 35 ] . however , these gene mutations explain less than 40 % of the familial predisposition to ovarian cancer . these data indicate that the missing heritability can be elucidated by a multigenic disease model with contribution of moderate and low - penetrance risk genes to ovarian malignancies . several intracellular pathways have been recognized in ovarian carcinogenesis . among them , the wnt/-catenin / t - cell factor ( tcf)/lymphoid enhancer factor ( lef ) signaling pathway has been considered to be essential for the growth and progression of ovarian cancers . the wnt/-catenin signaling pathway regulates a variety of elementary cellular functions , such as proliferation , polarity , adhesion , and motility during development , differentiation , and adult tissue homeostasis . this pathway is also essential for normal ovarian development , and different components of the canonical wnt signaling pathway are presented in the ovary [ 10 , 11 ] . wnt stimulation leads to a cytoplasmic accumulation of -catenin that is subsequently translocated to the nucleus , where it interacts with the tcf family and induces the transcription of wnt target genes . the absence of wnt stimulation results in -catenin phosphorylation by components of the cytosol multi - protein degradation complex and its subsequent proteosomal degradation . this multi - protein degradation complex contains adenomatous polyposis coli ( apc ) , conductin ( axin1 and axin2 ) , glycogen synthase kinase-3 ( gsk3 ) , and casein kinase 1 ( ck1 ) . the components of the wnt/-catenin / tcf / lef signaling pathway have been reported to be up - regulated in various cancer types , including ovarian cancers [ 13 , 14 ] . moreover , many studies have demonstrated that the presence of mutations or polymorphisms in ctnnb1 , apc , and axin2 can lead to aberrant activation of wnt/-catenin signaling at the onset of various types of malignancies , including ovarian cancer [ 1531 ] . in addition to these findings , inactivation of the apc gene in mice leads to the deregulation of wnt/-catenin signaling and the formation of adenocarcinomas that are morphologically similar to human ovarian endometrioid adenocarcinoma . therefore , we selected nine single nucleotide polymorphisms ( snps ) of ctnnb1 , apc , and axin2 located in distinct blocks of linkage disequilibrium ( ld ) in order to study whether these snps can be genetic risk factors of ovarian cancer ( online resource supplemental table 1 and supplemental fig . 1a , fig . selected snps were tested in the group of ovarian cancer patients and controls who did not carry the most common mutations of the brca1/brca2 genes . the patients include 258 women with histologically determined ovarian carcinoma according to the international federation of gynecology and obstetrics ( figo ) . they were inducted into the study from the university hospital , clinic of gynecological surgery and chair of gynecologic oncology at pozna university of medical sciences ( pozna , poland ) . histopathological classification , including the stage , grade , and tumor type , was performed by an experienced pathologist ( table 1 ) . the control group included 282 unrelated healthy female volunteers who were matched by age to the female cancer patients . controls were selected during medical examination at the university hospital , clinic of gynecological surgery at pozna university of medical sciences ( table 1 ) . the procedures of the study were approved by the local ethical committee of pozna university of medical sciences . all women with ovarian cancer and controls were caucasian from the wielkopolska area of poland.table 1clinical characteristics of ovarian cancer patients and healthy controlscharacteristicpatients ( n = 258)controls ( n = 282)mean age ( years ) sd58.4 9.757.4 7.5histological grade g183 ( 32.2 % ) g285 ( 32.9 % ) g390 ( 34.9 % ) gx0 ( 0.0 % ) clinical stage i96 ( 37.2 % ) ii40 ( 15.5 % ) iii88 ( 34.1 % ) iv34 ( 13.2 % ) histological type serous90 ( 34.9 % ) mucinous30 ( 11.6 % ) endometrioid48 ( 18.6 % ) clear cell24 ( 9.3 % ) brenne0 ( 0.0 % ) mixed22 ( 8.5 % ) solid18 ( 7.0 % ) untyped carcinoma26 ( 10.1 % ) data are given as no . ( % ) unless otherwise stated clinical characteristics of ovarian cancer patients and healthy controls data are given as no . ( % ) unless otherwise stated genomic dna was isolated from peripheral blood leukocytes by salt extraction . all patients and controls with ovarian cancer were tested for the three most common brca1 mutations ( 5382incc , c61 g , 4153dela ) affecting the polish population using the lightcycler 480 system with hybprobe assays ( roche , indianapolis , in , usa ) . in addition , the patient group was tested for the presence of the most common brca2 mutation ( 5946delt ) using high - resolution melting curve analysis ( hrm ) [ supplemental table 2 ] . the dna samples were then genotyped for the nine snps in ctnnb1 , apc , and axin2 ( supplemental table 1 and supplemental fig . 1a , fig . snps were selected with the use of the genome browsers of the international hapmap consortium ( http://www.hapmap.org/index.html.en ) , university of california santa cruz ( ucsc ; http://genome.ucsc.edu ) , and dbsnp database ( http://www.ncbi.nlm.nih.gov/projects/snp/ ) . snps were selected based on functional significance , location in different ld blocks , and minor allele frequency ( maf ) > 0.1 in the caucasian population . genotyping of the ctnnb1 rs4533622 , rs2953 , apc rs351771 , and axin2 rs4074947 , rs3923087 , rs2240308 snps was performed by hrm on the lightcycler 480 system ( roche diagnostics , mannheim , germany ) . genotyping of the apc rs11954856 , rs459552 , and axin2 rs7224837 snps was performed by pcr , followed by the appropriate restriction enzyme digestion [ pcr restriction fragment length polymorphism ( pcr - rflp ) ] according to the manufacturer s instructions ( fermentas , vilnius , lithuania ) . primer sequences and conditions for hrm and pcr - rflp analyses are presented in supplemental table 2 . genotyping quality was evaluated by repeated genotyping of 10 % randomly selected samples . for each snp , the hardy - weinberg equilibrium ( hwe ) was assessed by pearson s goodness - of - fit chi - square ( ) statistic . the differences in the allele and genotype frequencies between cases and controls were determined using standard or fisher tests . the odds ratio ( or ) and associated 95 % confidence intervals were also calculated . for the additive inheritance model , snps were tested for association with ovarian cancer using the cochran armitage trend test . to adjust for the multiple testing , pair - wise ld between selected snps was computed as both d and r values using haploview 4.0 software ( broad institute , cambridge , ma , usa ) . haplotype analysis was performed using the unphased 3.1.5 program with the following analysis options : all window sizes , full model , and uncertain haplotype . the p values for both global and individual tests of haplotype distribution between cases and controls were determined . gene interactions among all tested polymorphic loci were studied by the multifactor dimensionality reduction ( mdr ) approach ( mdr version 2.0 beta 5 ) . based on the obtained testing balanced accuracy and cross - validation consistency values , the best statistical gene a 1,000-fold permutation test was used to assess the statistical significance of mdr models ( mdr permutation testing module 0.4.9 alpha ) . the patients include 258 women with histologically determined ovarian carcinoma according to the international federation of gynecology and obstetrics ( figo ) . they were inducted into the study from the university hospital , clinic of gynecological surgery and chair of gynecologic oncology at pozna university of medical sciences ( pozna , poland ) . histopathological classification , including the stage , grade , and tumor type , was performed by an experienced pathologist ( table 1 ) . the control group included 282 unrelated healthy female volunteers who were matched by age to the female cancer patients . controls were selected during medical examination at the university hospital , clinic of gynecological surgery at pozna university of medical sciences ( table 1 ) . the procedures of the study were approved by the local ethical committee of pozna university of medical sciences . all women with ovarian cancer and controls were caucasian from the wielkopolska area of poland.table 1clinical characteristics of ovarian cancer patients and healthy controlscharacteristicpatients ( n = 258)controls ( n = 282)mean age ( years ) sd58.4 9.757.4 7.5histological grade g183 ( 32.2 % ) g285 ( 32.9 % ) g390 ( 34.9 % ) gx0 ( 0.0 % ) clinical stage i96 ( 37.2 % ) ii40 ( 15.5 % ) iii88 ( 34.1 % ) iv34 ( 13.2 % ) histological type serous90 ( 34.9 % ) mucinous30 ( 11.6 % ) endometrioid48 ( 18.6 % ) clear cell24 ( 9.3 % ) brenne0 ( 0.0 % ) mixed22 ( 8.5 % ) solid18 ( 7.0 % ) untyped carcinoma26 ( 10.1 % ) data are given as no . ( % ) unless otherwise stated clinical characteristics of ovarian cancer patients and healthy controls data are given as no . all patients and controls with ovarian cancer were tested for the three most common brca1 mutations ( 5382incc , c61 g , 4153dela ) affecting the polish population using the lightcycler 480 system with hybprobe assays ( roche , indianapolis , in , usa ) . in addition , the patient group was tested for the presence of the most common brca2 mutation ( 5946delt ) using high - resolution melting curve analysis ( hrm ) [ supplemental table 2 ] . the dna samples were then genotyped for the nine snps in ctnnb1 , apc , and axin2 ( supplemental table 1 and supplemental fig . 1a , fig . snps were selected with the use of the genome browsers of the international hapmap consortium ( http://www.hapmap.org/index.html.en ) , university of california santa cruz ( ucsc ; http://genome.ucsc.edu ) , and dbsnp database ( http://www.ncbi.nlm.nih.gov/projects/snp/ ) . snps were selected based on functional significance , location in different ld blocks , and minor allele frequency ( maf ) > 0.1 in the caucasian population . genotyping of the ctnnb1 rs4533622 , rs2953 , apc rs351771 , and axin2 rs4074947 , rs3923087 , rs2240308 snps was performed by hrm on the lightcycler 480 system ( roche diagnostics , mannheim , germany ) . genotyping of the apc rs11954856 , rs459552 , and axin2 rs7224837 snps was performed by pcr , followed by the appropriate restriction enzyme digestion [ pcr restriction fragment length polymorphism ( pcr - rflp ) ] according to the manufacturer s instructions ( fermentas , vilnius , lithuania ) . primer sequences and conditions for hrm and pcr - rflp analyses are presented in supplemental table 2 . for each snp , the hardy - weinberg equilibrium ( hwe ) was assessed by pearson s goodness - of - fit chi - square ( ) statistic . the differences in the allele and genotype frequencies between cases and controls were determined using standard or fisher tests . the odds ratio ( or ) and associated 95 % confidence intervals were also calculated . for the additive inheritance model , snps were tested for association with ovarian cancer using the cochran armitage trend test . to adjust for the multiple testing , pair - wise ld between selected snps was computed as both d and r values using haploview 4.0 software ( broad institute , cambridge , ma , usa ) . haplotype analysis was performed using the unphased 3.1.5 program with the following analysis options : all window sizes , full model , and uncertain haplotype . the p values for both global and individual tests of haplotype distribution between cases and controls were determined . gene interactions among all tested polymorphic loci were studied by the multifactor dimensionality reduction ( mdr ) approach ( mdr version 2.0 beta 5 ) . based on the obtained testing balanced accuracy and cross - validation consistency values , the best statistical gene a 1,000-fold permutation test was used to assess the statistical significance of mdr models ( mdr permutation testing module 0.4.9 alpha ) . in patients with ovarian cancer , 30 carriers of the most common brca1 mutations were identified . the brca1 5382insc mutation was identified in 22 individuals , c61 g in five individuals , and 4153dela in three individuals ( supplemental table 3 ) . the distribution of ctnnb1 , apc , and axin2 genotypes did not display deviation from hwe between patients and control groups ( p > 0.05 ) . the number of genotypes , ors , and 95 % confidence interval calculations for the nine ctnnb1 , apc , and axin2 polymorphisms are shown in table 2.table 2association of polymorphic variants of ctnnb1 , apc , and axin2 with the risk of ovarian cancergeners no.alleles maf genotypes cases genotypes controls p genotypic value p trend value p allelic valueordominant ( 95 % ci ) ; p valueorrecessive ( 95 % ci ) ; p value ctnnb1 rs4533622a / c0.4678/113/3790/122/700.0570.0920.0820.901 ( 0.6221.306 ) ; 0.5830.587 ( 0.3760.915 ) ; 0.018 ctnnb1 rs2953g / t0.4678/113/3790/122/700.0570.0920.0820.901 ( 0.6221.306 ) ; 0.5830.587 ( 0.3760.915 ) ; 0.018 apc rs11954856g / t0.4835/129/6376/141/640.0070.0070.009 2.034 ( 1.3023.178 ) ; 0.002 1.302 ( 0.8711.948 ) ; 0.198 apc rs351771c / t0.4588/114/2686/139/570.015 0.006 0.006 0.698 ( 0.4831.009 ) ; 0.0550.508 ( 0.3080.839 ) ; 0.007 apc rs459552a / t0.30129/86/13142/108/320.0640.0410.0340.778 ( 0.5481.106 ) ; 0.1610.472 ( 0.2420.923 ) ; 0.025 axin2 rs4074947c / t0.19137/80/10182/89/100.5770.2980.3021.208 ( 0.8411.734 ) ; 0.3061.249 ( 0.5103.056 ) ; 0.626 axin2 rs7224837a / g0.15161/61/6203/71/80.9170.8010.7991.069 ( 0.7271.573 ) ; 0.7330.926 ( 0.3162.708 ) ; 0.888 axin2 rs3923087a / g0.22133/84/10171/97/140.8140.7750.7771.089 ( 0.7631.555 ) ; 0.6400.882 ( 0.3842.026 ) ; 0.767 axin2 rs2240308a / g0.4967/115/4671/146/650.5100.2540.2600.809 ( 0.5461.197 ) ; 0.2880.844 ( 0.5511.292 ) ; 0.434statistically significant results are highlighted in boldexperiment - wide significance threshold required to keep type i error rate at 5 % ( nyholt correction ) : 0.006 ( effective number of independent marker loci : 8.388 ) maf minor allele frequency , or odds ratio , p trend p values of the trend test uppercase denotes the more frequent allele in the control samples calculated from the control samples the order of genotypes : dd / dd / dd ( d is the minor allele in the control samples ) dominant model : dd + dd vs. dd ( d is the minor allele ) recessive model : dd vs. dd + dd ( d is the minor allele ) association of polymorphic variants of ctnnb1 , apc , and axin2 with the risk of ovarian cancer statistically significant results are highlighted in bold experiment - wide significance threshold required to keep type i error rate at 5 % ( nyholt correction ) : 0.006 ( effective number of independent marker loci : 8.388 ) maf minor allele frequency , or odds ratio , p trend p values of the trend test uppercase denotes the more frequent allele in the control samples calculated from the control samples the order of genotypes : dd / dd / dd ( d is the minor allele in the control samples ) dominant model : dd + dd vs. dd ( d is the minor allele ) recessive model : dd vs. dd + dd ( d is the minor allele ) the lowest p values of the trend test ( ptrend ) were observed for the apc rs351771 and rs11954856 snps in patients with ovarian cancer ( ptrend = 0.006 and ptrend = 0.007 , respectively ) [ table 2 ] . moreover , we observed that , in a dominant inheritance model , the apc rs11954856 snp is associated with an increased risk of ovarian cancer development [ or = 2.034 ( 95 % ci 1.3023.178 ) ; p = 0.002 ] . we also found significant allelic differences for the apc rs351771 snp ( p = 0.006 ) between patients and controls . the experiment - wide significance threshold required to keep type i error rate at 5 % was 0.006 ( effective number of independent marker loci : 8.388 ) . there was no association of the apc rs11954856 snp ( supplemental table 4 ) or other tested snps ( not shown ) with any histologic subtypes of ovarian cancer . furthermore , none of the other eight ctnnb1 , apc , and axin2 polymorphisms displayed significant association with ovarian cancer development either in dominant , recessive , or additive inheritance models ( table 2 ) . haplotype analysis of the studied ctnnb1 , apc , and axin2 polymorphisms did not reveal snp combinations associated with the risk of ovarian cancer development ( table 3 ) . the lowest global p = 0.025 was observed for haplotypes composed of the apc rs11954856 and rs351771 snps ( table 3 ) . however , these results were not statistically significant when permutations were used to generate empiric p values . the empiric 5 % quintile of the best p value after 1,000 permutations was 0.03904 for ctnnb1 , 0.00748 for apc , and 0.00888 for axin2 haplotypes . the ctnnb1 rs4533622 and rs2953 snps were in perfect ld ( supplemental table 5 ) . the snps situated in distinct regions of apc and axin2 were either in strong or weak pairwise ld . this was calculated from the control samples , and had d ranges of 0.4010.988 for apc snps and 0.0021.000 for axin2 snps ( supplemental table 5).table 3results of haplotype analysis of the ctnnb1 , apc , and axin2 genes in patients with ovarian cancerpolymorphisms global p value ctnnb1 rs4533622_rs29533.0380.386 apc rs11954856_rs3517719.3520.025 rs351771_rs4595526.9450.074 rs11954856_rs351771_rs45955211.1410.133 axin2 rs4074947_rs72248371.7020.636 rs7224837_rs39230870.2450.970 rs3923087_rs22403082.9760.395 rs4074947_rs7224837_rs39230871.7390.973 rs7224837_rs3923087_rs22403084.0370.775 rs4074947_rs7224837_rs3923087_rs22403087.3830.946 chi - square empirical 5 % quantile of the best p value : 0.03904 empirical 5 % quantile of the best p value : 0.00748 empirical 5 % quantile of the best p value : 0.00888 results of haplotype analysis of the ctnnb1 , apc , and axin2 genes in patients with ovarian cancer empirical 5 % quantile of the best p value : 0.03904 empirical 5 % quantile of the best p value : 0.00748 empirical 5 % quantile of the best p value : 0.00888 exhaustive mdr analysis evaluating two- to four - loci combinations of all studied snps for each comparison did not demonstrate statistical significance in predicting susceptibility to ovarian cancer development ( table 4 ) . the best combination of possibly interactive polymorphisms was observed for rs4533622 of ctnnb1 , rs11954856 of apc , and rs7224837 and rs2240308 for axin2 ( testing balanced accuracy = 0.5719 , cross validation consistency of 10 out of 10 , permutation test p = 0.068).table 4results of gene gene interactions analyzed by multifactor dimensionality reduction methodpolymorphismstesting balanced accuracycross validation consistency ( % ) p value ctnnb1_rs4533622 , apc_rs119548560.5418600.356 ctnnb1_rs4533622 , apc_rs351771 , axin2_rs40749470.5041500.828 ctnnb1_rs4533622 , apc_rs11954856 , axin2_rs7224837 , axin2_rs22403080.57191000.068 significance of accuracy ( empirical p value based on 1,000 permutations ) results of gene gene interactions analyzed by multifactor dimensionality reduction method significance of accuracy ( empirical p value based on 1,000 permutations ) in patients with ovarian cancer , 30 carriers of the most common brca1 mutations were identified . the brca1 5382insc mutation was identified in 22 individuals , c61 g in five individuals , and 4153dela in three individuals ( supplemental table 3 ) . the distribution of ctnnb1 , apc , and axin2 genotypes did not display deviation from hwe between patients and control groups ( p > 0.05 ) . the number of genotypes , ors , and 95 % confidence interval calculations for the nine ctnnb1 , apc , and axin2 polymorphisms are shown in table 2.table 2association of polymorphic variants of ctnnb1 , apc , and axin2 with the risk of ovarian cancergeners no.alleles maf genotypes cases genotypes controls p genotypic value p trend value p allelic valueordominant ( 95 % ci ) ; p valueorrecessive ( 95 % ci ) ; p value ctnnb1 rs4533622a / c0.4678/113/3790/122/700.0570.0920.0820.901 ( 0.6221.306 ) ; 0.5830.587 ( 0.3760.915 ) ; 0.018 ctnnb1 rs2953g / t0.4678/113/3790/122/700.0570.0920.0820.901 ( 0.6221.306 ) ; 0.5830.587 ( 0.3760.915 ) ; 0.018 apc rs11954856g / t0.4835/129/6376/141/640.0070.0070.009 2.034 ( 1.3023.178 ) ; 0.002 1.302 ( 0.8711.948 ) ; 0.198 apc rs351771c / t0.4588/114/2686/139/570.015 0.006 0.006 0.698 ( 0.4831.009 ) ; 0.0550.508 ( 0.3080.839 ) ; 0.007 apc rs459552a / t0.30129/86/13142/108/320.0640.0410.0340.778 ( 0.5481.106 ) ; 0.1610.472 ( 0.2420.923 ) ; 0.025 axin2 rs4074947c / t0.19137/80/10182/89/100.5770.2980.3021.208 ( 0.8411.734 ) ; 0.3061.249 ( 0.5103.056 ) ; 0.626 axin2 rs7224837a / g0.15161/61/6203/71/80.9170.8010.7991.069 ( 0.7271.573 ) ; 0.7330.926 ( 0.3162.708 ) ; 0.888 axin2 rs3923087a / g0.22133/84/10171/97/140.8140.7750.7771.089 ( 0.7631.555 ) ; 0.6400.882 ( 0.3842.026 ) ; 0.767 axin2 rs2240308a / g0.4967/115/4671/146/650.5100.2540.2600.809 ( 0.5461.197 ) ; 0.2880.844 ( 0.5511.292 ) ; 0.434statistically significant results are highlighted in boldexperiment - wide significance threshold required to keep type i error rate at 5 % ( nyholt correction ) : 0.006 ( effective number of independent marker loci : 8.388 ) maf minor allele frequency , or odds ratio , p trend p values of the trend test uppercase denotes the more frequent allele in the control samples calculated from the control samples the order of genotypes : dd / dd / dd ( d is the minor allele in the control samples ) dominant model : dd + dd vs. dd ( d is the minor allele ) recessive model : dd vs. dd + dd ( d is the minor allele ) association of polymorphic variants of ctnnb1 , apc , and axin2 with the risk of ovarian cancer statistically significant results are highlighted in bold experiment - wide significance threshold required to keep type i error rate at 5 % ( nyholt correction ) : 0.006 ( effective number of independent marker loci : 8.388 ) maf minor allele frequency , or odds ratio , p trend p values of the trend test uppercase denotes the more frequent allele in the control samples calculated from the control samples the order of genotypes : dd / dd / dd ( d is the minor allele in the control samples ) dominant model : dd + dd vs. dd ( d is the minor allele ) recessive model : dd vs. dd + dd ( d is the minor allele ) the lowest p values of the trend test ( ptrend ) were observed for the apc rs351771 and rs11954856 snps in patients with ovarian cancer ( ptrend = 0.006 and ptrend = 0.007 , respectively ) [ table 2 ] . moreover , we observed that , in a dominant inheritance model , the apc rs11954856 snp is associated with an increased risk of ovarian cancer development [ or = 2.034 ( 95 % ci 1.3023.178 ) ; p = 0.002 ] . we also found significant allelic differences for the apc rs351771 snp ( p = 0.006 ) between patients and controls . the experiment - wide significance threshold required to keep type i error rate at 5 % was 0.006 ( effective number of independent marker loci : 8.388 ) . there was no association of the apc rs11954856 snp ( supplemental table 4 ) or other tested snps ( not shown ) with any histologic subtypes of ovarian cancer . furthermore , none of the other eight ctnnb1 , apc , and axin2 polymorphisms displayed significant association with ovarian cancer development either in dominant , recessive , or additive inheritance models ( table 2 ) . haplotype analysis of the studied ctnnb1 , apc , and axin2 polymorphisms did not reveal snp combinations associated with the risk of ovarian cancer development ( table 3 ) . the lowest global p = 0.025 was observed for haplotypes composed of the apc rs11954856 and rs351771 snps ( table 3 ) . however , these results were not statistically significant when permutations were used to generate empiric p values . the empiric 5 % quintile of the best p value after 1,000 permutations was 0.03904 for ctnnb1 , 0.00748 for apc , and 0.00888 for axin2 haplotypes . the ctnnb1 rs4533622 and rs2953 snps were in perfect ld ( supplemental table 5 ) . the snps situated in distinct regions of apc and axin2 were either in strong or weak pairwise ld . this was calculated from the control samples , and had d ranges of 0.4010.988 for apc snps and 0.0021.000 for axin2 snps ( supplemental table 5).table 3results of haplotype analysis of the ctnnb1 , apc , and axin2 genes in patients with ovarian cancerpolymorphisms global p value ctnnb1 rs4533622_rs29533.0380.386 apc rs11954856_rs3517719.3520.025 rs351771_rs4595526.9450.074 rs11954856_rs351771_rs45955211.1410.133 axin2 rs4074947_rs72248371.7020.636 rs7224837_rs39230870.2450.970 rs3923087_rs22403082.9760.395 rs4074947_rs7224837_rs39230871.7390.973 rs7224837_rs3923087_rs22403084.0370.775 rs4074947_rs7224837_rs3923087_rs22403087.3830.946 chi - square empirical 5 % quantile of the best p value : 0.03904 empirical 5 % quantile of the best p value : 0.00748 empirical 5 % quantile of the best p value : 0.00888 results of haplotype analysis of the ctnnb1 , apc , and axin2 genes in patients with ovarian cancer empirical 5 % quantile of the best p value : 0.03904 empirical 5 % quantile of the best p value : 0.00748 empirical 5 % quantile of the best p value : 0.00888 exhaustive mdr analysis evaluating two- to four - loci combinations of all studied snps for each comparison did not demonstrate statistical significance in predicting susceptibility to ovarian cancer development ( table 4 ) . the best combination of possibly interactive polymorphisms was observed for rs4533622 of ctnnb1 , rs11954856 of apc , and rs7224837 and rs2240308 for axin2 ( testing balanced accuracy = 0.5719 , cross validation consistency of 10 out of 10 , permutation test p = 0.068).table 4results of gene gene interactions analyzed by multifactor dimensionality reduction methodpolymorphismstesting balanced accuracycross validation consistency ( % ) p value ctnnb1_rs4533622 , apc_rs119548560.5418600.356 ctnnb1_rs4533622 , apc_rs351771 , axin2_rs40749470.5041500.828 ctnnb1_rs4533622 , apc_rs11954856 , axin2_rs7224837 , axin2_rs22403080.57191000.068 significance of accuracy ( empirical p value based on 1,000 permutations ) results of gene gene interactions analyzed by multifactor dimensionality reduction method significance of accuracy ( empirical p value based on 1,000 permutations ) we found that the apc rs11954856 polymorphism may be a risk factor of ovarian cancer in polish population . to date , several polymorphisms in apc have been demonstrated to be risk factors of different cancers [ 24 , 25 , 30 , 31 ] . 1b ) has been associated with increased breast cancer risk . the apc i1307k rs1801155 polymorphism ( supplemental fig . 1b ) has been observed to be associated with an increased risk for colorectal cancer but not breast / ovarian cancers [ 25 , 36 , 37 ] . moreover , a significant interaction has been found between the d1822v rs459552 ( supplemental fig . 1b ) polymorphism and dietary intakes of high fat , cholesterol , calcium , and fiber for colorectal cancer risk [ 30 , 31 ] . the -catenin is the main wnt pathway effector for which aberrant action has been demonstrated in various cancers . the role of mutations in ctnnb1 leading to aberrant functioning of -catenin at the onset of ovarian cancer have been highlighted . there are several studies demonstrating the presence of an oncogenic mutation , mainly located in exon 3 of ctnnb1 , in upwards of 40 % ovarian endometrioid adenocarcinomas [ 1517 , 38 , 39 ] . additionally , several polymorphisms in ctnnb1 ( rs4135385 , rs11564475 , rs2293303 ) have been correlated with gastric cancer susceptibility and favorable gastric cancer survival ( rs4135385 ) ( supplemental fig . however , in our studies we did not observe a significant association of the selected ctnnb1 snps with the development of ovarian cancer . axin is a scaffold protein in the destruction complex , enabling the phosphorylation of -catenin by gsk-3 [ 40 , 41 ] . recently , a 64 % allele - specific loss of heterozygosity in axin2 was observed in epithelial ovarian cancer . however , in our studies we did not find an association of the studied axin2 snps with ovarian cancer development . certain polymorphisms located in axin2 have been shown to be risk factors of astrocytoma ( rs1133683 ) , lung cancer ( rs2240308 ) , and prostate cancer ( rs35285779 ) [ supplemental fig . in addition to these findings , the five axin2 snps rs7210356 , rs4791171 , rs11079571 , rs3923087 , and rs3923086 were associated with an increased risk of breast cancer ( supplemental fig . our study demonstrated significantly increased apc rs11954856 and rs351771 snps frequencies in polish women with ovarian cancer . these snps were not identified in recently conducted genome - wide association studies ( gwas ) as risk factors for ovarian cancer , probably due to genome - wide significance thresholds required for a conventional gwas ( p value < 5 10 ) [ 4345 ] . the association of intronic rs11954856 and synonymous rs351771 ( ala545ala ) snps with ovarian cancer may be due to ld with one or more functional polymorphisms of apc . the lack of association of ctnnb1 and axin2 snps with ovarian cancer might be due to the small sample size . therefore , to confirm the role of the studied snps in ovarian cancer , this study should be replicated in a larger and independent cohort , and functional studies of these snps must be performed . plot of hapmap snps within the ctnnb1 ( a ) , apc ( b ) and axin2 ( c ) regions . the plot was generated using the genotype data from hapmap ceu samples and the haploview 4.0 software ( broad institute , cambridge , ma ) . the numbers in the squares indicate percentage of ld between a given pair of snps ( d values ) . the names of the studied snps are enclosed in boxes and the red asterix correspond to hapmap avaible snps cited in discussion section ( jpg 3355 kb ) supplementary material 1 ( jpg 5528 kb ) supplementary material 1 ( jpg 1913 kb ) supplementary material 5 ( docx 15 kb ) supplementary material 6 ( docx 19 kb ) supplementary material 7 ( docx 14 kb ) supplementary material 8 ( docx 16 kb ) supplementary material 9 ( docx 15 kb )

background and objectivethe wnt/-catenin signaling pathway has been considered to be a factor in the development and progression of ovarian cancer.methodsall patients with ovarian cancer and controls were tested for brca1 mutations ( 5382incc , c61 g , 4153dela ) with hybprobe assays and for brca2 mutation ( 5946delt ) using high - resolution melting curve analysis ( hrm ) . mutation carriers were excluded from the association analysis . we studied nine single nucleotide polymorphisms ( snps ) located in ctnnb1 ( -catenin ) [ rs4533622 , rs2953 ] , apc ( rs11954856 , rs351771 , rs459552 ) , and axin2 ( rs4074947 , rs7224837 , rs3923087 , rs2240308 ) in women with ovarian cancer without brca1/brca2 mutations ( n = 228 ) and controls ( n = 282 ) . genotyping of ctnnb1 rs4533622 , rs2953 , apc rs351771 , axin2 rs4074947 , rs3923087 , and rs2240308 was performed by hrm , while that of apc rs11954856 , rs459552 and axin2 rs7224837 was conducted by pcr followed by the appropriate restriction enzyme digestion [ pcr restriction fragment length polymorphism ( pcr - rflp)].resultsthe most common brca1/brca2 mutations were identified in 30 patients with ovarian cancer . these mutations were not found in controls . the lowest p values of the trend test ( ptrend ) were observed for the apc rs351771 and rs11954856 snps in patients with ovarian cancer ( ptrend = 0.006 and ptrend = 0.007 , respectively ) . using a dominant inheritance model , we found that the apc rs11954856 snp is associated with an increased risk of ovarian cancer development [ odds ratio = 2.034 ( 95 % ci 1.3023.178 ) ; p = 0.002 ] . we also observed significant allelic differences for the apc rs351771 snp between patients and controls ( p = 0.006).conclusionour study demonstrated significantly increased apc rs11954856 and rs351771 snp frequencies in polish women with ovarian cancer.electronic supplementary materialthe online version of this article ( doi:10.1007/s40291 - 013 - 0059-y ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Material and Methods Patients and Controls Genotyping Statistical Analysis Results None Association of Association of Multifactor Dimensionality Reduction Analysis of GeneGene Interactions Among the Studied Discussion Conclusion Electronic supplementary material Conflict of Interest

the online version of this article ( doi:10.1007/s40291 - 013 - 0059-y ) contains supplementary material , which is available to authorized users . therefore , we selected nine single nucleotide polymorphisms ( snps ) of ctnnb1 , apc , and axin2 located in distinct blocks of linkage disequilibrium ( ld ) in order to study whether these snps can be genetic risk factors of ovarian cancer ( online resource supplemental table 1 and supplemental fig . all women with ovarian cancer and controls were caucasian from the wielkopolska area of poland.table 1clinical characteristics of ovarian cancer patients and healthy controlscharacteristicpatients ( n = 258)controls ( n = 282)mean age ( years ) sd58.4 9.757.4 7.5histological grade g183 ( 32.2 % ) g285 ( 32.9 % ) g390 ( 34.9 % ) gx0 ( 0.0 % ) clinical stage i96 ( 37.2 % ) ii40 ( 15.5 % ) iii88 ( 34.1 % ) iv34 ( 13.2 % ) histological type serous90 ( 34.9 % ) mucinous30 ( 11.6 % ) endometrioid48 ( 18.6 % ) clear cell24 ( 9.3 % ) brenne0 ( 0.0 % ) mixed22 ( 8.5 % ) solid18 ( 7.0 % ) untyped carcinoma26 ( 10.1 % ) data are given as no . all patients and controls with ovarian cancer were tested for the three most common brca1 mutations ( 5382incc , c61 g , 4153dela ) affecting the polish population using the lightcycler 480 system with hybprobe assays ( roche , indianapolis , in , usa ) . in addition , the patient group was tested for the presence of the most common brca2 mutation ( 5946delt ) using high - resolution melting curve analysis ( hrm ) [ supplemental table 2 ] . genotyping of the ctnnb1 rs4533622 , rs2953 , apc rs351771 , and axin2 rs4074947 , rs3923087 , rs2240308 snps was performed by hrm on the lightcycler 480 system ( roche diagnostics , mannheim , germany ) . genotyping of the apc rs11954856 , rs459552 , and axin2 rs7224837 snps was performed by pcr , followed by the appropriate restriction enzyme digestion [ pcr restriction fragment length polymorphism ( pcr - rflp ) ] according to the manufacturer s instructions ( fermentas , vilnius , lithuania ) . all women with ovarian cancer and controls were caucasian from the wielkopolska area of poland.table 1clinical characteristics of ovarian cancer patients and healthy controlscharacteristicpatients ( n = 258)controls ( n = 282)mean age ( years ) sd58.4 9.757.4 7.5histological grade g183 ( 32.2 % ) g285 ( 32.9 % ) g390 ( 34.9 % ) gx0 ( 0.0 % ) clinical stage i96 ( 37.2 % ) ii40 ( 15.5 % ) iii88 ( 34.1 % ) iv34 ( 13.2 % ) histological type serous90 ( 34.9 % ) mucinous30 ( 11.6 % ) endometrioid48 ( 18.6 % ) clear cell24 ( 9.3 % ) brenne0 ( 0.0 % ) mixed22 ( 8.5 % ) solid18 ( 7.0 % ) untyped carcinoma26 ( 10.1 % ) data are given as no . all patients and controls with ovarian cancer were tested for the three most common brca1 mutations ( 5382incc , c61 g , 4153dela ) affecting the polish population using the lightcycler 480 system with hybprobe assays ( roche , indianapolis , in , usa ) . in addition , the patient group was tested for the presence of the most common brca2 mutation ( 5946delt ) using high - resolution melting curve analysis ( hrm ) [ supplemental table 2 ] . genotyping of the ctnnb1 rs4533622 , rs2953 , apc rs351771 , and axin2 rs4074947 , rs3923087 , rs2240308 snps was performed by hrm on the lightcycler 480 system ( roche diagnostics , mannheim , germany ) . genotyping of the apc rs11954856 , rs459552 , and axin2 rs7224837 snps was performed by pcr , followed by the appropriate restriction enzyme digestion [ pcr restriction fragment length polymorphism ( pcr - rflp ) ] according to the manufacturer s instructions ( fermentas , vilnius , lithuania ) . the number of genotypes , ors , and 95 % confidence interval calculations for the nine ctnnb1 , apc , and axin2 polymorphisms are shown in table 2.table 2association of polymorphic variants of ctnnb1 , apc , and axin2 with the risk of ovarian cancergeners no.alleles maf genotypes cases genotypes controls p genotypic value p trend value p allelic valueordominant ( 95 % ci ) ; p valueorrecessive ( 95 % ci ) ; p value ctnnb1 rs4533622a / c0.4678/113/3790/122/700.0570.0920.0820.901 ( 0.6221.306 ) ; 0.5830.587 ( 0.3760.915 ) ; 0.018 ctnnb1 rs2953g / t0.4678/113/3790/122/700.0570.0920.0820.901 ( 0.6221.306 ) ; 0.5830.587 ( 0.3760.915 ) ; 0.018 apc rs11954856g / t0.4835/129/6376/141/640.0070.0070.009 2.034 ( 1.3023.178 ) ; 0.002 1.302 ( 0.8711.948 ) ; 0.198 apc rs351771c / t0.4588/114/2686/139/570.015 0.006 0.006 0.698 ( 0.4831.009 ) ; 0.0550.508 ( 0.3080.839 ) ; 0.007 apc rs459552a / t0.30129/86/13142/108/320.0640.0410.0340.778 ( 0.5481.106 ) ; 0.1610.472 ( 0.2420.923 ) ; 0.025 axin2 rs4074947c / t0.19137/80/10182/89/100.5770.2980.3021.208 ( 0.8411.734 ) ; 0.3061.249 ( 0.5103.056 ) ; 0.626 axin2 rs7224837a / g0.15161/61/6203/71/80.9170.8010.7991.069 ( 0.7271.573 ) ; 0.7330.926 ( 0.3162.708 ) ; 0.888 axin2 rs3923087a / g0.22133/84/10171/97/140.8140.7750.7771.089 ( 0.7631.555 ) ; 0.6400.882 ( 0.3842.026 ) ; 0.767 axin2 rs2240308a / g0.4967/115/4671/146/650.5100.2540.2600.809 ( 0.5461.197 ) ; 0.2880.844 ( 0.5511.292 ) ; 0.434statistically significant results are highlighted in boldexperiment - wide significance threshold required to keep type i error rate at 5 % ( nyholt correction ) : 0.006 ( effective number of independent marker loci : 8.388 ) maf minor allele frequency , or odds ratio , p trend p values of the trend test uppercase denotes the more frequent allele in the control samples calculated from the control samples the order of genotypes : dd / dd / dd ( d is the minor allele in the control samples ) dominant model : dd + dd vs. dd ( d is the minor allele ) recessive model : dd vs. dd + dd ( d is the minor allele ) association of polymorphic variants of ctnnb1 , apc , and axin2 with the risk of ovarian cancer statistically significant results are highlighted in bold experiment - wide significance threshold required to keep type i error rate at 5 % ( nyholt correction ) : 0.006 ( effective number of independent marker loci : 8.388 ) maf minor allele frequency , or odds ratio , p trend p values of the trend test uppercase denotes the more frequent allele in the control samples calculated from the control samples the order of genotypes : dd / dd / dd ( d is the minor allele in the control samples ) dominant model : dd + dd vs. dd ( d is the minor allele ) recessive model : dd vs. dd + dd ( d is the minor allele ) the lowest p values of the trend test ( ptrend ) were observed for the apc rs351771 and rs11954856 snps in patients with ovarian cancer ( ptrend = 0.006 and ptrend = 0.007 , respectively ) [ table 2 ] . moreover , we observed that , in a dominant inheritance model , the apc rs11954856 snp is associated with an increased risk of ovarian cancer development [ or = 2.034 ( 95 % ci 1.3023.178 ) ; p = 0.002 ] . we also found significant allelic differences for the apc rs351771 snp ( p = 0.006 ) between patients and controls . the best combination of possibly interactive polymorphisms was observed for rs4533622 of ctnnb1 , rs11954856 of apc , and rs7224837 and rs2240308 for axin2 ( testing balanced accuracy = 0.5719 , cross validation consistency of 10 out of 10 , permutation test p = 0.068).table 4results of gene gene interactions analyzed by multifactor dimensionality reduction methodpolymorphismstesting balanced accuracycross validation consistency ( % ) p value ctnnb1_rs4533622 , apc_rs119548560.5418600.356 ctnnb1_rs4533622 , apc_rs351771 , axin2_rs40749470.5041500.828 ctnnb1_rs4533622 , apc_rs11954856 , axin2_rs7224837 , axin2_rs22403080.57191000.068 significance of accuracy ( empirical p value based on 1,000 permutations ) results of gene gene interactions analyzed by multifactor dimensionality reduction method significance of accuracy ( empirical p value based on 1,000 permutations ) in patients with ovarian cancer , 30 carriers of the most common brca1 mutations were identified . the number of genotypes , ors , and 95 % confidence interval calculations for the nine ctnnb1 , apc , and axin2 polymorphisms are shown in table 2.table 2association of polymorphic variants of ctnnb1 , apc , and axin2 with the risk of ovarian cancergeners no.alleles maf genotypes cases genotypes controls p genotypic value p trend value p allelic valueordominant ( 95 % ci ) ; p valueorrecessive ( 95 % ci ) ; p value ctnnb1 rs4533622a / c0.4678/113/3790/122/700.0570.0920.0820.901 ( 0.6221.306 ) ; 0.5830.587 ( 0.3760.915 ) ; 0.018 ctnnb1 rs2953g / t0.4678/113/3790/122/700.0570.0920.0820.901 ( 0.6221.306 ) ; 0.5830.587 ( 0.3760.915 ) ; 0.018 apc rs11954856g / t0.4835/129/6376/141/640.0070.0070.009 2.034 ( 1.3023.178 ) ; 0.002 1.302 ( 0.8711.948 ) ; 0.198 apc rs351771c / t0.4588/114/2686/139/570.015 0.006 0.006 0.698 ( 0.4831.009 ) ; 0.0550.508 ( 0.3080.839 ) ; 0.007 apc rs459552a / t0.30129/86/13142/108/320.0640.0410.0340.778 ( 0.5481.106 ) ; 0.1610.472 ( 0.2420.923 ) ; 0.025 axin2 rs4074947c / t0.19137/80/10182/89/100.5770.2980.3021.208 ( 0.8411.734 ) ; 0.3061.249 ( 0.5103.056 ) ; 0.626 axin2 rs7224837a / g0.15161/61/6203/71/80.9170.8010.7991.069 ( 0.7271.573 ) ; 0.7330.926 ( 0.3162.708 ) ; 0.888 axin2 rs3923087a / g0.22133/84/10171/97/140.8140.7750.7771.089 ( 0.7631.555 ) ; 0.6400.882 ( 0.3842.026 ) ; 0.767 axin2 rs2240308a / g0.4967/115/4671/146/650.5100.2540.2600.809 ( 0.5461.197 ) ; 0.2880.844 ( 0.5511.292 ) ; 0.434statistically significant results are highlighted in boldexperiment - wide significance threshold required to keep type i error rate at 5 % ( nyholt correction ) : 0.006 ( effective number of independent marker loci : 8.388 ) maf minor allele frequency , or odds ratio , p trend p values of the trend test uppercase denotes the more frequent allele in the control samples calculated from the control samples the order of genotypes : dd / dd / dd ( d is the minor allele in the control samples ) dominant model : dd + dd vs. dd ( d is the minor allele ) recessive model : dd vs. dd + dd ( d is the minor allele ) association of polymorphic variants of ctnnb1 , apc , and axin2 with the risk of ovarian cancer statistically significant results are highlighted in bold experiment - wide significance threshold required to keep type i error rate at 5 % ( nyholt correction ) : 0.006 ( effective number of independent marker loci : 8.388 ) maf minor allele frequency , or odds ratio , p trend p values of the trend test uppercase denotes the more frequent allele in the control samples calculated from the control samples the order of genotypes : dd / dd / dd ( d is the minor allele in the control samples ) dominant model : dd + dd vs. dd ( d is the minor allele ) recessive model : dd vs. dd + dd ( d is the minor allele ) the lowest p values of the trend test ( ptrend ) were observed for the apc rs351771 and rs11954856 snps in patients with ovarian cancer ( ptrend = 0.006 and ptrend = 0.007 , respectively ) [ table 2 ] . moreover , we observed that , in a dominant inheritance model , the apc rs11954856 snp is associated with an increased risk of ovarian cancer development [ or = 2.034 ( 95 % ci 1.3023.178 ) ; p = 0.002 ] . we also found significant allelic differences for the apc rs351771 snp ( p = 0.006 ) between patients and controls . the best combination of possibly interactive polymorphisms was observed for rs4533622 of ctnnb1 , rs11954856 of apc , and rs7224837 and rs2240308 for axin2 ( testing balanced accuracy = 0.5719 , cross validation consistency of 10 out of 10 , permutation test p = 0.068).table 4results of gene gene interactions analyzed by multifactor dimensionality reduction methodpolymorphismstesting balanced accuracycross validation consistency ( % ) p value ctnnb1_rs4533622 , apc_rs119548560.5418600.356 ctnnb1_rs4533622 , apc_rs351771 , axin2_rs40749470.5041500.828 ctnnb1_rs4533622 , apc_rs11954856 , axin2_rs7224837 , axin2_rs22403080.57191000.068 significance of accuracy ( empirical p value based on 1,000 permutations ) results of gene gene interactions analyzed by multifactor dimensionality reduction method significance of accuracy ( empirical p value based on 1,000 permutations ) we found that the apc rs11954856 polymorphism may be a risk factor of ovarian cancer in polish population . our study demonstrated significantly increased apc rs11954856 and rs351771 snps frequencies in polish women with ovarian cancer .

oxidative degradation of recalcitrant nitroaromatic pollutants is initiated by the formation of a cis - dihydroxylated metabolite that spontaneously rearranges to catechol with accompanying release of nitrite ( figure 1 ) . an understanding of the cis - dihydroxylation mechanism is thus valuable in the development of bioremediation measures , particularly protein engineering of dioxygenases to modify selectivity and enhance efficiency . the theoretical kinetic isotope effect derived using knowledge of the transition state structure can also serve as a reference value for compound - specific isotope analysis , which enables identification of the predominant transformation pathway of pollutants at a particular contaminated site . the reaction is catalyzed by nitrobenzene 1,2-dioxygenase ( nbdo ) , as well as 2-nitrotoluene dioxygenase ( 2ntdo ) , which differ in catalytic site residue at positions 204 , 251 , and 293 . these two enzymes are members of the naphthalene family of rieske non - heme iron dioxygenases ( rdos ) , which consist of a rieske [ 2fe2s ] ferredoxin , an nadh - dependent flavoprotein reductase , and an 33 oxygenase . rdos also catalyze monohydroxylation , desaturation , sulfoxidation , o- and n - dealkylation , and amine oxidation . nbdo , in particular , can oxidize both the aromatic ring and the alkyl side chain of nitroaromatic compounds to yield a mixture of catechol and nitrobenzyl alcohol . monohydroxylation is an undesirable competing oxidation pathway , as it does not eliminate the nitro group that prevents further degradation . the active site of rdos is a high - spin mononuclear fe bound to two histidines and a bidentate aspartate residue , forming the recurring 2-his-1-carboxylate facial triad motif of non - heme iron - containing oxygenases . a rieske [ 2fe2s ] cluster located within 12 of fe in an adjacent subunit transfers two electrons from nad(p)h to the active site during the catalytic cycle . o2 binding and activation is triggered by one - electron reduction of the rieske cluster and formation of the enzyme substrate complex . extensive studies on naphthalene 1,2-dioxygenase ( ndo ) , including crystallographic data , single turnover and peroxide shunt experiments , and theoretical calculations , indicate a side - on bound ferric ( hydro)peroxo complex as a key reaction intermediate . o - labeling studies on nbdo , ndo , 2ntdo , and toluene dioxygenase ( tdo ) further suggest that both hydroxyl groups of the product mainly originate from a single o2/h2o2 molecule . a theoretical study of naphthalene cis - dihydroxylation by ndo proposed that this fe ooh complex directly oxidizes the substrate via an epoxide intermediate . subsequent ring - opening leads to a carbocation , which is then attacked by the hydroxo ligand to yield cis - diol . the active site remains in the ferric state throughout the process , indicating that fe is not involved in the redox reaction . on the other hand , an indole o2 adduct fitted to an electron density map of ndo crystals suggests that the substrate is attacked by a peroxo ligand bound end - on to fe . an fe = o oxidant is implicated in the activity of enzymes such as 2-oxo acid dioxygenases and aromatic amino acid hydroxylases , which also possess the 2-his-1-carboxylate facial triad motif . this was confirmed by spectroscopic studies on taurine--ketoglutarate dioxygenase ( taud ) , tyrosine hydroxylase ( tyrh ) , and phenylalanine hydroxylase ( pheh ) . o bond cleavage to form this high - valent intermediate is facilitated by additional electron donation from a cofactor or cosubstrate in contrast to rdos , where only one electron is provided by the rieske center . oxygen bond heterolysis in the latter yields ho fe = o , for which the calculated energy barrier is quite high ( 26.5 kcal mol ) . rdos have a high - spin ligand environment , and theoretical studies on similar fe oor complexes show that the strong o o bond can be attributed to -donation from o moreover , the non - heme ligands of rdos do not stabilize high oxidation states ( through spin delocalization and ligand oxidation ) as porphyrin and thiolate ligands do for heme enzymes , which could explain the endothermicity of heterolytic o o bond cleavage . nevertheless , minor o incorporation ( 3% ) observed in naphthalene dioxygenation in the presence of h2o indicates the involvement of the ho fe = o intermediate , which would allow solvent exchange to occur . monooxygenation of indan by tdo was also found to yield o - labeled products . heme enzymes and methane monooxygenase , which also formally have an fe = o oxidant , catalyze olefin epoxidation instead of cis - dihydroxylation , which is a reaction unique to rdos . an insight into the cis - dihydroxylation mechanism of high - valent iron complexes can be gained instead from functional models , which are designed to mimic the ligand environment and reactivity of rdos . these biomimetic complexes are being developed as green alternatives to toxic or expensive oxidation catalysts . notable examples are [ fe(6-me3-tpa)(ch3cn)2](clo4)2 ( tpa being tris(2-pyridylmethyl)amine ) , which has a tetradentate ligand and two cis labile sites and is the first reported biomimetic catalyst for olefin oxidation ; [ fe(l1)(cl ) ] , where the hl1 [ 3-(dipyridin-2-ylmethyl)-1,5,7-trimethyl-2,4-dioxo-3-azabicyclo[3.3.1]nonane-7-carboxylic acid ] ligand is modeled after the 2-his-1-carboxylate motif ; and [ fe(tpa)(ncme)2 ] , which catalyzes the dihydroxylation of an aromatic double bond ( naphthalene ) . fe = o oxidant in olefin cis - dihydroxylation has been reported for the synthetic system [ fe(o)(oh)(pytacn)](cf3so3 ) ( tacn = triazacyclononane ) using variable - temperature mass spectrometry . oxygen oxygen bond cleavage is mediated by water as inferred from the significant oxygen exchange with labeled water in the cis - diol product . an important difference between these biomimetic catalysts and rdos is the low - spin ligand environment in the former , which leads to a weaker oxygen oxygen bond and , consequently , a lower barrier for heterolysis . theoretical studies on fe pytacn and fe tpa suggest an unsymmetrical concerted mechanism for cis - dihydroxylation initiated by the attack of the hydroxo ligand on the olefin to form a radical , followed by barrierless diol formation . the cis - dihydroxylation of nitroaromatic compounds , represented by nitrobenzene and 2-nitrotoluene , is examined in the present study by means of hybrid density functional theory ( dft ) . nitrobenzene dioxygenation via the previously proposed epoxide pathway is investigated and compared with that of naphthalene and benzene . alternative mechanisms arising from direct reaction with fe ooh are also explored to compare the activation barriers of substrate oxidation ( with or without concerted oxygen oxygen bond cleavage ) with that of the oxygen oxygen bond cleavage to form ho finally , the mechanism of cis - dihydroxylation by ho fe = o is determined on the basis of experimentally observed o incorporation in the cis - diol product . the possible reaction mechanisms for cis - dihydroxylation of nitrobenzene and 2-nitrotoluene were analyzed using four models of the nitrobenzene dioxygenase active site , presented in figure 2 , which were constructed based on the available crystal structure of nbdo with nitrobenzene bound in the active site ( pdb i d : 2bmq ) . the smallest model included the metal ion and side chains of its first - shell ligands , namely , his211 , his206 , and asp360 ( model 1 , figure 2a ) . the active site model was then expanded to include ( a ) asn258 , which forms a hydrogen bond with the substrate ( model 2 , figure 2b ) ; ( b ) h2o molecule , which forms a hydrogen bond with the hydroperoxo ligand ( model 3 , figure 2c ) ; and ( c ) asn199 ( model 3 , figure 2c ) . a water molecule was incorporated in the active site model based on the results of the molecular dynamics simulation of nbdo ( data not shown ) , which indicated the presence of water at the entrance to the substrate pocket . the position of the dioxygen bound side - on to the mononuclear iron was determined through an overlay with the crystal structure of the ndo - o2-indole complex ( pdb i d : 1o7n ) . in all calculations , constraints were imposed on the atomic coordinates of c or c carbon atoms of the included residues , to account for the rigidity of the protein backbone . calculations for the other substrate were performed using the two smallest models of the active site , by replacing nitrobenzene with 2-nitrotoluene . four models of nbdo active site used in this study : model 1 ( a ) comprising only first - shell iron ligands and a substrate ; model 2 ( b ) additionally including asn258 residue ; model 3 ( c ) and model 4 ( d ) constructed by adding a water molecule and asn199 residue , respectively . atoms marked with an asterisk were frozen according to the nitrobenzene dioxygenase crystal structure during all calculations . relaxed potential energy surface ( pes ) scans were carried out to approximately locate possible transition states . all stationary points were then optimized , and vibrational analysis was performed for all minima and transition states to confirm that they have zero and exactly one imaginary frequency , respectively . all optimizations were performed using the dft hybrid b3lyp functional and lacvp * basis set , combining the 6 - 31 g * basis set on c , h , n , o atoms and hay and wadt effective core potential ( ecp ) for the description of the fe atom . to mimic the polarization effects of the protein environment , the smd continuum solvent model with a dielectric constant of 5.7 was used in all reported calculations , unless stated otherwise . the final energies were recomputed using the b97d grimme functional including dispersion and lacv3p+ * basis set ( ecp for fe and 6 - 311+g * for all other atoms ) . the final energies were also computed using dispersion - corrected b3lyp - d2 and b3lyp - d3 functionals ( see table 1 and tables s1 , s2 , and s3 in the supporting information ) . despite visible differences , the overall conclusions drawn from the values obtained using different functionals are similar . the largest discrepancy between the barriers calculated using the b97d and b3lyp functionals is observed in the case of the o o cleavage ( table 1 ) . as can be seen through comparison between the dispersion - corrected and noncorrected b3lyp values , dispersion effects are negligible in this particular reaction . from this it can be also inferred that significantly different description of the energy of the system provided by the b97d functional is not related to dispersion effects , but rather to the nature of the functional itself . b97d is a general - purpose functional parametrized for various systems , including those containing transition metals , and it has been shown to outperform other commonly used functionals , including b3lyp , especially in the case of the reactions involving transition metals . thus , we believe it is more suitable for the description of the reaction under study , and therefore the final energies calculated using this functional will be mainly used in the discussion of the results , with b3lyp - d2 and b3lyp - d3 values referred to when formulating relevant conclusions . the effect of reoptimization of the stationary points using the dispersion - corrected functional was examined using model 1 with nitrobenzene . the geometries of the transition states optimized with or without dispersion correction were found to be very similar and energy barriers calculated for the species optimized at b97d / lacvp * level close to the values obtained through single - point corrections calculated for the b3lyp / lacvp * geometries ( see table s4 and s5 in the supporting information ) . spin populations derived from the natural bond orbital analysis from triple- basis set are reported . where needed , mulliken atomic spin densities are also provided for comparison of the presented results with the data reported in the literature . iron complex was modeled in the sextet state ( s = 5/2 ) as a positively charged protonated species , i.e. [ fe ooh ] , which was found to be the most energetically favorable arrangement of the dioxygen enzyme complex in naphthalene dioxygenase . additionally , the feasibility of the attack of the ho fe = o species ( s = 2 ) on the aromatic ring of nitrobenzene was assessed using the smallest model of the active site . all calculations were performed using the gaussian 09 electronic structure package , and structures were produced with vmd . in their computational study of the cis - dihydroxylation mechanism in naphthalene 1,2-dioxygenase ( ndo ) , bassan et al . iron(iii ) species may directly attack the substrate , naphthalene , to form a cis - diol . based on the results obtained for the three different models of the active site using gas phase optimization at the b3lyp / lacvp level , they concluded that the most feasible reaction pathway involves the formation of an epoxide intermediate from the direct attack of the hydroperoxo moiety on the substrate double bond . in their proposed mechanism , the proximal o atom of the hydroperoxo ligand initially attacks the c = c bond , with distances of 2.16 and 2.20 . o * antibonding orbital directly as confirmed by the lack of significant change in the fe mulliken spin density at the transition state . thus , the o o bond is broken concomitantly with c o bond formation . an epoxide intermediate is formed , but the reaction is not complete , as indicated by the long c o bond distances ( 1.56 and 1.58 ) and the residual spin density ( 0.08 ) on the reacting o atom . the reaction further evolves toward a carbocation , which originates from the cleavage of the epoxide c o bond . at this stationary point , the spin density of fe remains at around 4 while that of the proximal o atom increases to 0.26 . eventually the product , cis - diol , is formed by binding of the cation to the oh ligand of the iron intermediate . the attack of oh on the other carbon to form the cis - diol has a very small barrier ( 1 kcal / mol ) , consistent with the early transition state ( c oh bond distance of 2.60 ) . the rate - determining step was found to be the formation of an epoxide , with the concomitant cleavage of the oxygen oxygen bond in the ferric hydroperoxo moiety . the activation energy for this concerted step calculated for the medium - size model was 17.5 kcal mol ( g = 19.1 kcal mol ) , which was confirmed using the larger model . nitrobenzene dioxygenase shares 80% sequence identity with naphthalene 1,2-dioxygenase and is also capable of cis - dihydroxylation of naphthalene . hence , we decided to investigate whether the chemical pattern proposed for the reaction occurring in the ndo active site is feasible for the formation of cis - dihydrodiol from nitrobenzene catalyzed by nbdo . the transition state leading to the formation of an epoxide was located for the two smallest models of the nbdo active site used in this study , namely , the one including only first shell iron ligands ( model 1 ) and a larger system , which additionally contains asn258 residue ( model 2 ) . model 1 used here corresponds to the active site model used in ref ( 27 ) , but unlike the latter study geometry optimizations for both models were performed at the b3lyp / lacvp * level with continuum solvation ( dielectric constant of 5.7 ) to account for electrostatic effects from the protein environment . using model 1 , we also obtained the transition state involving concerted o o bond cleavage and c o bond formation . in our case , c o bond distances are 1.86 and 2.45 while the fe mulliken spin density is 4.07 . in contrast to bassan et al . , epoxide formation is complete ( as indicated by c o bond distances of 1.39 and 1.45 , and zero spin density on the o atom ) . the activation barrier for the formation of an epoxide was computed to be 20.6 kcal mol ( 15.3 kcal mol relative to separate reactants ) . similarly to what was observed for naphthalene , the reaction is exothermic , with the epoxide intermediate lying 14.9 kcal mol lower in energy than the reactant complex ( 20.1 kcal mol lower relative to separate reactants ) , but contrary to the results reported for ndo , we do not observe the formation of an epoxide intermediate bound to mononuclear iron , which may further evolve into a carbocation . instead , in the intermediate state originating from the direct attack of fe ooh on the nitrobenzene c1c2 double bond ( see figure 4 for atom numbering ) , the o fe bond is broken and the epoxide species dissociates from the iron cluster . dissociation of the epoxide results in a change of the iron coordination sphere , as illustrated in figure 3a . the geometry of the optimized intermediate is confirmed using model 2 . even in the presence of the asn258 residue , which forms a hydrogen bond with the nitro group of nitrobenzene , the distance between the arene oxide and the iron atom elongates upon optimization , leaving the hydroxo group as the only ligand coordinating the face of the fe cluster . optimized geometries , relevant spin populations , and bond lengths ( ) of ( a ) epoxide intermediate obtained for the smallest model of nbdo active site ; ( b ) epoxide intermediate for model 2 , additionally containing asn258 residue . in order to examine whether our findings are independent of the choice of the methodology , i.e. , gas phase vs continuum solvent optimization , and to compare the feasibility of the mechanism involving an epoxide intermediate for nitroaromatic and nonsubstituted aromatic substrates , we additionally reoptimized the stationary points for both models in the gas phase using the b3lyp functional and lacvp basis set as in ref ( 27 ) . we have reproduced the transition state preceding epoxide formation obtained by bassan et al . for naphthalene , and additionally investigated the same step using benzene as a substrate , both in the gas phase and continuum solvent model . in the two cases where the substrate does not contain a nitro group , the reaction proceeds via the chemical pattern reported for ndo . in the case of the gas phase reaction of nitrobenzene , the epoxide dissociation from the fe center is still observed and the activation energy calculated at the b3lyp / lacv3p**//b3lyp / lacvp level is 33.2 and 38.7 kcal mol for model 1 and model 2 , respectively these activation barriers are much higher than the value reported by bassan et al . ( 17.5 kcal mol ) , which may be partially attributed to neglecting solvent effects in energy calculations and spurious interactions found in the gas phase optimized reactant complexes , especially in model 2 . the b3lyp activation barriers calculated for the species optimized in continuum solvent model are 31.2 and 33.6 kcal mol for model 1 and model 2 , respectively , and further decrease to 2126 kcal mol when dispersion correction is included in b3lyp calculations ( tables s1 , s2 , and s3 in the supporting information ) . at the same time , gas phase activation energies calculated for nonsubstituted aromatic substrates , benzene and naphthalene , are much lower than the barrier obtained for nitrobenzene and in close agreement with the values previously calculated for naphthalene ( for details see the supporting information ) . as already mentioned , in the epoxide mechanism , two electrons from the substrate are transferred to the o o * antibonding orbital leading to concerted o o bond cleavage and c o bond formation . the increased barrier for our substrate can be attributed to the nitro substituent , which tends to make electrons less available for transfer to the active site . the dissociation of the epoxide intermediate from the active site suggests that the previously proposed mechanism for naphthalene dihydroxylation by ndo is not feasible for nitroaromatic compounds . however , in principle one could consider a possible return of the epoxide intermediate to the metal cluster , or its hydrolysis , which could take place outside the active site . the latter reaction would yield nonstereospecific products , as water can attack at any side of the ring , hence it is an unlikely scenario for the cis - diol formation from the dissociated epoxide intermediate . the fact that o labeling studies show that both o atoms originate from the same source also discounts the possibility of cis - diol formation from the hydrolysis of the dissociated epoxide . on the other hand , the return of the epoxide intermediate to the active site could possibly allow the reaction to proceed to the next step , i.e. , formation of a cation intermediate through the opening of the epoxide ring ( scheme 1 ) . indeed , using model 1 we were able to locate a transition state for the epoxide ring - opening ( see figure s2 in the supporting information ) , which lies 18.6 kcal mol above the preceding ( dissociated ) epoxide intermediate and is lower in energy than the transition state for the epoxide formation by 16.8 kcal mol . the reaction pathway followed from this transition state by integrating the intrinsic reaction coordinate leads to an intermediate with a single c o bond , optimization of which , however , does not yield an expected cation intermediate . instead , we observe a barrierless hydride shift resulting in the formation of a ketone ( scheme 2 , figure s2 in the supporting information ) . such transformation was also observed by bassan et al . , who also mentioned that epoxide ring - opening can lead to a 1,2-hydride shift to form a ketone , for which the barrier is almost zero . they attributed formation of the cis - diol through a cationic species , despite the slightly larger barrier ( 1 kcal / mol ) , to protein effects . possibly due to the presence of the electron - withdrawing nitro group , the cation intermediate proposed by bassan et al . is even less stable in the case of nitrobenzene , and we were not able to obtain such in our calculations . hence , the final product could not be obtained from a nitroaromatic substrate through an epoxide pathway . it should be noted , however , that we acknowledge the possibility of the current findings being related to the limitations of the adopted model that only partially accounts for the protein environment , which in this particular case might be crucial for the stabilization of the intermediate products formed along the reaction pathway . considering the discussed differences found between the results presented here , and those reported for naphthalene , and a relatively low barrier of the epoxide formation compared to alternative ways of the attack on the ring ( see the next section ) , the discussed pathway should and will be examined as a possible cis - dihydroxylation mechanism in the hybrid qm / mm calculations , which will follow the current study . finally , an important consideration in the discussion of the epoxide pathway is that our results are in fact consistent with an observation that rdos generally do not catalyze epoxidation , even as an intermediate step . this was shown through the oxidation of styrene , which was used as a probe for epoxide formation since it is oxidized to styrene 1,2-oxide by cytochrome p-450 and other monooxygenases . moreover , styrene 1,2-oxide was not a substrate for ndo , indicating that the diol is formed directly and not through epoxide hydrolysis . since the mechanism involving an epoxide intermediate was found not to be plausible for the cis - dihydroxylation of nitroaromatic substrates , it was necessary to probe other chemical routes for such transformation , initiated by the direct attack of the fe three possible ways of the initial attack on the ring were considered here : the attack of the hydroxyl group on the c2 carbon , and the attack of the second oxygen atom of the hydroperoxo group on either c1 or c2 carbon , as shown in figure 4 . for each of the considered pathways , we located the corresponding transition state for the concerted attack ( with concomitant o o cleavage ) and the transition state leading to the formation of a single c o bond without the cleavage of the oxygen oxygen bond in the ferric hydroperoxo moiety ( nonconcerted attack ) . c1 attack leads to the formation of an epoxide intermediate , which was discussed in the previous section . three routes of the attack of fe ooh ( a ) or ho fe = o ( b ) intermediate on the substrate considered as possible initial steps of its cis - dihydroxylation . see text for description . in each of the probed mechanisms , the attack of the oxygen atom on the aromatic ring without the oxygen oxygen bond cleavage was found to have a higher energetic barrier than the corresponding concerted attack . c2 bond or an epoxide intermediate with c2 and c3 carbon bonded to o atom , from neither of which we were able to obtain a final product . we also probed whether the nonconcerted o c2 attack can initiate a reaction pathway leading to the formation of a diol , as in the case of model 3 we were able to locate a transition state with a relatively low barrier , compared to other nonconcerted attacks . in this particular case , an intermediate with a single c o bond formed in the first step of the reaction can evolve toward an epoxide , which is formed upon an o o bond cleavage . similarly to what was observed for the pathway discussed earlier , the epoxide species dissociates from the iron cluster , despite the presence of asn258 residue , as it is the case in model 3 . hence , within the scope of the current study , this pathway does not seem viable for cis - dihydroxylation of nitroaromatic substrates . it is worth mentioning that it resembles to a large extent an alternative mechanism for the formation of the cis - diol from naphthalene presented by bassan et al . , which involves an iron(ii)hydroperoxyl species . in the reaction presented therein , a similar , nonconcerted attack of the hydroperoxo ligand on the substrate leads to the formation of a radical intermediate with a single c o bond , this step being associated with the barrier of 1920 kcal mol and endothermicity of around 18 kcal mol . the subsequent step of the reaction yields a cis - diol , but the associated transition state is higher in energy than the preceding one , based on which the mechanism was considered as rather unlikely . in our case , a radical is also delocalized on the substrate , evidenced by mulliken spin density on nitrobenzene of 0.35 and 0.76 in the transition state and intermediate product , respectively . similarly to what was observed for naphthalene , this radical intermediate has a very high energy compared to reactant complex , exhibiting a large negative driving force of this step of the reaction . the estimated energy of the transition state associated with the cleavage of the o o bond is similar to the transition state for the formation of a radical intermediate . a concerted c o bond formation and oxygen oxygen bond cleavage mechanism for the three possible modes of fe ooh attack was also examined using model 1 with 2-nitrotoluene as a substrate . activation energies are in agreement with the barriers obtained for the other substrate , with the values of 23.6 , 21.2 , and 21.1 kcal mol for the o1c2 , o similarly to what was found for nitrobenzene , the resulting intermediates did not lead to the formation of a diol . barriers calculated using the dispersion - corrected b3lyp functional ( tables s2 and s3 in the supporting information ) show a similar picture of the plausibility of direct reaction between the substrate and fe ooh . in general , nonconcerted fe ooh attacks on the ring have higher activation energies than corresponding reactions coupled with the o o cleavage . an exception to this , as in the case of b97d results , is a barrier for the nonconcerted o c1 attack yielding an epoxide intermediate of around 2526 kcal mol is larger than the one obtained with the b97d functional , but at the same time according to b3lyp - d results it is the most favorable pathway involving the fe as mentioned above , a similar barrier was found for a nonconcerted o c2 attack , and a concerted o c2 attack , which was excluded from further consideration as not leading to the final product , is also not energetically favored over the concerted o subsequently , we considered the possibility of the formation of a high - valent intermediate , ho fe = o , prior to substrate oxidation . in low - spin biomimetic non - heme iron complexes , oxygen oxygen bond cleavage is facilitated by the formation of a five - membered intermediate with water , as evident from exchange with o - labeled solvent . however , this would always lead to a mixed product , whereas in the cis - diol obtained from the enzyme - catalyzed reaction , both hydroxyl groups originate predominantly from a single oxygen source . it is thus more likely that ho fe = o is formed directly via heterolytic o o bond cleavage in rdos . this involves initial transfer of an -electron from fe to the o o bond * orbital , as evidenced by the decreased spin density on fe and increased spin density on o1 . at the transition state , the o o bond increases to 2 while the fe = o double bond starts to form . transfer of the second electron to the * orbital completes o o bond cleavage and formation of ho fe = o . the gibbs free energy of activation for o o bond heterolysis was also determined in the presence of the substrate using different models of the enzyme . the barrier is only 15.5 kcal mol for the smallest model ( model 1 ) and is decreased to 14.6 kcal mol upon inclusion of asn258 in model 2 . interestingly , in the case of model 3 , which additionally contains a water molecule , the barrier slightly increases to 16.7 kcal mol , while in turn , model 4 , which includes asn199 , which is within h bonding distance of the hydroperoxo ligand and is postulated to be connected to a water channel that facilitates proton transfer , yields a lower barrier of 15.0 kcal mol . fe = o is more energetically favorable than substrate oxidation by fe ooh . this suggests that the reaction proceeds through the formation of the high - valent oxo species and its subsequent attack on the ring , rather than the direct attack of the ferric hydroperoxo ligand on the substrate . it should be noted , however , that current results do not firmly elucidate the role of protein environment , including asn258 or asn199 residues , in facilitating heterolytic oxygen oxygen bond cleavage , and further study of this problem is desired . the activation energies for the o o cleavage calculated using b3lyp - d functionals are much higher than those obtained with the b97d functional and range from 24.1 to 27.8 kcal mol ( table 2 ) . depending on the model used , these values become comparable or larger by up to 3.9 kcal mol than the barriers for the initial attack of fe ooh on the substrate ring , which , considering the accuracy of the calculated energies , still allows us to consider the formation of ho fe = o species and its subsequent reaction with the substrate as a plausible scenario for its cis - dihydroxylation . fe = o , in comparison with a direct reaction between fe ooh and the substrate , suggests that the high - valent intermediate is a possible oxidant . this would be consistent with minor o incorporation ( 3% ) observed in naphthalene dioxygenation in the presence of h2o . a separate experiment with naphthalene cis - diol and h2o confirms that solvent exchange occurs with a reactive intermediate and not with the product.o - labeled products may be attributed to rapid intermolecular exchange between ho therefore , we probed possible dioxygenation pathways starting from a direct attack of ho fe = o on the substrate ( figure 4b ) . the barriers computed for the initial formation of an oxygen carbon bond presented in table 3 prove this step to be in general more energetically favorable when the substrate is attacked by an ho fe = o species rather than by the ferric hydroperoxo intermediate discussed before . in parentheses , the results obtained for the two smallest models indicate that the o1c2 attack is the first step of the reaction , while for models 3 and 4 , a noticeably smaller barrier is found for the o the latter findings may be , however , affected by the presence of a water molecule in the active site model . it can be expected that the lowering of the activation barrier for the two largest models , compared to the values computed for models 1 and 2 , might be the effect of the inclusion of a water molecule and its stabilization in the continuum solvent used in this study . moreover , in the current study , we were not able to obtain a final product in the reaction starting with the o a comparison between the barriers for all possible initial steps of the reaction also shows that the formation of a single c o bond initiated by the attack of hydroxyl oxygen of the iron(v)oxo complex on the c2 carbon is the most feasible transformation , from which the reaction can evolve toward the final product . similar activation energies were computed for the initial step of 2-nitrotoluene oxidation , with g of 0.4 and 2.4 kcal mol for the o1c2 attack in models 1 and 2 , respectively . a barrier of 4.8 kcal mol calculated for the o c2 attack in model 1 of 2-nitrotoluene also agrees well with the ordering of the possible transition states located for nitrobenzene . the barriers calculated using b3lyp - d functionals agree very well with the results presented in table 3 , and confirm low activation energies for the ho fe = o attack compared to a direct reaction of fe ooh with the substrate ( tables s2 and s3 in the supporting information ) . likewise , the b3lyp - d results demonstrate that the energetically most favorable step initiating cis - dihydroxylation is the formation of an o1c2 bond through an ho fe = o attack on the aromatic ring . the mechanism of cis - dihydroxylation , as shown in figure 5 , was studied in detail using models 1 and 2 . initial attack of the hydroxo ligand on the c2 carbon is the rate - determining step with a gibbs free energy of activation of 7.0 kcal / mol in model 1 and 2.4 kcal / mol in model 2 . the higher barrier in model 1 may be partly due to artificial hydrogen bonding between the substrate and the hydroperoxo ligand in the reactant complex , which is eliminated in the larger models upon inclusion of asn258 . the increase in the barrier to 8.0 and 7.4 kcal / mol in models 3 and 4 , respectively , suggests that structural contributions from the protein environment have a significant effect on reaction energetics . steric , as well as electrostatic , effects will be investigated further in a qm / mm study . as the c2o1 bond forms in the transition state ( dc o = 2.12 in both models , figures 6 and 7 ) , an electron is transferred to the active site , resulting in radical formation in the substrate ( spin density of 0.5 ) . radical formation , as confirmed by the spin density of 1 on the substrate in the intermediate state , is exothermic by 5.5 and 7.4 kcal / mol in models 1 and 2 ( table 4 ) . fe is reduced to the + 4 oxidation state in the first step of the reaction , leading to a spin density of 2.9 . in the final step , the oxo ligand attacks the c1 carbon with an activation barrier of 3.2 and 2.8 kcal / mol in models 1 and 2 ( table 4 ) . the low barrier is consistent with an early transition state as evidenced by the long c1o bond distance ( 2.48 in model 1 , figure 6 , and 2.49 in model 2 ) . the other electron from the substrate is transferred to the active site during this step , which is reflected in the decreased spin density on nitrobenzene ( 0.8 ) and increased spin density on fe ( 3.1 ) . cis - diol formation is exothermic by 42.9 and 47.1 kcal / mol in models 1 and 2 ( table 4 ) . this is accompanied by reduction of the active site back to the initial ferric state , where the spin density on fe is about 3.9 . upon the formation of the diol , the distance between the nitro group and c1 carbon elongates to 1.6 , with the substituent still bonded to the aromatic ring , which agrees with the general reaction scheme of rieske dioxygenases suggested in the literature , according to which the nitrite release occurs in the subsequent step of the catalytic cycle . reaction profile of cis - dihydroxylation of nitrobenzene through a direct attack of the ho fe = o species on the aromatic ring . the figure shows gibbs free energy diagram obtained for the smallest model of the nbdo active site . optimized geometries , relevant spin populations , and bond lengths ( ) of the key stationary points located along the reaction pathway depicted in figure 5 : ts1 , transition state for the initial attack of the iron(v)oxo intermediate on the carbon carbon double bond ; int1 radical intermediate formed upon the formation of a single c o bond ; ts2 transition state for the oxygen attack on the c1 carbon yielding the cis - dihydrodiol pc . spin population for nitrobenzene ( nbz ) was summed over all atoms of the ring and the nitro group . transition state for the attack of the iron(v)oxo species on nitrobenzene ring obtained for model 2 of nbdo active site , containing first - shell iron ligands and asn258 residue . the gibbs free energy values were obtained using two different models of the active site ( see text for details ) . table 4 also shows the energy profiles calculated for 2-nitrotoluene undergoing the same reaction mechanism . the energy diagrams for both substrates are very similar , but in the case of 2-nitrotoluene , we do not observe lowering of the activation barrier for the first step of the reaction while moving from model 1 to model 2 . the geometries of the stationary points obtained for 2-nitrotoluene in the two smallest models are analogous to the ones located for nitrobenzene , and we do not observe significant differences between the two compounds undergoing the suggested chemical pattern . the cis - dihydroxylation mechanism of nitrobenzene by nbdo is similar to that of olefins catalyzed by the biomimetic complexes fe pytacn and fe tpa . however , the second c o bond formation is not barrierless in the present case possibly due to the stability of the nitrobenzyl radical intermediate . oxo active site , catalyze epoxidation of the c = c bond instead of cis - dihydroxylation due to the absence of two cis labile sites . other enzymes with a 2-his-1-carboxylate facial triad motif have an fe = o oxidant due to the presence of a cofactor or cosubstrate that donates an additional electron . as in principle this species can also be formed during the nbdo catalytic process , provided that a second electron is supplied to the active site , we have calculated the activation barriers for the possible attacks of this intermediate on the substrate as well . if ho fe = o is used as an oxidant , the barriers for the o1c2 , o c2 attack on the nitrobenzene ring become 24.8 , 25.3 , and 14.7 kcal mol , respectively , which indicates that ho fe = o is in general less reactive than the ho fe = o complex . nevertheless , the possibility of the product being formed in the most favorable pathway involving ho fe = o species , i.e. , the one starting from the o c2 attack , was explored . the first step of the reaction yields a radical intermediate with a single c o bond ( scheme 3 ) , formed with endothermicity of 12.1 kcal mol . subsequently , an epoxide intermediate is formed with a barrier of 13.7 kcal mol , which is comparable to the barrier associated with the first step of the reaction . the epoxide intermediate is very high in energy , lying 22.4 kcal mol above the initial reactant complex , and similarly to what was observed for the epoxide mechanism described before , it dissociates from the iron center ( see figure s3 in the supporting information ) . assuming the return to the active site was possible , the transition state for the epoxide ring - opening was located and the barrier associated with this step of the reaction was computed to be 4.3 kcal mol . in the resulting intermediate , upon the formation of a single c1o bond , the nitro group of the substrate is released , as evidenced by the c1n bond elongated to 3.1 . the final step of the reaction involves the attack of hydroxyl oxygen on the c2 carbon , which is associated with the activation barrier of 13.7 kcal mol and yields the final product , cis - dihydrodiol , with the nitro substituent rebound to the substrate ring . we have employed dft methods to investigate the cis - dihydroxylation mechanism of nitrobenzene dioxygenase . possible chemical patterns for dioxygenation of nitrobenzene and 2-nitrotoluene were probed using four models of nbdo active site . based on the literature data , we assumed the iron dioxygen ground state to be a sextet ( s = 5/2 ) with + 2 charge . iron(iii ) and iron(v)oxo intermediate on the aromatic ring of the substrate were considered , and activation energies of the corresponding initial steps of the cis - dihydroxylation were calculated . the gibbs free energy barriers where then used as a guideline in assessing the feasibility of a given mechanism . oxygen cleavage leading from the fe ooh to the high - valent ho fe = o species was also evaluated . finally , the reactivity of the ho fe = o species ( s = 2 ) toward the aromatic ring of nitrobenzene was assessed and compared with that of the iron(v)oxo intermediate . we have found that the gibbs free energies of activation for the direct attack of the ferric hydroperoxo intermediate on the ring , occurring either with or without concomitant oxygen oxygen bond rupture , are comparable to or higher than the activation barrier for the formation of the iron(v)oxo species from its ferric precursor . this suggests the ho moreover , the two cis - dihydroxylation pathways starting with a direct attack of the fe ooh species on the ring , including the mechanism involving the epoxidation step suggested previously for ndo , which were recognized as energetically plausible , did not lead to the formation of the expected product , cis - diol . the reason for this is an instability of the intermediate products formed in these reactions , stemming from the presence of the nitro substituent , which might be overcome in the explicit protein environment . thus , these mechanisms will be reconsidered as potentially viable and further investigated in our future qm / mm study . at the same time , the activation energies for the possible attacks of ho fe = o on the substrate aromatic ring were found to be much lower than the ones calculated for the fe ooh intermediate , proving ho fe = o to be a stronger oxidant in the studied process . furthermore , the most favorable reaction pathway found for this oxidant leads to the formation of a cis - dihydrodiol from both of the considered nitroaromatic substrates . the said mechanism starts with the rate - limiting attack of the hydroxyl oxygen on the c2 carbon of the substrate to form a radical intermediate , which is stabilized by the nitro substituent through spin delocalization . in the following step of the reaction , the second oxygen of the ho fe = o moiety attacks the c1 carbon forming a highly stabilized final product , a cis - dihydrodiol . our proposed mechanism is also consistent with experimentally observed o incorporation in the cis - diol product , and will be used as a starting point in the investigation of steric and electrostatic effects accompanying the reaction taking place in the full enzyme environment . as stated above , our conclusions were verified by performing calculations for both nitrobenzene and 2-nitrotoluene , with no significant differences found between the reactions modeled for both substrates . the calculations performed here for 2-nitrotoluene will also allow for the future comparison between the cis - dihydroxylation and monohydroxylation reactions of this substrate , both of which are catalyzed by nbdo . lastly , the results obtained for the ho fe = o species proved it to be in general less reactive than the ho fe = o complex . however , the pathway starting from the most favorable attack of the ho fe = o on the substrate was also found to be yielding the final product .

the mechanism of cis - dihydroxylation of nitrobenzene and 2-nitrotoluene catalyzed by nitrobenzene 1,2-dioxygenase ( nbdo ) , a member of the naphthalene family of rieske non - heme iron dioxygenases , was studied by means of the density functional theory method using four models of the enzyme active site . different possible reaction pathways for the substrate dioxygenation initiated either by the feiii ooh or ho fev = o attack on the aromatic ring were considered and the computed activation barriers compared with the gibbs free energy of activation for the oxygen oxygen cleavage leading to the formation of the iron(v)oxo species from its ferric hydroperoxo precursor . the mechanism of the substrate cis - dihydroxylation leading to the formation of a cis - dihydrodiol was then investigated , and the most feasible mechanism was found to be starting with the attack of the high - valent iron oxo species on the substrate ring yielding a radical intermediate , which further evolves toward the final product .

Introduction Computational Methods Results and Discussion Conclusions

oxidative degradation of recalcitrant nitroaromatic pollutants is initiated by the formation of a cis - dihydroxylated metabolite that spontaneously rearranges to catechol with accompanying release of nitrite ( figure 1 ) . the reaction is catalyzed by nitrobenzene 1,2-dioxygenase ( nbdo ) , as well as 2-nitrotoluene dioxygenase ( 2ntdo ) , which differ in catalytic site residue at positions 204 , 251 , and 293 . these two enzymes are members of the naphthalene family of rieske non - heme iron dioxygenases ( rdos ) , which consist of a rieske [ 2fe2s ] ferredoxin , an nadh - dependent flavoprotein reductase , and an 33 oxygenase . rdos have a high - spin ligand environment , and theoretical studies on similar fe oor complexes show that the strong o o bond can be attributed to -donation from o moreover , the non - heme ligands of rdos do not stabilize high oxidation states ( through spin delocalization and ligand oxidation ) as porphyrin and thiolate ligands do for heme enzymes , which could explain the endothermicity of heterolytic o o bond cleavage . an insight into the cis - dihydroxylation mechanism of high - valent iron complexes can be gained instead from functional models , which are designed to mimic the ligand environment and reactivity of rdos . theoretical studies on fe pytacn and fe tpa suggest an unsymmetrical concerted mechanism for cis - dihydroxylation initiated by the attack of the hydroxo ligand on the olefin to form a radical , followed by barrierless diol formation . the cis - dihydroxylation of nitroaromatic compounds , represented by nitrobenzene and 2-nitrotoluene , is examined in the present study by means of hybrid density functional theory ( dft ) . alternative mechanisms arising from direct reaction with fe ooh are also explored to compare the activation barriers of substrate oxidation ( with or without concerted oxygen oxygen bond cleavage ) with that of the oxygen oxygen bond cleavage to form ho finally , the mechanism of cis - dihydroxylation by ho fe = o is determined on the basis of experimentally observed o incorporation in the cis - diol product . the possible reaction mechanisms for cis - dihydroxylation of nitrobenzene and 2-nitrotoluene were analyzed using four models of the nitrobenzene dioxygenase active site , presented in figure 2 , which were constructed based on the available crystal structure of nbdo with nitrobenzene bound in the active site ( pdb i d : 2bmq ) . a water molecule was incorporated in the active site model based on the results of the molecular dynamics simulation of nbdo ( data not shown ) , which indicated the presence of water at the entrance to the substrate pocket . [ fe ooh ] , which was found to be the most energetically favorable arrangement of the dioxygen enzyme complex in naphthalene dioxygenase . additionally , the feasibility of the attack of the ho fe = o species ( s = 2 ) on the aromatic ring of nitrobenzene was assessed using the smallest model of the active site . based on the results obtained for the three different models of the active site using gas phase optimization at the b3lyp / lacvp level , they concluded that the most feasible reaction pathway involves the formation of an epoxide intermediate from the direct attack of the hydroperoxo moiety on the substrate double bond . the attack of oh on the other carbon to form the cis - diol has a very small barrier ( 1 kcal / mol ) , consistent with the early transition state ( c oh bond distance of 2.60 ) . the rate - determining step was found to be the formation of an epoxide , with the concomitant cleavage of the oxygen oxygen bond in the ferric hydroperoxo moiety . hence , we decided to investigate whether the chemical pattern proposed for the reaction occurring in the ndo active site is feasible for the formation of cis - dihydrodiol from nitrobenzene catalyzed by nbdo . the transition state leading to the formation of an epoxide was located for the two smallest models of the nbdo active site used in this study , namely , the one including only first shell iron ligands ( model 1 ) and a larger system , which additionally contains asn258 residue ( model 2 ) . similarly to what was observed for naphthalene , the reaction is exothermic , with the epoxide intermediate lying 14.9 kcal mol lower in energy than the reactant complex ( 20.1 kcal mol lower relative to separate reactants ) , but contrary to the results reported for ndo , we do not observe the formation of an epoxide intermediate bound to mononuclear iron , which may further evolve into a carbocation . considering the discussed differences found between the results presented here , and those reported for naphthalene , and a relatively low barrier of the epoxide formation compared to alternative ways of the attack on the ring ( see the next section ) , the discussed pathway should and will be examined as a possible cis - dihydroxylation mechanism in the hybrid qm / mm calculations , which will follow the current study . since the mechanism involving an epoxide intermediate was found not to be plausible for the cis - dihydroxylation of nitroaromatic substrates , it was necessary to probe other chemical routes for such transformation , initiated by the direct attack of the fe three possible ways of the initial attack on the ring were considered here : the attack of the hydroxyl group on the c2 carbon , and the attack of the second oxygen atom of the hydroperoxo group on either c1 or c2 carbon , as shown in figure 4 . for each of the considered pathways , we located the corresponding transition state for the concerted attack ( with concomitant o o cleavage ) and the transition state leading to the formation of a single c o bond without the cleavage of the oxygen oxygen bond in the ferric hydroperoxo moiety ( nonconcerted attack ) . three routes of the attack of fe ooh ( a ) or ho fe = o ( b ) intermediate on the substrate considered as possible initial steps of its cis - dihydroxylation . in each of the probed mechanisms , the attack of the oxygen atom on the aromatic ring without the oxygen oxygen bond cleavage was found to have a higher energetic barrier than the corresponding concerted attack . in the reaction presented therein , a similar , nonconcerted attack of the hydroperoxo ligand on the substrate leads to the formation of a radical intermediate with a single c o bond , this step being associated with the barrier of 1920 kcal mol and endothermicity of around 18 kcal mol . the subsequent step of the reaction yields a cis - diol , but the associated transition state is higher in energy than the preceding one , based on which the mechanism was considered as rather unlikely . the estimated energy of the transition state associated with the cleavage of the o o bond is similar to the transition state for the formation of a radical intermediate . activation energies are in agreement with the barriers obtained for the other substrate , with the values of 23.6 , 21.2 , and 21.1 kcal mol for the o1c2 , o similarly to what was found for nitrobenzene , the resulting intermediates did not lead to the formation of a diol . an exception to this , as in the case of b97d results , is a barrier for the nonconcerted o c1 attack yielding an epoxide intermediate of around 2526 kcal mol is larger than the one obtained with the b97d functional , but at the same time according to b3lyp - d results it is the most favorable pathway involving the fe as mentioned above , a similar barrier was found for a nonconcerted o c2 attack , and a concerted o c2 attack , which was excluded from further consideration as not leading to the final product , is also not energetically favored over the concerted o subsequently , we considered the possibility of the formation of a high - valent intermediate , ho fe = o , prior to substrate oxidation . in low - spin biomimetic non - heme iron complexes , oxygen oxygen bond cleavage is facilitated by the formation of a five - membered intermediate with water , as evident from exchange with o - labeled solvent . the gibbs free energy of activation for o o bond heterolysis was also determined in the presence of the substrate using different models of the enzyme . this suggests that the reaction proceeds through the formation of the high - valent oxo species and its subsequent attack on the ring , rather than the direct attack of the ferric hydroperoxo ligand on the substrate . depending on the model used , these values become comparable or larger by up to 3.9 kcal mol than the barriers for the initial attack of fe ooh on the substrate ring , which , considering the accuracy of the calculated energies , still allows us to consider the formation of ho fe = o species and its subsequent reaction with the substrate as a plausible scenario for its cis - dihydroxylation . fe = o , in comparison with a direct reaction between fe ooh and the substrate , suggests that the high - valent intermediate is a possible oxidant . a separate experiment with naphthalene cis - diol and h2o confirms that solvent exchange occurs with a reactive intermediate and not with the product.o - labeled products may be attributed to rapid intermolecular exchange between ho therefore , we probed possible dioxygenation pathways starting from a direct attack of ho fe = o on the substrate ( figure 4b ) . the barriers computed for the initial formation of an oxygen carbon bond presented in table 3 prove this step to be in general more energetically favorable when the substrate is attacked by an ho fe = o species rather than by the ferric hydroperoxo intermediate discussed before . in parentheses , the results obtained for the two smallest models indicate that the o1c2 attack is the first step of the reaction , while for models 3 and 4 , a noticeably smaller barrier is found for the o the latter findings may be , however , affected by the presence of a water molecule in the active site model . moreover , in the current study , we were not able to obtain a final product in the reaction starting with the o a comparison between the barriers for all possible initial steps of the reaction also shows that the formation of a single c o bond initiated by the attack of hydroxyl oxygen of the iron(v)oxo complex on the c2 carbon is the most feasible transformation , from which the reaction can evolve toward the final product . the barriers calculated using b3lyp - d functionals agree very well with the results presented in table 3 , and confirm low activation energies for the ho fe = o attack compared to a direct reaction of fe ooh with the substrate ( tables s2 and s3 in the supporting information ) . likewise , the b3lyp - d results demonstrate that the energetically most favorable step initiating cis - dihydroxylation is the formation of an o1c2 bond through an ho fe = o attack on the aromatic ring . the mechanism of cis - dihydroxylation , as shown in figure 5 , was studied in detail using models 1 and 2 . initial attack of the hydroxo ligand on the c2 carbon is the rate - determining step with a gibbs free energy of activation of 7.0 kcal / mol in model 1 and 2.4 kcal / mol in model 2 . upon the formation of the diol , the distance between the nitro group and c1 carbon elongates to 1.6 , with the substituent still bonded to the aromatic ring , which agrees with the general reaction scheme of rieske dioxygenases suggested in the literature , according to which the nitrite release occurs in the subsequent step of the catalytic cycle . reaction profile of cis - dihydroxylation of nitrobenzene through a direct attack of the ho fe = o species on the aromatic ring . optimized geometries , relevant spin populations , and bond lengths ( ) of the key stationary points located along the reaction pathway depicted in figure 5 : ts1 , transition state for the initial attack of the iron(v)oxo intermediate on the carbon carbon double bond ; int1 radical intermediate formed upon the formation of a single c o bond ; ts2 transition state for the oxygen attack on the c1 carbon yielding the cis - dihydrodiol pc . transition state for the attack of the iron(v)oxo species on nitrobenzene ring obtained for model 2 of nbdo active site , containing first - shell iron ligands and asn258 residue . the gibbs free energy values were obtained using two different models of the active site ( see text for details ) . the cis - dihydroxylation mechanism of nitrobenzene by nbdo is similar to that of olefins catalyzed by the biomimetic complexes fe pytacn and fe tpa . oxo active site , catalyze epoxidation of the c = c bond instead of cis - dihydroxylation due to the absence of two cis labile sites . as in principle this species can also be formed during the nbdo catalytic process , provided that a second electron is supplied to the active site , we have calculated the activation barriers for the possible attacks of this intermediate on the substrate as well . if ho fe = o is used as an oxidant , the barriers for the o1c2 , o c2 attack on the nitrobenzene ring become 24.8 , 25.3 , and 14.7 kcal mol , respectively , which indicates that ho fe = o is in general less reactive than the ho fe = o complex . assuming the return to the active site was possible , the transition state for the epoxide ring - opening was located and the barrier associated with this step of the reaction was computed to be 4.3 kcal mol . in the resulting intermediate , upon the formation of a single c1o bond , the nitro group of the substrate is released , as evidenced by the c1n bond elongated to 3.1 . the final step of the reaction involves the attack of hydroxyl oxygen on the c2 carbon , which is associated with the activation barrier of 13.7 kcal mol and yields the final product , cis - dihydrodiol , with the nitro substituent rebound to the substrate ring . possible chemical patterns for dioxygenation of nitrobenzene and 2-nitrotoluene were probed using four models of nbdo active site . iron(iii ) and iron(v)oxo intermediate on the aromatic ring of the substrate were considered , and activation energies of the corresponding initial steps of the cis - dihydroxylation were calculated . oxygen cleavage leading from the fe ooh to the high - valent ho fe = o species was also evaluated . finally , the reactivity of the ho fe = o species ( s = 2 ) toward the aromatic ring of nitrobenzene was assessed and compared with that of the iron(v)oxo intermediate . we have found that the gibbs free energies of activation for the direct attack of the ferric hydroperoxo intermediate on the ring , occurring either with or without concomitant oxygen oxygen bond rupture , are comparable to or higher than the activation barrier for the formation of the iron(v)oxo species from its ferric precursor . this suggests the ho moreover , the two cis - dihydroxylation pathways starting with a direct attack of the fe ooh species on the ring , including the mechanism involving the epoxidation step suggested previously for ndo , which were recognized as energetically plausible , did not lead to the formation of the expected product , cis - diol . at the same time , the activation energies for the possible attacks of ho fe = o on the substrate aromatic ring were found to be much lower than the ones calculated for the fe ooh intermediate , proving ho fe = o to be a stronger oxidant in the studied process . furthermore , the most favorable reaction pathway found for this oxidant leads to the formation of a cis - dihydrodiol from both of the considered nitroaromatic substrates . the said mechanism starts with the rate - limiting attack of the hydroxyl oxygen on the c2 carbon of the substrate to form a radical intermediate , which is stabilized by the nitro substituent through spin delocalization . in the following step of the reaction , the second oxygen of the ho fe = o moiety attacks the c1 carbon forming a highly stabilized final product , a cis - dihydrodiol . however , the pathway starting from the most favorable attack of the ho fe = o on the substrate was also found to be yielding the final product .

the motion and associated emission of gravitational waves ( gw ) of self - gravitating systems have been a main research interest in general relativity . the problem is complicated conceptually as well as mathematically because of the nonlinearity of the einstein equations . there is no hope in any foreseeable future to have exact solutions describing motions of arbitrarily shaped , massive bodies , so we have to adopt some sort of approximation schemes for solving the einstein equations to study such problems . in the past years many types of approximation schemes have been developed depending on the nature of the system under consideration . here we shall focus on a particular scheme called the post - newtonian ( pn ) approximation . there are many systems in astrophysics where newtonian gravity is dominant , but general relativistic gravity plays also an important role in their evolution . for such systems it would be nice to have an approximation scheme which gives a newtonian description in the lowest order and general relativistic effects as higher order perturbations . the post - newtonian approximation is perfectly suited for this purpose . historically einstein computed first the post - newtonian effects , e.g. the precession of the perihelion . studies of the post - newtonian approximation were made by lorentz and droste , einstein , infeld , and hoffmann , fock , plebansky and bazanski , and chandrasekhar and associates [ 40 , 41 , 42 , 43 ] . now it is widely known that the post - newtonian approximation is important in analyzing a number of relativistic problems , such as the equations of motion of binary pulsars [ 34 , 61 , 74 , 89 ] , solar - system tests of general relativity [ 159 , 160 , 161 , 164 ] , and gravitational radiation reaction [ 39 , 42 ] . any approximation scheme necessitates one or several small parameters characterizing the nature of the system under consideration . a typical parameter which most of the schemes adopt is the magnitude of the metric deviation from a certain background metric . in particular if the background is minkowski spacetime and there is no other parameter , the scheme is sometimes called the post - minkowskian approximation in the sense that the constructed spacetime reduces to minkowski spacetime in the limit that the parameter tends to zero . this limit is called the weak field limit . in the case of the post - newtonian approximation the background spacetime is also minkowski spacetime , but there is another small parameter , that is , the typical velocity of the system divided by the speed of light . we introduce a non - dimensional parameter to express the slowness of the system . these two parameters ( the deviation from the flat metric and the velocity ) have to have a certain relation in the following sense . as the gravitational field gets weaker , all velocities and forces characteristic of the material systems become smaller , in order to permit the weakening of gravity to remain an important effect in the system s dynamics . for example in the case of a binary system , the typical velocity would be the orbital velocity /c and the deviation from the flat metric would be the newtonian potential , say . then these are related by /c /c which guarantees that the system is bounded by its own gravity . in the post - newtonian approximation , the equations of general relativity take the form of newton s equations in an appropriate limit as 0 . such a limit is called the newtonian limit and it will be the basis of constructing the post - newtonian approximation . however , the limit is not in any sense trivial since it may be thought of as two limits tied together as just described . it is also worth noting that the newtonian limit can not be uniform everywhere for all time . for example any compact binary system , no matter how weak the gravity between its components and slow its orbital motion is , will eventually spiral together due to backreaction from the emission of gravitational waves . as the result the effects of its newtonian gravity will be swamped by those of its gravitational waves . this will mean that higher order effects of the post - newtonian approximation eventually dominate the lowest order newtonian dynamics and thus if the post - newtonian approximation is not carefully constructed , this effect can lead to many formal problems , such as divergent integrals . it has been shown that such divergences may be avoided by carefully defining the newtonian limit . moreover , the use of such a limit provides us a strong indication that the post - newtonian hierarchy is an asymptotic approximation to general relativity . therefore we shall first discuss in this paper the newtonian limit and how to construct the post - newtonian hierarchy before attacking practical problems in later sections . before going into the details , we mention the reason for the growth of interest in the post - newtonian approximation in recent years . certainly the discovery of the binary neutron star system psr 1913 + 16 was a strong reason to have renewed interest in the post - newtonian approximation , since it is the first system found in which general relativistic gravity plays a fundamental role in its evolution . particularly the indirect discovery of gravitational waves by the observation of the period shortening led to many fruitful studies of the equations of motion with gravitational radiation emission in 1980s ( see [ 47 , 48 , 49 ] for a review ) . the effect of radiation reaction appears in the form of a potential force at the order of higher than the newtonian force in the equations of motion . ehlers and colleagues critically discussed the foundation of the so - called quadrupole formula for the radiation reaction ( see also the introduction of ) . there have been various attempts to show the validity of the quadrupole formula [ 5 , 47 , 79 , 90 , 104 , 105 , 106 , 107 , 135 , 138 , 139 , 155 , 157 , 156 ] . namely , damour proved the formula for compact binaries with the help of the dominant schwarzschild condition . at that time , however , no serious attempts with direct detection of gravitational waves in mind had been made for the study of higher order effects in the equations of motion . the situation changed gradually in the late 1980s because of the increasing expectation of a direct detection of gravitational waves by kilometer - size interferometric gravitational wave detectors , such as ligo [ 1 , 116 ] , virgo [ 35 , 153 ] , geo [ 62 , 83 ] , and tama [ 114 , 149 ] . coalescing binary neutron stars are the most promising candidates of sources of gravitational waves for such detectors . the reasons are that ( i ) we expect , say , the ( initial ) ligo to detect the signal of coalescence of binary neutron stars about once per year to once per hundreds of years [ 38 , 103 , 102 ] , and ( ii ) the waveform from coalescing binaries can be predicted with high accuracy compared to other sources [ 1 , 151 , 161 ] . information carried by gravitational waves tells us not only various physical parameters of neutron stars , but also the cosmological parameters [ 75 , 119 , 141 , 142 , 158 ] if and only if we can make a detailed comparison between the observed signal with theoretical predictions during the epoch of the so - called inspiraling phase where the orbital separation is much larger than the radius of the component stars . this is the place where the post - newtonian approximation may be applied to make theoretical templates for gravitational waves . the problem is that in order to make any meaningful comparison between theory and observation we need to know the detailed waveforms generated by the motion up to , say , 4 pn order which is of order higher than the newtonian order [ 2 , 3 , 10 , 147 ] . this request from gravitational wave astronomy forces us to construct higher order post - newtonian equations of motion and waveform templates . replying to this request , there have been various works studying the equations of motion for a compact binary system and developing higher order post - newtonian gravitational waveform templates for such a system . the most systematic among those works that have succeeded in achieving higher order iteration are the ones by blanchet , damour , and iyer who have developed a scheme to calculate the waveform at a higher order , where the post - minkowskian approximation is used to construct the external field and the post - newtonian approximation is used to construct the field near the material source . they and their collaborators have obtained the waveform up to 3.5 pn order which is of order higher than the lowest quadrupole wave [ 23 , 24 , 29 , 31 , 32 ] by using the equations of motion up to that order [ 22 , 91 , 93 , 111 , 123 , 130 ] . blanchet and schfer have investigated a spectral ( fourier ) decomposition of the tail and computed the contribution of the tail to the gravitational wave luminosity emitted by a binary system having a general eccentric orbit . asada and futamase showed that the dominant part of the tail term originates from the phase shift of the wave due to the coulomb part of the gravitational field . in this paper , we mainly discuss the foundation of the newtonian limit and the post - newtonian equations of motion for relativistic compact binaries in an inspiralling phase . in the next section 1.2 many authors have derived equations of motion up to the 1 pn order [ 66 , 73 , 76 , 94 , 117 , 152 ] , up to 2.5 pn order [ 30 , 47 , 50 , 51 , 52 , 46 , 87 , 95 , 113 , 130 ] , and up to 3 pn order [ 22 , 91 , 93 ] . the 3.5 pn correction to the equations of motion has been derived by [ 111 , 123 , 130 ] . the post - newtonian equations of motion are now available in harmonic coordinates up to 3.5 pn order inclusively . see also [ 17 , 85 , 86 , 96 ] for the 3.5 pn and the 4.5 pn correction based on the energy balance . besides the equations of motion , attempts have been made to derive a hamiltonian for a binary system . the second order post - newtonian computation of the hamiltonian was tackled by [ 125 , 126 , 127 ] and completed in [ 56 , 135 , 136 , 137 ] . so far , the post - newtonian arnowitt - deser - misner ( adm ) hamiltonian is available in adm transverse - traceless coordinates up to 3.5 pn order inclusively . the equations of motion for a two point mass binary in harmonic coordinates up to 2.5 pn order , at which the radiation reaction effect first appears , were derived by damour and deruelle [ 52 , 46 ] based on the post - minkowskian approach . these works used dirac delta distributions to express the point masses mathematically , therefore they inevitably resorted to a purely mathematical regularization procedure to deal with divergences arising from the nonlinearity of general relativity . damour addressed the applicability of the use of a dirac delta distribution to self - gravitating objects . by investigating the tidal effect exerted by the companion star on the main star , he gave a plausible argument known as the dominant schwarzschild condition which supports that up to 2.5 pn order , the field around the main star is recovered by the energy momentum tensor expressed in terms of a dirac delta distribution . direct validations of the 2.5 pn equations of motion by damour and deruelle , where a dirac delta distribution was not used , have been obtained in several works [ 87 , 95 , 113 , 130 ] . grishchuk and kopeikin and kopeikin worked on extended but intrinsically spherical bodies with weak internal gravity using the post - newtonian approximation both inside and outside the stars . they volume - integrated the equations of the conservation of the stress - energy tensor of the matter for an ideal fluid with two compact supports and obtained their equations of motion . the present authors and asada on the other hand derived the 2.5 pn equations of motion for point particles with arbitrarily strong internal gravity using a regular point particle limit called the strong field point particle limit . these authors also used the local conservation law but adopted a surface integral approach , and they did not specify an explicit form of the stress - energy tensor but assumed that it satisfies some scaling on the initial hypersurface . blanchet , faye , and ponsot also derived the 2.5 pn equations of motion using dirac delta distributions for which hadamard s partie finie regularization was employed to handle the divergences due to their use of dirac delta distributions . in this approach , they have assumed that the two point masses follow regularized geodesic equations . ( more precisely , they have assumed that the dynamics of two point masses are described by a regularized action , from which a regularized geodesic equation was shown to be derived . ) they also derived the gravitational field up to 2.5 pn order in an explicit form which may help constructing initial data for numerical simulations of compact binaries . namely , our work shows the applicability of the damour and deruelle 2.5 pn equations of motion to a relativistic compact binary consisting of regular stars with strong internal gravity . we mention here that stars consisting of relativistic compact binaries , for which we are searching as gravitational wave sources , have a strong internal gravitational field , and that it is a nontrivial question whether a star follows the same orbit regardless of the strength of its internal gravity . currently we have the equations of motion for relativistic compact binaries through the 3.5 pn approximation of general relativity in hand . actually the 3.5 pn correction to the equations of motion is relatively easily derived [ 111 , 123 , 130 ] . at 3 pn order , an issue on undetermined coefficient associated with the regularization procedures was found which we now briefly discuss . a 3 pn iteration result was first reported by jaranowski and schfer [ 98 , 99 ] . there a 3 pn adm hamiltonian in the adm transverse traceless ( admtt ) gauge for two point masses expressed as two dirac delta distributions was derived based on the adm canonical approach [ 125 , 135 ] . however , it was found in [ 98 , 99 ] that the regularization they had used caused one coefficient kinetic to be undetermined in their framework . origins of these two coefficients were attributed to some unsatisfactory features of regularization they had used , such as violation of the leibniz rule . the former coefficient , which appears as a numerical multiplier of the term that depends on the momenta of the point particles , was then fixed as kinetic = 41/24 by a posteriori imposing poincare invariance on their 3 pn hamiltonian . as for the latter coefficient , damour et al . succeeded in fixing it as static = 0 , adopting dimensional regularization1 . moreover , with this method they found the same value of kinetic as in , which ensures lorentz invariance of their hamiltonian . on the other hand , blanchet and faye have tackled the derivation of the 3 pn equations of motion for two point masses expressed as two dirac delta distributions in harmonic coordinates [ 25 , 27 ] based on their previous work . the divergences due to their use of dirac delta distributions were systematically regularized with the help of lorentz invariant generalized hadamard partie finie regularization . they have extended the notion of the hadamard partie finie regularization to regularize divergent integrals and a singular function which does not permit a power - like expansion near its singularities . furthermore , the regularization is carefully constructed in so that it respects lorentz invariance . their equations of motion respect the lorentz invariance in the post - newtonian pertubative sense and admits a conserved energy of orbital motion modulo the 2.5 pn radiation reaction effect . they found , however , that there exists one and only one undetermined coefficient ( which they call ) . interestingly , the two groups independently constructed a transformation between the two gauges and found that these two results coincide with each other when there exists a relation [ 55 , 64 ] 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ { { \rm{static } } } } = - { { 11 } \over 3}\lambda - { { 1987 } \over { 840}}.$$\end{document } therefore , it is possible to fix the parameter from the result of as 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda = - { { 1987 } \over { 3080}}.$$\end{document } however , the applicability of mathematical regularization to the current problem is not a trivial issue , but an assumption to be verified , or at least supported by convincing arguments . there is no argument such as the dominant schwarzschild condition at 3 pn order . the present authors have derived the 3 pn equations of motion for relativistic compact binaries [ 91 , 92 , 93 ] in harmonic coordinates based on their previous work . , they did not find any undetermined coefficient at all in the equations of motion and found the same value of the parameter as equation ( 2 ) . physically equivalent 3 pn equations of motion in harmonic coordinates were also completed by blanchet , damour , and esposito - farse based on their previous works [ 25 , 27 , 30 ] . there , they have used the dimensional regularization to overcome the problem with the ( generalized ) hadamard partie finie . the physical equivalence between the two results , blanchet - damour - esposito - farse s and our equations of motion , suggests that , at least up to 3 pn order , a particle ( with a strong internal gravity ) follows a regularized ( in some sense ) geodesic equation in a dynamical spacetime , a part of whose gravitational field the particle itself generates . will and his collaborators [ 130 , 129 , 163 ] have been tackling the 3 pn iteration of the equations of motion where they take into account the density profile of the stars explicitly . their result may ( or may not ) give a direct check of the effacement principle up to 3 pn order which states that the motion of the objects depends only on their masses and not on their internal structures up to the order where the tidal effect comes into play . this article is organized as follows : in section 2 we introduce the newtonian limit in general relativity and present how to construct the post - newtonian hierarchy . there we mention how to avoid divergent integrals which appear at a higher order in the previous treatments . we shall then give a formulation of the dynamics of a newtonian perfect fluid as the lowest order post - newtonian approximation in harmonic coordinates . we focus our attention on the derivation of the 3 pn equations of motion based on the work by the authors of this article from section 3 to section 8 . in section 3 , we discuss the basics of our derivation of the post - newtonian equations of motion for relativistic compact binaries . there we discuss how to incorporate strong internal gravity in the post - newtonian approximation . in section 4 , , we derive the newtonian and the 1 pn equations of motion in our formalism . in section 5 , we briefly explain how to derive the 3 pn gravitational field when possible , and how to derive equations of motion when the gravitational field is unavailable in an explicit closed form . in sections 6 and 7 , we derive the mass - energy relation and the momentum - velocity relation up to 3 pn order , and finally in section 8 , we derive the 3 pn equations of motion . the resulting equations of motion respect lorentz invariance , admit a conserved energy ( modulo the 2.5 pn radiation reaction effect ) , and are free from any ambiguity . section 8 ends with a summary and some remarks on possible future study of the 4 pn order iteration . in appendix a , we give a brief sketch of the direct integration of the relaxed einstein equations ( dire ) method by will and wiseman [ 129 , 162 , 165 ] which we have used in this article . although in the main body of this article we focus our attention on spherical massive bodies ( or point particles ) , we shall apply our formalism to extended bodies in appendix b. in appendix c , using the strong field point particle limit and the surface integral approach , we discuss that a particle with strong internal gravity moves on a geodesic of some smooth metric part which is produced by the particle itself . this work is done by the authors in the collaboration with takashi fukumoto . throughout this article since the newtonian limit is the basis of the post - newtonian approximation , we shall first formulate the newtonian limit . we will not mention other formulations of the post - newtonian approximation [ 63 , 70 , 134 ] . this formulation is based on the observation that any asymptotic approximation of any theory needs a sequence of solutions of the basic equations of the theory [ 146 , 154 ] . namely , if we write the equations in abstract form as 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e(g ) = 0$$\end{document } for an unknown function g , one would like to have a one - parameter ( or possibly multi - parameter ) family of solutions , 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e(g(\lambda ) ) = 0,$$\end{document } where is some parameter . asymptotic approximation then says that a function f( ) approximates g( ) to order if f( ) g()/ 0 as 0 . we choose the sequence of solutions with appropriate properties in such a way that the properties reflect the character of the system under consideration . we shall formulate the post - newtonian approximation according to the general idea just described . as stated in the introduction , we would like to have an approximation which applies to the systems whose motions are described almost by newtonian theory . thus we need a sequence of solutions of the einstein equations parameterized by ( the typical velocity of the system divided by the speed of light ) which has newtonian character as 0 . the newtonian equations involve six variables , namely the density , the pressure p , the gravitational potential , and the velocity , i = 1 , 2 , 3 ) : 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla ^2}\phi - 4\pi \rho = 0,$$\end{document } 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}\rho + { \nabla _ i}(\rho { v^i } ) = 0,$$\end{document } 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho { \partial _ t}{v^i } + \rho { v^j}{\nabla _ j}{v^i } + { \nabla ^i}p + \rho { \nabla ^i}\phi = 0,$$\end{document } supplemented by an equation of state . for simplicity we have considered a perfect fluid . it can be seen that the variables { (x , t ) , p(x , t ) , (x , t ) , (x , t ) } obeying the above equations satisfy the following scaling law : 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \rho ( { x^i},t ) \rightarrow { \epsilon ^2}\rho ( { x^i},\epsilon t ) , } \\ { p({x^i},t ) \rightarrow { \epsilon ^4}p({x^i},\epsilon t ) , } \\ { { v^i}({x^k},t ) \rightarrow \epsilon { v^i}({x^k},\epsilon t ) , } \\ { \phi ( { x^i},t ) \rightarrow { \epsilon ^2}\phi ( { x^i},\epsilon t ) . } \\ \end{array}$$\end{document } one can easily understand the meaning of this scaling by noticing that is the magnitude of the typical velocity ( divided by the speed of light ) . then the magnitude of the gravitational potential will be of order because of the balance between gravity and the centrifugal force . the scaling of the time variable expresses the fact that the weaker gravity is ( 0 ) the longer the time scale is . thus we wish to have a sequence of solutions of the einstein equations which has the above scaling as 0 . we shall also take the point of view that the sequence of solutions is determined by the appropriate sequence of initial data . this has a practical advantage because there will be no solutions of the einstein equations which satisfy the above scaling ( 8) even as 0 . this is because the einstein equations are nonlinear in the field variables , so it will not be possible to enforce the scaling everywhere in spacetime . we shall therefore impose it only on the initial data for the solution of the sequence . here we first give a general discussion on the formulation of the post - newtonian approximation independent of any initial value formalism and then present the concrete treatment in harmonic coordinates . the condition is used because of its popularity and some advantages in the generalization to the systems with strong internal gravity . as the initial data for the matter we take the same data set in the newtonian case , namely the density , the pressure p , and the coordinate velocity . in most of the application , we usually assume a simple equation of state which relates the pressure to the density . since general relativity is an overdetermined system , there will be constraint equations among the initial data for the field . we shall write the free data for the field as ( qij , pij ) whose explicit forms depend on the coordinate condition one assumes . in any coordinates we shall assume these data for the field vanish since we are interested in the evolution of an isolated system by its own gravitational interaction . it is expected that this choice corresponds to the absence of radiation far away from the source . thus we choose the following initial data which is indicated by the newtonian scaling : 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \rho ( t = 0,{x^i},\epsilon ) = { \epsilon ^2}a({x^i } ) , } \\ { p(t = 0,{x^i},\epsilon ) = { \epsilon ^4}b({x^i } ) , } \\ { { v^i}(t = 0,{x^k},\epsilon ) = \epsilon { c^i}({x^k } ) , } \\ { { q_{ij}}(t = 0,{x^i},\epsilon ) = 0,\quad \quad \,\,\,\ , } \\ { { p_{ij}}(t = 0,{x^i},\epsilon ) = 0,\quad \quad \,\,\ , } \\ \end{array}$$\end{document } where the functions a , b , and c are c functions that have compact support contained entirely within a sphere of a finite radius . corresponding to the above data , we have a one - parameter set of spacetime parameterized by . it may be helpful to visualize the set as a fiber bundle , with base space r being the real line ( coordinate ) and fiber r being the spacetime ( coordinates t , x ) . the fiber = 0 is minkowski spacetime since it is defined by zero data . in the following we shall assume that the solutions are sufficiently smooth functions of for small 0 . the limit is , however , not unique and is defined by giving a smooth nowhere vanishing vector field on the fiber bundle which is nowhere tangent to each fiber [ 84 , 146 ] . the integral curves of the vector field give a correspondence between points in different fibers , namely events in different spacetimes with different values of . remembering the newtonian scaling of the time variable in the limit , we introduce the newtonian dynamical time , 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tau = \epsilon t,$$\end{document } and define the integral curve as the curve on which and x stay constant2 . in fact if we take the limit 0 along this curve , the orbital period of the binary system with = 0.01 is 10 times that of the system with = 0.1 as expected from the newtonian scaling . notice that this map never reaches the fiber = 0 ( minkowski spacetime ) . we assume the existence of such a sequence of solutions constructed by the initial data satisfying the above scaling with respect to . we shall call such a sequence a regular asymptotically newtonian sequence . we will not go into details partly because in order to prove the assumptions we need a deep understanding of the existence and uniqueness properties of the cauchy problem of the einstein equations with perfect fluids of compact support which are not available at present . we shall now define the newtonian , post - newtonian , and higher approximations of various quantities as the appropriate higher tangents of the corresponding quantities to the above integral curve at = 0 . for example the hierarchy of approximations for the spacetime metrics can be expressed as follows : 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { g_{\mu \nu}}(\epsilon , \tau , { x^i } ) = { g_{\mu \nu}}(0 , \tau , { x^i } ) + \epsilon ( { { \mathcal l}_v}{g_{\mu \nu}})(0 , \tau , { x^i})\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + { 1 \over 2}{\epsilon ^2}({\mathcal l}_v^2{g_{\mu \nu}})(0,\tau,{x^i } ) + \cdots + { { { \epsilon ^n } } \over { n!}}({\mathcal l}_v^n{g_{\mu \nu}})(0,\tau,{x^i } ) + { r_{n + 1 } } , } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal l_v}$\end{document } is the lie derivative with respect to the tangent vector of the curves defined above , and the remainder term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_{n + 1}^{\mu \nu}$\end{document } is 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_{n + 1}^{\mu \nu } = { { { \epsilon ^{n + 1 } } } \over { ( n + 1)!}}\int\nolimits_0 ^ 1 d \ell \,{(1 - \ell)^{n + 1}}({\mathcal l}_v^{n + 1}{g_{\mu \nu}})(\ell \epsilon , \tau , { x^i}).$$\end{document } taylor s theorem guarantees that the series is an asymptotic expansion about = 0 under certain assumptions mentioned above . it may be useful to point out that the above definition of the approximation scheme may be formulated purely geometrically in terms of a jet bundle . the above definition of the post - newtonian hierarchy gives us an asymptotic series in which each term in the series is manifestly finite . this is based on the dependence of the domain of dependence of the field point ( x ) . the region is finite with finite values of , and the diameter of the region increases like as 0 . without this linkage of the region with the expansion parameter , the post - newtonian approximation leads to divergences in the higher orders . namely , it is assumed that the slow motion assumption enables one to taylor expand the retarded integrals in retarded time such as 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int d r\,f(\tau - \epsilon r , \ldots ) = \int d r\,f(\tau , \ldots ) - \epsilon \int d r\,rf{(\tau , \ldots)_{,\tau } } + \ldots,$$\end{document } and assign the second term to a higher order because of its explicit in front . this is incorrect because r as 0 and thus r is not uniformly small in the newtonian limit . but at some higher order there appear many terms which do not fall off sufficiently fast because of the nonlinearity of the einstein equations . it turns out that such a divergence appears at 4 pn order , indicating a breakdown of the pn approximation in harmonic coordinates 3 . this sort of divergence may be eliminated if we remember that the upper bound of the integral does depend on as . thus we would get something like ln instead of ln in the usual approach . this shows that the asymptotic newtonian sequence is not differentiable in at = 0 , but there is no divergence in the expansion and it has still an asymptotic approximation in that involves logarithms . other than the initial value formulation method [ 79 , 82 ] mentioned above , various methods have been proposed to solve this problem of the divergent integrals . it is known that a higher order post - newtonian metric does not respect the asymptotically flat condition . this does not mean that the post - newtonian approximation is useless at such a high order . the problem is related to the fact that a simple post - newtonian iteration is meaningful only in the near zone about one wavelength distance away from the material source and is not useful outside of the near zone , called far zone , where the wave effect ( retardation effect ) is manifest . so roughly speaking , if a far zone metric satisfying proper boundary conditions at infinity is solved so that we have a boundary condition to the field equations for a post - newtonian metric in the buffer zone , we can find a post - newtonian metric which is meaningful in the sense that it respects the correct behaviour at the near zone boundary . the far zone metric satisfies a stationarity condition and is parametrized by radiative multipole moments . on the other hand , they solve a post - newtonian near zone metric up to a homogeneous solution . they then establish an association between those radiative multipole moments and the source multipole moments that characterize a post - newtonian near zone metric to fix the homogeneous solution , and find a post - newtonian metric which satisfies the correct behaviour in the buffer zone . will and wiseman have developed the dire method [ 129 , 162 , 165 ] where they split the integral region in the retarded integral into two one being the near zone and the other being the far zone . the retarded integral over the far zone is directly evaluated with the assumption of sufficient stationarity of the system in the infinite past . in fact , both the blanchet - damour method and the will - wiseman method are proved to give a physically equivalent result . in this paper , for our computation of the 3 pn equations of motion , we will use the will and wiseman method to solve the problem of the breakdown of the post - newtonian approximation in the near zone . here the reduced einstein equations in the harmonic condition are written as 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde g^{\alpha \beta}}{\tilde g^{\mu \nu}}{\,_{,\alpha \beta } } = 16\pi { \theta ^{\mu \nu } } - { \tilde g^{\mu \alpha}}{\,_{,\beta}}{\tilde g^{\nu \beta}}{\,_{,\alpha}},$$\end{document } 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ \nu}{x^\alpha } ] = 0,$$\end{document } where 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde g^{\mu \nu } } = { ( - g)^{1/2}}{g^{\mu \nu}},$$\end{document } 17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\theta ^{\alpha \beta } } = ( - g)({t^{\alpha \beta } } + t_{{\rm{ll}}}^{\alpha \beta}),$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_{ll}^{\mu \nu}$\end{document } is the landau - lifshitz pseudotensor . in this section we shall choose an isentropic perfect fluid for t which is enough for most applications , 18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t^{\alpha \beta } } = ( \rho + \rho \pi + p){u^\alpha}{u^\beta } + p{g^{\alpha \beta}},$$\end{document } where is the rest mass density , ii the internal energy , p the pressure , and u the four - velocity of the fluid with normalization 19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\alpha \beta}}{u^\alpha}{u^\beta } = - 1.$$\end{document } the conservation of energy and momentum is expressed as 20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ \beta}{t^{\alpha \beta } } = 0.$$\end{document } defining the gravitational field variable as 21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{\mu \nu } } = { \eta ^{\mu \nu } } - { ( - g)^{1/2}}{g^{\mu \nu}},$$\end{document } where is the minkowski metric , the reduced einstein equations ( 14 ) and the gauge condition ( 15 ) take the following form : 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$({\eta ^{\alpha \beta } } - { h^{\alpha \beta}}){h^{\mu \nu}}_{,\alpha \beta } = - 16\pi { \theta ^{\mu \nu } } + { h^{\mu \alpha}}_{,\beta}{h^{\nu \beta}}_{,\alpha},$$\end{document } 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{\mu \nu}}_{,\nu } = 0.$$\end{document } thus the characteristics are determined by the operator ( h) , and thus the light cone deviates from that in the flat spacetime . we may use this form of the reduced einstein equations in the calculation of the waveform far away from the source because the deviation plays a fundamental role there . in the study of the gravitational field near the source it is not necessary to consider the deviation of the light cone from the flat one and thus it is convenient to use the following form of the reduced einstein equations : 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\eta ^{\mu \nu}}{h^{\alpha \beta}}_{,\mu \nu } = - 16\pi { \lambda ^{\alpha \beta}},$$\end{document } where 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\lambda ^{\alpha \beta } } = { \theta ^{\alpha \beta } } + { \chi ^{\alpha \beta \mu \nu}}_{,\mu \nu},$$\end{document } 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\chi ^{\alpha \beta \mu \nu } } = { ( 16\pi)^{- 1}}({h^{\alpha \nu}}{h^{\beta \mu } } - { h^{\alpha \beta}}{h^{\mu \nu}}).$$\end{document } equations ( 23 ) and ( 24 ) together imply the conservation law 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\lambda ^{\alpha \beta}}_{,\beta } = 0.$$\end{document } we shall take as our variables the set { , p , , h } , with the definition 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${v^i } = { u^i}/{u^0}.$$\end{document } the time component of four - velocity u is determined from equation ( 19 ) . to make a well - defined system of equations we must add the conservation law for the number density n , which is some function of the density and pressure p : 29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ \alpha}(n{u^\alpha } ) = 0.$$\end{document } equations ( 27 ) and ( 29 ) imply that the flow is adiabatic . the role of the equation of state is played by the arbitrary function n( , p ) . initial data for the above set of equations are h , h,0 , , p , and , but not all these data are independent because of the existence of the constraint equations . equations ( 23 ) and ( 24 ) imply the four constraint equations among the initial data for the field , 30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { h^{\alpha 0 } } + 16\pi { \lambda ^{\alpha 0 } } - { \delta ^{ij}}h_{i , j}^\alpha { \,^{,0 } } = 0,$$\end{document } where is the laplacian in the flat space . we shall choose h and h,0 as free data and solve equation ( 30 ) for h( = 0 , , 3 ) and equation ( 23 ) for h,0 . of course these constraints can not be solved explicitly , since contains h , but they can be solved iteratively as explained below . as discussed above , we shall assume that the free data h and h,0 for the field vanish . one can show that such initial data satisfy the omurchadha and york criterion for the absence of radiation far away from the source . in the actual calculation , it is convenient to use an expression with explicit dependence of . the harmonic condition allows us to have such an expression in terms of the retarded integral , 31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{\mu \nu}}(\epsilon , \tau , { x^i } ) = 4\int\nolimits_{c(\epsilon , \tau , { x^i } ) } { { d^3 } } y\,{\lambda ^{\mu \nu}}(\tau - \epsilon r,{y^i},\epsilon)/r + h_{\rm{h}}^{\mu \nu}(\epsilon , \tau , { x^i}),$$\end{document } where r = y x and c( , , x ) is the past flat light cone of the event ( , x ) in the spacetime given by , truncated where it intersects with the initial hypersurface = 0 . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{\rm{h}}^{\mu \nu}$\end{document } is the unique solution of the homogeneous wave equation in the flat spacetime , 32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\square h_{\rm{h}}^{\mu \nu } = { \eta ^{\alpha \beta}}h_h^{\mu \nu}{\,_{,\alpha \beta } } = 0.$$\end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{\rm{h}}^{\mu \nu}$\end{document } evolves from a given initial data on the = 0 initial hypersurface which are subject to the constraint equations ( 30 ) . the explicit form of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{\rm{h}}^{\mu \nu}$\end{document } is available via the poisson formula ( see e.g. ) , 33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{\rm{h}}^{\mu \nu}(\tau , { x^i } ) = { \tau \over { 4\pi}}\oint\nolimits_{\partial c(\tau , { x^i } ) } { { h^{\mu \nu}}_{,\tau } } ( \tau = 0,{y^i})\,d{\omega _ y } + { 1 \over { 4\pi}}{\partial \over { \partial \tau}}\left [ { \tau \oint\nolimits_{\partial c(\tau , { x^i } ) } { { h^{\mu \nu } } } ( \tau = 0,{y^i})\,d{\omega _ y } } \right].$$\end{document } we shall henceforth ignore the homogeneous solutions because they play no important role . because of the dependence of the integral region , the domain of integral is finite as long as 0 and their diameter increases like as 0 . given the formal expression ( 31 ) in terms of initial data ( 9 ) , we can take the lie derivative and evaluate these derivatives at = 0 . the lie derivative is nothing but a partial derivative with respect to in the coordinate system for the fiber bundle given by ( , , x ) . t = t which is of order , since t is of order . similarly the other components of stress - energy tensor t = t and t are of order as well . thus we expect that the first nonvanishing derivative in equation ( 31 ) will be the forth derivative . in fact we find 34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_4{h^{\tau \tau}}(\tau , { x^i } ) = 4\int\nolimits_{{r^3 } } { { { 2\rho ( \tau , { y^i } ) } \over r } } \,{d^3}y,$$\end{document } 35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_4{h^{\tau i}}(\tau , { x^i } ) = 4\int\nolimits_{{r^3 } } { { { 2\rho { { ( \tau , { y^k})}_1}{v^i}(\tau , { x^k } ) } \over r } } \,{d^3}y,$$\end{document } 36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_4{h^{ij}}(\tau , { x^k } ) = 4\int\nolimits_{{r^3 } } { { { 2\rho { { ( \tau , { y^k})}_1}{v^i}{{(\tau , { y^k})}_1}{v^j}(\tau , { y^k}){+ _ 4}t_{{\rm{ll}}}^{ij}(\tau , { y^k } ) } \over r } } \,{d^3}y,$$\end{document } where we have adopted the notation 38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_nf(\tau , { x^i } ) = { 1 \over { n!}}\underset { \epsilon \rightarrow 0 } { \lim } { { { \partial ^n } } \over { \partial { \epsilon ^n}}}f(\epsilon , \tau , { x^i}),$$\end{document } and 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_4t_{{\rm{ll}}}^{ij } = { 1 \over { 64\pi}}\left({{}_4{h^{\tau \tau , i}}{}_4{h^{\tau \tau , j } } - { 1 \over 2}{\delta ^{ij}}_4{h^{\tau \tau , k}}_4{h^{\tau \tau}}_{,k } } \right).$$\end{document } in the above calculation we have taken the point of view that h is a tensor field , defined by giving its components in the assumed harmonic coordinates as the difference between the tensor density \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { - g } { g^{\mu \nu}}$\end{document } and . the conservation law ( 27 ) also has its first nonvanishing derivatives at this order , which are 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_2{\rho _ { , \tau } } + { { ( _ 2}{\rho _ 1}{v^i})_{,i } } = 0,$$\end{document } 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{(_2}{\rho _ 1}{v^i})_{,\tau } } + { { ( _ 2}{\rho _ 1}{v^i}_1{v^j})_{,j}}{+ _ 4}{p^{,i } } - { 1 \over 4}{\,_2}{\rho _ 4}{h^{\tau \tau , i } } = 0.$$\end{document } equations ( 34 ) , ( 40 ) , and ( 41 ) constitute newtonian theory of gravity . thus the lowest nonvanishing derivative with respect to is indeed newtonian theory , and the 1 pn and 2 pn equations emerge from the sixth and eighth derivatives , respectively , in the conservation law ( 27 ) . at the next derivative , in this section we review briefly the strong field point particle limit , the surface integral approach for the evaluation of the gravitational force , and scalings of matter and field variables on an initial hypersurface . also we revisit some elementary but useful equations , the newtonian velocity momentum relation and the newtonian equations of motion for extended bodies . these are the basic ideas of our formalism . on the base of our formalism , see also [ 11 , 91 , 92 , 93 , 94 , 95 , 140 ] . on the post - newtonian approximation and related issues from different viewpoints , see [ 47 , 48 , 49 , 16 , 18 , 19 ] and references therein . in a relativistic compact binary system in an inspiralling phase , each star is well approximated by a point particle with a few low order multipole moments . this is because in the inspiralling phase , the stellar size divided by the orbital separation is much smaller than unity . if we wish to apply the post - newtonian approximation to the inspiraling phase of binary neutron stars , the strong internal gravity must be taken into account . the usual post - newtonian approximation explicitly assumes the weakness of the gravitational field everywhere including inside the material source . it is argued by applying the strong equivalence principle that the external gravitational field which governs the orbital motion of the binary system is independent of the internal structure of the components up to tidal interaction . thus it is expected that the results obtained under the assumption of weak gravity also apply for the case of a neutron star binary . experimental evidence for the strong equivalence principle is obtained only for systems with weak gravity [ 159 , 160 , 161 , 164 ] , but at present no experiment is available in a case with strong internal gravity . in the theoretical aspect , the theory of extended objects in general relativity is still in a preliminary stage for an application to realistic systems . the matched asymptotic expansion technique has been used to treat a system with strong gravity in certain situations [ 47 , 65 , 66 , 104 , 105 ] . another way to handle strong internal gravity is by the use of a dirac delta distribution type source with a fixed mass . physically , there is no such source in general relativity because of the existence of black holes . before a body shrinks to a point , it forms a black hole whose size is fixed by its mass . for this reason we can shrink the body keeping the compactness ( m / r ) , i.e. the strength of the internal field fixed . namely we should scale the mass m just like the radius r. this can be fitted nicely into the concept of the regular asymptotic newtonian sequence defined in section 2 because there the mass also scales along the sequence of solutions . in fact , if we take the masses of two stars as m , and the separation between two stars as l , then thus the mass m scales as if we fix the separation4 . in the above we have assumed that the density scales as to guarantee this scaling for the mass while keeping the size of the body fixed . now we shrink the size as to keep the compactness of each component . then the density should scale as . we shall call such a scheme the strong field point particle limit since the limit keeps the strength of internal gravity5 . the above consideration suggests the following initial data to define a regular asymptotic newtonian sequence which describes a nearly newtonian system with strong internal gravity . the initial data are two uniformly rotating fluids with compact spatial support whose stress - energy tensor and size scales as and , respectively . we also assume that each of these fluid configurations would be a stationary equilibrium solution of the einstein equations if the other were absent . any remaining motions are tidal effects caused by the other body , which will be of order smaller than the internal self - force . these data allow us to use the newtonian time = t as a natural time coordinate everywhere including the interior region of the stars . as for multipole moments , the scaling \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r = \mathcal o({\epsilon ^2})$\end{document } enables us to incorporate the multipole expansion of the stars into the post - newtonian approximation . in inspiralling compact binaries the tidally and rotationally induced quadrupole moments are too small to affect the binary orbital motion . the time to coalesce is too short for the binary to be tidally locked and corotate [ 15 , 44 , 110 ] . the phase shift due to the quadrupole - orbit couplings may be negligible in the ligo bandwidth . however , to detect gravitational waves from inspiralling binaries , we have to have highly accurate prior knowledge about the binary orbital motion , say , 4 pn equations of motion or so . the effect of quadrupole moments on the orbital motion can be of about the same order as that of spin - spin interactions , while the spin - spin interactions appear at about 2.5 pn order for slowly rotating stars . also , at the late inspiralling phase , an effect of extendedness of the stars on the motion will be important . thus it is important to take the multipole moments of the stars into account in a way that is suitable for compact stars when we derive the equations of motion for an inspiralling compact binary . one way to evaluate the gravitational force acting on a star is the volume integral approach . in the newtonian case , the gravitational force \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f_1^i$\end{document } on star 1 becomes 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f_1^i = \int\nolimits_{{b_1 } } { { d^3 } } x\,\rho { { \partial \phi } \over { \partial { x^i}}}.$$\end{document } the integral region b1 covers star 1 but does not cover the star 2 ( the companion star ) . to evaluate the above integral we must know the internal structure , which is generally a difficult task even in the newtonian dynamics , needless to say in general relativity . by means of the surface integral approach we can put off dealing with the internal structure problem until tidal effects affect the orbital motion . in the newtonian case , using the poisson equation and gauss s law , we can rewrite the above volume integral into a surface integral , 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f_1^i = - \oint\nolimits_{\partial { b_1 } } d { s_j}\,{t^{ij}},$$\end{document } 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t^{ij } } \equiv { 1 \over { 4\pi}}\left({{{\partial \phi } \over { \partial { x^i}}}{{\partial \phi } \over { \partial { x^j } } } - { { { \delta ^{ij } } } \over 2}{{\partial \phi } \over { \partial { x^k}}}{{\partial \phi } \over { \partial { x^k } } } } \right).$$\end{document } note that the sphere b1 has no intersection , neither with star 1 nor star 2 . thus , we can evaluate the gravitational force acting on star 1 without knowledge about the internal structure of star 1 . the surface integral approach was used by einstein , infeld , and hoffmann in general relativity . they used the vacuum einstein equations only , and their method can be applied to any object including a black hole . but in our formalism , we shall treat only regular objects like a neutron star . now let us introduce the body zone to provide the surfaces of the surface integral approach . the scalings of r and m motivate us to define the body zone of star a(a = 1 , 2 ) as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${b_a } \equiv \{{x^i}\vert \vert \vec x - { \vec z_a}(\tau)\vert \ ; < \epsilon { r_a}\}$\end{document } and the body zone coordinates of the star a as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ a^{\underset{-}{i } } \equiv { \epsilon ^{- 2}}({x^i } - z_a^i(\tau))$\end{document}. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i(\tau)$\end{document } is a representative point of the star a , e.g. the center of the mass of the star a. ra , called the body zone radius , is an arbitrary length scale ( much smaller than the orbital separation and not identical to the radius of the star ) and constant ( i.e. dra / d = 0 ) . with the body zone coordinates , the star does not shrink when 0 , while the boundary of the body zone goes to infinity ( see figure 1 ) . figure 1body zone coordinates and near zone coordinates . in the near zone coordinates ( , x ) , both the body zone and the star shrink as ( thin dotted arrow ) and ( thick dotted arrow ) respectively . both the thin and thick arrows point inside . in the body zone coordinates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\tau , \alpha _ a^{\underset{-}{i}})$\end{document } , the star does not shrink while the body zone boundary goes to infinity as ( thin dotted line pointing outside ) . body zone coordinates and near zone coordinates . in the near zone coordinates ( , x ) , both the body zone and the star shrink as ( thin dotted arrow ) and ( thick dotted arrow ) respectively . both the thin and thick arrows point inside . in the body zone coordinates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\tau , \alpha _ a^{\underset{-}{i}})$\end{document } , the star does not shrink while the body zone boundary goes to infinity as ( thin dotted line pointing outside ) . then it is appropriate to define the star s characteristic quantities such as the mass , the spin , and so on with the body zone coordinates . on the other hand the body zone serves us with a surface ba , through which gravitational energy momentum flux flows , and in turn it amounts to the gravitational force acting on star a ( see figure 2 ) . since the body zone boundary ba is far away from the surface of star a , we can evaluate the gravitational energy momentum flux over ba with the post - newtonian gravitational field . in fact we shall express our equations of motion in terms of integrals over ba and be able to evaluate them explicitly . the arrows around star 1 represent the gravitational energy momentum flux flowing through the body zone boundary . gravitational energy momentum flux through the body zone boundary . the arrows around star 1 represent the gravitational energy momentum flux flowing through the body zone boundary . following [ 79 , 82 , 138 ] , we use the initial value formulation to solve the einstein equations . as initial data for the matter variables and the gravitational field , we take a set of nearly stationary solutions of the exact einstein equations representing two widely separated fluid balls , each of which rotates uniformly . we assume that these solutions are parametrized by and that the matter and field variables have the following scalings . the density scales as ( in the ( t , x ) coordinates ) , implied by the scalings of m and r. the scaling of the density suggests that the natural dynamical time ( free fall time ) inside the star may be = t . then if we can not assume almost stationary condition on the stars , it is difficult to use the post - newtonian approximation [ 80 , 81 ] . in practice , however , our formalism is still applicable to pulsating stars if the effect of pulsation is not important in the orbital motion . the velocity of stellar rotation is assumed to be \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal o(\epsilon)$\end{document}. in other words , we assume that the star rotates slowly and is pressure supported6 . by this assumption , the spin - orbit coupling force appears at 2 pn order rather than the usual 1.5 pn order . the slowly spinning motion assumption is not crucial : in fact , it is straightforward to incorporate a rapidly spinning compact body into our formalism . from these initial data we have the following scalings of the star a s stress - energy tensor components \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_a^{\mu \nu}$\end{document } in the body zone coordinates : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_a^{\tau \tau } = \mathcal o({\epsilon ^{- 2}}),\;t_a^{\tau { \underset{-}{i } } } = \mathcal o({\epsilon ^{- 4}}),t_a^{i{\underset{-}{j } } } = \mathcal o({\epsilon ^{- 8}})$\end{document}. here the underlined indices mean that for any tensor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${a^i},\;{a^{\underset{-}{i } } } = { \epsilon ^{- 2}}{a^i}$\end{document}. in , we have transformed \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_n^{\mu \nu}$\end{document } , the components of the stress - energy tensor of the matter in the near zone coordinates , to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_a^{\mu \nu}$\end{document } using the transformation from the near zone coordinates to the ( generalized ) fermi normal coordinates at 1 pn order . it is difficult , however , to construct the ( generalized ) fermi normal coordinates at an high post - newtonian order . we simply assume that for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_n^{\mu \nu}$\end{document } ( or rather \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\lambda _ n^{\mu \nu}$\end{document } , the source term of the relaxed einstein equations ; see equation ( 63 ) ) , 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_n^{\tau \tau } = { \mathcal o}({\epsilon ^{- 2}}),$$\end{document } 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_n^{\tau \underline i } = { \mathcal o}({\epsilon ^{- 4}}),$$\end{document } 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_n^{\underline i \underline j } = { \mathcal o}({\epsilon ^{- 8}}),$$\end{document } as their leading scalings . as for the field variables on the initial hypersurface , we simply assume that the field is of 2.5 pn order except for the field determined by the constraint equations . note that the radiation reaction effect to the stars first appears at the 2.5 pn order . futamase showed that even if one takes the field of order 1 pn , initial value of the field does not affect the subsequent motion of the system up to 2.5 pn order . thus , we expect that the initial value of the field does not affect the orbital motion of the system up to 3 pn order , though a detailed calculation has not been done yet . first , by using the initial value formulation one can avoid the famous runaway solution problem in a radiation reaction problem . second , one can construct an initial condition on some spacelike hypersurface rather than at past null infinity . putting an initial condition for the field in the past null infinity requires a prior knowledge about the spacetime , which is obtained through the time evolution of the field from the initial condition in our universe there may be no past null infinity because of the big bang . the binary system is in the background gravitational radiation bath for which we know only its statistical properties . for example , the phase of the radiation is assumed to be random and irrelevant to the motion of the binary . the origins of the radiation are cosmological , or related to the evolution of the system before the initial hypersurface . then we can evaluate the expected time evolution of the binary system by letting the system evolve from a set of possible initial conditions and taking a statistical ensemble average over the initial conditions . before ending this section , we present some equations for newtonian extended bodies ( stars ) . these equations will give a useful guideline when we develop our formalism . the basic equations are the equation of continuity , the euler equation , and the poisson equation , respectively : 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\partial \rho } \over { \partial \tau } } + { { \partial \rho { v^i } } \over { \partial { x^i } } } = 0,$$\end{document } 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho { { d{v^i } } \over { d\tau } } = \rho { { \partial \phi } \over { \partial { x_i}}},$$\end{document } 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \phi = 4\pi \rho.$$\end{document } we define the mass , the dipole moment , the quadrupole moment , and the momentum of the star a as 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_a } \equiv \int\nolimits_{{b_a } } { { d^3 } } x\,\rho,$$\end{document } 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d_a^i \equiv \int\nolimits_{{b_a } } { { d^3 } } x\,\rho ( { x^i } - z_a^i),$$\end{document } 53\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i_a^{ij } \equiv \int\nolimits_{{b_a } } { { d^3 } } x\,\rho ( { x^i } - z_a^i)({x^j } - z_a^j),$$\end{document } 54\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_a^i \equiv \int\nolimits_{{b_a } } { { d^3 } } x\,\rho { v^i}.$$\end{document } here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } is a representative point of the star a. the time derivative of the mass vanishes . setting the time derivative of the dipole moment to zero gives the velocity momentum relation and a definition of the center of mass , 55\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{d^i } } \over { d\tau } } = p_a^i - { m_a}v_a^i = 0,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$v_a^i = dz_a^i / d\tau$\end{document}. using the velocity momentum relation , we calculate the time derivative of the momentum , 56\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_1}{{{d^2}z_1^i } \over { d{\tau ^2 } } } = f_1^i,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f_1^i$\end{document } is defined by equation ( 43 ) . the newtonian potential can be expressed by the mass and multipole moment as 57\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi = \sum\limits_{a = 1,2 } { \left [ { { { { m_a } } \over { \vert \vec x - { { \vec z}_a}\vert } } + { 1 \over 2}i_a^{ij}{{{\partial ^2 } } \over { \partial { x^i}\,\partial { x^j}}}\left({{1 \over { \vert \vec x - { { \vec z}_a}\vert } } } \right ) } \right]}.$$\end{document } substituting into equation ( 56 ) , we obtain the equations of motion , 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_1}{{{d^2}z_1^i } \over { d{\tau ^2 } } } = { \partial \over { \partial z_1^i}}{{{m_1}{m_2 } } \over { \vert { { \vec z}_1 } - { { \vec z}_2}\vert } } + { 1 \over 2}(i_1^{ij } + i_2^{ij}){{{\partial ^3 } } \over { \partial z_1^i\partial z_2^j\partial z_2^k}}\left({{1 \over { \vert { { \vec z}_1 } - { { \vec z}_2}\vert } } } \right).$$\end{document } here we ignored the mass multipole moments of the stars that are of higher order than the quadrupole moments . actually , it is straightforward to formally include all the newtonian mass multipole moments in the surface integral approach , 59\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_1}{{{d^2}z_1^i } \over { d{\tau ^2 } } } = \sum\limits_{p = 0 } { \sum\limits_{q = 0 } { { { { { ( - 1)}^{p + 1}}(2p + 2q + 1 ) ! ! } \over { p!q ! } } } } { { i_1^{\langle { m_p}\rangle}i_2^{\langle { n_q}\rangle } } \over { r_{12}^{p + q + 2}}}{n^{\langle i{m_p}{n_q}\rangle}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec r_{12 } } = { \vec z_1 } - { \vec z_2}$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${n^i } = r_{12}^i/{r_{12}}$\end{document } , mp = m1m2 mp is a corrective index , denotes the symmetric - tracefree operation on the indices between the brackets , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$i_a^{{m_p}}$\end{document } are the newtonian mass multipole moments of order 2p . following the basic idea explained in the previous section 3 , we develop now our formalism for the derivation of the post - newtonian equations of motion suitable for relativistic compact binaries . see also [ 94 , 95 ] . as discussed in the previous section 3 , we express our equations of motion in terms of surface integrals over the body zone boundary where it is assumed that the metric slightly deviates from the flat metric = diag( , 1 , 1 , 1 ) ( in the near zone coordinates ( , x ) ) . thus we define a deviation field h as 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{\mu \nu } } \equiv { \eta ^{\mu \nu } } - \sqrt { - g } \,{g^{\mu \nu}},$$\end{document } where g is the determinant of the metric . indices are raised or lowered by the flat ( auxiliary ) metric unless otherwise stated . now we impose the harmonic coordinate condition on the metric , 61\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{\mu \nu}}_{,\nu } = 0,$$\end{document } where the comma denotes the partial derivative . in the harmonic gauge , we can recast the einstein equations into the relaxed form , 62\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\square{h^{\mu \nu } } = - 16\pi { \lambda ^{\mu \nu}},$$\end{document } where = is the flat dalembertian and 63\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\lambda ^{\mu \nu } } \equiv { \theta ^{\mu \nu } } + { \chi ^{\mu \nu \alpha \beta}}_{,\alpha \beta},$$\end{document } 64\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\theta ^{\mu \nu } } \equiv ( - g)({t^{\mu \nu } } + t_{{\rm{ll}}}^{\mu \nu}),$$\end{document } 65\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\chi ^{\mu \nu \alpha \beta } } \equiv { 1 \over { 16\pi}}({h^{\alpha \nu}}{h^{\beta \mu } } - { h^{\alpha \beta}}{h^{\mu \nu}}).$$\end{document } here , t and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_{{\rm{ll}}}^{\mu \nu}$\end{document } denote the stress - energy tensor of the stars and the landau - lifshitz pseudotensor . the explicit form of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_{{\rm{ll}}}^{\mu \nu}$\end{document } in the harmonic gauge is 66\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { ( - 16\pi g)t_{{\rm{ll}}}^{\mu \nu } = { g_{\alpha \beta}}{g^{\gamma \delta}}{h^{\mu \alpha}}_{,\gamma}{h^{\nu \beta}}_{,\delta } + { 1 \over 2}{g^{\mu \nu}}{g_{\alpha \beta}}{h^{\alpha \gamma}}_{,\delta}{h^{\beta \delta}}_{,\gamma } - 2{g_{\alpha \beta}}{g^{\gamma ( \mu}}{h^{\nu)\alpha}}_{,\delta}{h^{\delta \beta}}_{,\gamma}\quad \quad \quad \quad } \\ { + { 1 \over 2}\left({{g^{\mu \alpha}}{g^{\nu \beta } } - { 1 \over 2}{g^{\mu \nu}}{g^{\alpha \beta } } } \right)\left({{g_{\gamma \delta}}{g_{\epsilon \zeta } } - { 1 \over 2}{g_{\gamma \epsilon}}{g_{\delta \zeta } } } \right){h^{\gamma \epsilon}}_{,\alpha}{h^{\delta \zeta}}_{,\beta}. } \\ \end{array}$$\end{document } originates from our use of the flat dalembertian instead of the curved space dalembertian . in consistency with the harmonic condition , the conservation law is expressed as 67\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\lambda ^{\mu \nu}}_{,\nu } = 0.$$\end{document } note that the divergence of itself vanishes identically due to the symmetry of its indices . now we rewrite the relaxed einstein equations into an integral form , 68\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{\mu \nu}}(\tau , { x^i } ) = 4\int\nolimits_{c(\tau , { x^k } ) } { { d^3 } } y\,{{{\lambda ^{\mu \nu}}(\tau - \epsilon \vert \vec x - \vec y\vert , { y^k};\epsilon ) } \over { \vert \vec x - \vec y\vert } } + h_{\rm{h}}^{\mu \nu}(\tau , { x^i}),$$\end{document } where c( , x ) means the past light cone emanating from the event ( , x ) . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{\rm{h}}^{\mu \nu}$\end{document } is a homogeneous solution of the homogeneous wave equation in the flat spacetime , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\square h_{\rm{h}}^{\mu \nu } = 0$\end{document}. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{\rm{h}}^{\mu \nu}$\end{document } evolves from a given initial data on the = 0 initial hypersurface . the explicit form of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{\rm{h}}^{\mu \nu}$\end{document } is available via the poisson formula ( see e.g. ) 69\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{\rm{h}}^{\mu \nu}(\tau , { x^i } ) = { \tau \over { 4\pi}}{\oint\nolimits_{\partial c(\tau , { x^i } ) } { { h^{\mu \nu } } } _ { , \tau}}(\tau = 0,{y^i})\,d{\omega _ y } + { 1 \over { 4\pi}}{\partial \over { \partial \tau}}\left [ { \tau \oint\nolimits_{\partial c(\tau , { x^i } ) } { { h^{\mu \nu } } } ( \tau = 0,{y^i})\,d{\omega _ y } } \right].$$\end{document } we solve the einstein equations as follows . first we split the integral region into two zones : the near zone and the far zone . the near zone is the region containing the gravitational wave source where the wave character of the gravitational radiation is not manifest . in other words , in the near zone the retardation effect on the field is negligible . the size of the near zone is about a little larger than one wave length of the gravitational wave emitted by the source . in this paper we take the near zone as a sphere centered at some fixed point and enclosing the binary system . the radius of this sphere is set to be \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal r/\epsilon$\end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document } is arbitrary but larger than the size of the binary and the wave length of the gravitational radiation . the scaling of the near zone radius is derived from the dependence of the wavelength of the gravitational radiation emitted due to the orbital motion of the binary . note that roughly speaking the frequency of such a wave is about twice the keplerian frequency of the binary . the center of the near zone sphere would be determined , if necessary , for example , to be the center of mass of the near zone . the outside of the near zone is the far zone where the retardation effect of the field is crucial . for the near zone field point pn , we write the field as 70\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{\mu \nu}}({{\rm{p}}_{\rm{n } } } \in n ) = h_{{p_n}(n)}^{\mu \nu } + h_{{p_n}(f)}^{\mu \nu } + h_{\rm{h}}^{\mu \nu},$$\end{document } 71\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{{p_n}(n)}^{\mu \nu } \equiv 4\int\nolimits_{n = \{y:\vert y\vert \leq r/\epsilon \ } } { { d^3 } } y\,{{{\lambda ^{\mu \nu}}(\tau - \epsilon \vert \vec x - \vec y\vert , { y^k};\epsilon ) } \over { \vert \vec x - \vec y\vert}},$$\end{document } 72\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{{p_n}(f)}^{\mu \nu } \equiv 4\int\nolimits_{f = \{y:\vert y\vert > r/\epsilon \ } } { { d^3 } } y\,{{{\lambda ^{\mu \nu}}(\tau - \epsilon \vert \vec x - \vec y\vert , { y^k};\epsilon ) } \over { \vert \vec x - \vec y\vert}}.$$\end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{{p_n}(n)}^{\mu \nu}$\end{document } is the near zone integral contribution to the near zone field , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{{p_n}(f)}^{\mu \nu}$\end{document } is the far zone integral contribution to the near zone field . an explicit calculation shows that apparently there are the far zone contributions to the near zone field at 3 pn order . pati and will showed that the far zone contribution does not affect the equations of motion up to 3 pn order inclusively and that the far zone contribution first appears at 4 pn order . this result is consistent with the earlier result of blanchet and damour who used the multipolar - post - minkowskian formalism . we follow the dire method and checke that the far zone contribution does not affect the equations of motion up to 3 pn order in appendix a. henceforth we shall focus our attention on the near zone contribution \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{{p_n}(n)}^{\mu \nu}$\end{document } and do not write down the far zone contribution in the following calculation of the field . as for the homogeneous solution , we shall ignore it for simplicity . if we take random initial data for the field supposed to be of 1 pn order , they are irrelevant to the dynamics of the binary system up to the radiation reaction order . as we have assumed in the previous section 3.3 that the magnitude of the free data of the gravitational field on the initial hypersurface is 2.5 pn order , we expect that the homogeneous solution does not affect the equations of motion up to 3 pn order . we leave a full implementation of the initial value formulation on the field as future work . it is worth noticing that when we let become sufficiently large , then the condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{\rm{h}}^{\mu \nu}(\tau , { x^i } ) = 0$\end{document } corresponds to a no - incoming radiation condition at ( minkowskian ) past null infinity ( see e.g. ) . equation ( 69 ) can be written down as kirchhoff s formula , 73\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{\rm{h}}^{\mu \nu}(\tau , { x^i } ) = \oint\nolimits_{\partial c(\tau , { x^i } ) } { { { d{\omega _ y } } \over { 4\pi } } } { \left . { \left [ { { \partial \over { \partial \rho}}(\rho { h^{\mu \nu}}(\tau{\prime},{y^i } ) ) + { \partial \over { \partial \tau{\prime}}}(\rho { h^{\mu \nu}}(\tau{\prime},{y^i } ) ) } \right ] } \right\vert _ { \tau{\prime } = 0,\,\rho = \vert \vec x - \vec y\vert = \tau}}.$$\end{document } then the no - incoming radiation condition at ( minkowskian ) past null - infinity , 74\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underset { \underset { r \rightarrow \infty } { \tau = r , } } { \lim } \left [ { { \partial \over { \partial r}}(r{h^{\mu \nu } } ) + { \partial \over { \partial \tau}}(r{h^{\mu \nu } } ) } \right ] = 0,$$\end{document } is a sufficient condition to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{\rm{h}}^{\mu \nu}(\tau , { x^i } ) = 0$\end{document } when goes to infinity . now we shall devote ourselves to the evaluation of the near zone contribution to the near zone field , 75\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{\mu \nu}}(\tau , { x^i } ) = 4\int\nolimits_{n(\tau , { x^k } ) } { { d^3 } } y\,{{{\lambda ^{\mu \nu}}(\tau - \epsilon \vert \vec x - \vec y\vert , { y^k};\epsilon ) } \over { \vert \vec x - \vec y\vert}}.$$\end{document } henceforth we shall omit the subscript pn(n ) of the field h for notational simplicity . we shall evaluate the near zone contribution as follows . first , we make a retarded expansion of equation ( 75 ) and change the integral region to a = constant spatial hypersurface , 76\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{\mu \nu } } = 4\sum\limits_{n = 0 } { { { { { ( - \epsilon)}^n } } \over { n ! } } } { \left({{\partial \over { \partial \tau } } } \right)^n}\int\nolimits_n { { d^3 } } y\,\vert \vec x - \vec y{\vert ^{n - 1}}\lambda _ n^{\mu \nu}(\tau , { y^k};\epsilon).$$\end{document } note that the above integral depends on the arbitrary length \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document } in general . the cancellation between the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document } dependent terms in the far zone contribution and those in the near zone contribution was shown by through all the post - newtonian order . in the following , we shall omit the terms which have negative powers of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r\;({\mathcal r^{- k}};k > 0)}$\end{document}. in other words , we simply let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r } \rightarrow 0$\end{document } whenever it gives a convergent result . on the other hand , we shall retain terms having positive powers of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}\;({{\mathcal r}^k};k > 0)$\end{document } and logarithmic terms \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\ln \;\mathcal r)$\end{document } to confirm that the final result , in the end of calculation , is independent of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document } ( and for logarithmic terms to keep the arguments of logarithm non - dimensional ) . second we split the integral into two parts : a contribution from the body zone ba , and from elsewhere , n / b . schematically we evaluate the following two types of integrals ( we omit indices of the field ) , 77\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}{c } } { h = { h_b } + { h_{n / b}},\quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { { h_b } = { ^6}\sum\limits_{a = 1,2 } { \int_{{b_a } } { { d^3 } } } { \alpha _ a}{\mkern 1mu } \frac{{f(\tau , { { \vec z}_a } + { ^2}{{\vec \alpha } _ a})}}{{|{{\vec r}_a } - { ^2}{{\vec \alpha } _ a}{|^{1 - n}}}},{\mkern 1mu } { \mkern 1mu } { \mkern 1mu } \quad } \\ { { h_{n / b } } = \int_{n / b } { { d^3}y\frac{{f(\tau , \vec y)}}{{|\vec x - \vec y{|^{1 - n } } } } , } \quad \quad \quad \quad \quad { \mkern 1mu } { \mkern 1mu } { \mkern 1mu } { \mkern 1mu } \quad } \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec r_a } \equiv \vec x - { \vec z_a}$\end{document}. we shall deal with these two contributions successively . as for the body zone contribution , we make a multipole expansion using the scaling of the integrand , i.e. in the body zone . for example , the n = 0 part in equation ( 76 ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{b\;n = 0}^{\mu \nu}$\end{document } gives 78\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{b\,n = 0}^{\tau \tau } = 4{\epsilon ^4}\sum\limits_{a = 1,2 } { \left({{{p_a^\tau } \over { { r_a } } } + { \epsilon ^2}{{d_a^kr_a^k } \over { r_a^3 } } + { \epsilon ^4}{{3i_a^{\langle kl\rangle}r_a^kr_a^l } \over { 2r_a^5 } } + { \epsilon ^6}{{5i_a^{\langle klm\rangle}r_a^kr_a^lr_a^m } \over { 2r_a^7 } } } \right ) } + { \mathcal o}({\epsilon ^{12}}),$$\end{document } 79\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{b\,n = 0}^{\tau i } = 4{\epsilon ^4}\sum\limits_{a = 1,2 } { \left({{{p_a^i } \over { { r_a } } } + { \epsilon ^2}{{j_a^{ki}r_a^k } \over { r_a^3 } } + { \epsilon ^4}{{3j_a^{kli}r_a^kr_a^l } \over { 2r_a^5 } } } \right ) } + { \mathcal o}({\epsilon ^{10}}),$$\end{document } 80\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{b\,n = 0}^{ij } = 4{\epsilon ^2}\sum\limits_{a = 1,2 } { \left({{{z_a^{ij } } \over { { r_a } } } + { \epsilon ^2}{{z_a^{kij}r_a^k } \over { r_a^3 } } + { \epsilon ^4}{{3z_a^{\langle kl\rangle ij}r_a^kr_a^l } \over { 2r_a^5 } } + { \epsilon ^6}{{5z_a^{\langle klm\rangle ij}r_a^kr_a^lr_a^m } \over { 2r_a^7 } } } \right ) } + { \mathcal o}({\epsilon ^{10}}).$$\end{document } here the operator denotes a symmetric and tracefree ( stf ) operation on the indices in the brackets . see [ 129 , 150 , 165 ] for some useful formulas of stf tensors . also we define \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_a } \equiv \vert { \vec r_a}\vert$\end{document}. to derive the 3 pn equations of motion , h up to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^{10}})$\end{document } and h as well as h up to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^8})$\end{document } are required . in the above equations we define the multipole moments as 81\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i_a^{{k_l } } \equiv { \epsilon ^2}\int\nolimits_{{b_a } } { { d^3 } } { \alpha _ a}\,\lambda _ n^{\tau \tau}\,\alpha _ a^{{{\underline k}_l}},$$\end{document } 82\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_a^{{k_l}i } \equiv { \epsilon ^4}\int\nolimits_{{b_a } } { { d^3 } } { \alpha _ a}\,\lambda _ n^{\tau \underline i}\,\alpha _ a^{{{\underline k}_l}},$$\end{document } 83\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z_a^{{k_l}ij } \equiv { \epsilon ^8}\int\nolimits_{{b_a } } { { d^3 } } { \alpha _ a}\,\lambda _ n^{\underline a^{{{\underline k}_l}},$$\end{document } where we introduced a collective multi - index \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${i_l } \equiv { i_1}{i_2 } \ldots { i_l}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ a^{{{\underset{-}{i}}_l } } \equiv \alpha _ a^{{{\underset{-}{i}}_1}}\alpha _ a^{{{\underset{-}{i}}_2 } } \ldots \alpha _ a^{{{\underset{-}{i}}_l}}$\end{document}. then \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau \equiv i_a^{{k_0}}$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^{{k_1 } } \equiv i_a^{{k_1}}$\end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^{{k_1 } } \equiv j_a^{{k_1}}$\end{document}. we simply call \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\mu$\end{document } the four - momentum of the star a , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^i$\end{document } the momentum , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document } the energy7 . also we call \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^k$\end{document } the dipole moment of the star a , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$i_a^{kl}$\end{document } the quadrupole moment of the star a. then we transform these moments into more convenient forms . by the conservation law ( 67 ) , we have 84\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda _ n^{\tau i } = { ( \lambda _ n^{\tau \tau}y_a^i)_{,\tau } } + { ( \lambda _ n^{\tau j}y_a^i)_{,j } } + v_a^i\lambda _ n^{\tau \tau},$$\end{document } 85\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda _ n^{ij } = { ( \lambda _ n^{\tau ( i}y_a^{j)})_{,\tau } } + { ( \lambda _ n^{k(i}y_a^{j)})_{,k } } + v_a^{(i}\lambda _ n^{j)\tau},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$v_a^i \equiv \dot z_a^i$\end{document } an overdot denotes a time derivative with respect to , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec y_a } \equiv \vec y - { \vec z_a}$\end{document}. using these equations and noticing that the body zone remains unchanged ( in the near zone coordinates ) , i.e. a = 0 , we have 86\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_a^i = p_a^\tau v_a^i + q_a^i + { \epsilon ^2}{{dd_a^i } \over { d\tau}},$$\end{document } 87\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_a^{ij } = { 1 \over 2}\left({m_a^{ij } + { \epsilon ^2}{{di_a^{ij } } \over { d\tau } } } \right ) + v_a^{(i}d_a^{j ) } + { 1 \over 2}{\epsilon ^{- 2}}q_a^{ij},$$\end{document } 88\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z_a^{ij } = { \epsilon ^2}p_a^\tau v_a^iv_a^j + { 1 \over 2}{\epsilon ^6}{{{d^2}i_a^{ij } } \over { d{\tau ^2 } } } + 2{\epsilon ^4}v_a^{(i}{{dd_a^{j ) } } \over { d\tau } } + { \epsilon ^4}{{dv_a^{(i } } \over { d\tau}}d_a^{j ) } + { \epsilon ^2}q_a^{(i}v_a^{j ) } + { \epsilon ^2}r_a^{(ij ) } + { 1 \over 2}{\epsilon ^2}{{dq_a^{ij } } \over { d\tau}},$$\end{document } 89\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z_a^{kij } = { 3 \over 2}a_a^{kij } - a_a^{(ij)k},$$\end{document } where 90\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_a^{ij } \equiv 2{\epsilon ^4}\int\nolimits_{{b_a } } { { d^3 } } { \alpha _ a}\,\alpha _ a^{\left [ { \underline i } \right.}\lambda _ n^{{}^{\left . { \underline j } \right]}\tau},$$\end{document } 91\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q_a^{{k_l}i } \equiv { \epsilon ^{- 4}}\oint\nolimits_{\partial { b_a } } d { s_k}\,\left({\lambda _ n^{\tau k } - v_a^k\lambda _ n^{\tau \tau } } \right)y_a^{{k_l}}y_a^i,$$\end{document } 92\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_a^{{k_l}\,i\,j } \equiv { \epsilon ^{- 4}}\oint\nolimits_{\partial { b_a } } d { s_k}\,\left({\lambda _ n^{k\,j } - \upsilon _ a^k\lambda _ n^{\tau \,j } } \right)y_a^{{k_l}}y_a^i,$$\end{document } and 93\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_a^{kij } \equiv { \epsilon ^2}j_a^{k(i}v_a^{j ) } + { \epsilon^2}v_a^kj_a^{(ij ) } + r_a^{k(ij ) } + { \epsilon ^4}{{dj_a^{k(ij ) } } \over { d\tau}}.$$\end{document } the operators [ ] and ( ) attached to the indices denote anti - symmetrization and symmetrization . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_a^{ij}$\end{document } is the spin of the star a and equation ( 86 ) is the momentum - velocity relation . thus our momentum - velocity relation is a direct analog of the newtonian momentum - velocity relation ( see section 3.4 ) . in general , we have 94\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_a^{{k_l}i } = j_a^{({k_l}i ) } + { { 2l } \over { l + 1}}j_a^{({k_{l - 1}}[{k_l})i]},$$\end{document } 95\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z_a^{{k_l}ij } = { 1 \over 2}\left [ { z_a^{({k_l}i)j } + { { 2l } \over { l + 1}}z_a^{({k_{l - 1}}[{k_l})i]j } + z_a^{({k_l}j)i } + { { 2l } \over { l + 1}}z_a^{({k_{l - 1}}[{k_l})j]i } } \right],$$\end{document } and 96\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j_a^{({k_l}i ) } = { 1 \over { l + 1}}{\epsilon ^2}{{di_a^{{k_l}i } } \over { d\tau } } + v_a^{(i}i_a^{{k_l } ) } + { 1 \over { l + 1}}{\epsilon ^{- 2l}}q_a^{{k_l}i},$$\end{document } 97\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z_a^{({k_l}i)j } + z_a^{({k_l}j)i } = { \epsilon ^2}v_a^{(i}j_a^{{k_l})j } + { \epsilon ^2}v_a^{(j}j_a^{{k_l})i } + { 2 \over { l + 1}}{\epsilon ^4}{{dj_a^{{k_l}(ij ) } } \over { d\tau } } + { \epsilon ^{- 2l + 2}}r_a^{{k_l}(ij)},$$\end{document } where l is the number of indices in the multi - index kl . now , from the above equations , especially equation ( 88 ) , we find that the body zone contributions , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{b\;n = 0}^{\mu \nu}$\end{document } , are of order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^4})$\end{document}. note that if we can not or do not assume the ( nearly ) stationarity of the initial data for the stars , then , instead of equation ( 88 ) we have 98\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z_a^{ij } = { \epsilon ^2}p_a^\tau v_a^iv_a^j + { 1 \over 2}{{{d^2}i_a^{ij } } \over { d{\eta ^2 } } } + \ldots,$$\end{document } where we used the dynamical time ( see section 3.3 ) . in this case the lowest order metric differs from the newtonian form . from our ( almost ) stationarity assumption the remaining motion inside a star , apart from the spinning motion , is caused only by the tidal effect by the companion star and from equation ( 88 ) ; it appears at 3 pn order . to obtain the lowest order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{b\;n = 0}^{\mu \nu}$\end{document } , we have to evaluate the surface integrals \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^{{k_l}i}$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_a^{{k_l}ij}$\end{document}. generally , in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{b\;n = 0}^{\mu \nu}$\end{document } the moments \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j_a^{{k_l}i}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^{{k_l}ij}$\end{document } appear formally at the order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\epsilon ^{2l + 4}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\epsilon ^{2l + 2}}$\end{document}. thus \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^{{k_l}i}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_a^{{k_l}ij}$\end{document } appear as 99\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{b\,n = 0}^{\tau i } \sim \cdots + { \epsilon ^4}{{r_a^{{k_l}}q_a^{\langle { k_l}\rangle i } } \over { r_a^{2l + 1 } } } + \ldots,$$\end{document } 100\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{b\,n = 0}^{ij } \sim \cdots + { \epsilon ^4}{{r_a^{{k_l}}r_a^{\langle { k_l}\rangle ( ij ) } } \over { r_a^{2l + 1 } } } + \ldots,$$\end{document } where we omitted irrelevant terms and numerical coefficients . thus one may expect that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^{{k_l}i}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_a^{{k_l}ij}$\end{document } appear at the order for any l and we have to calculate an infinite number of moments . in fact , this is not the case and it was shown in that only l = 0 , 1 terms of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_a^{{k_l}ij}$\end{document } contribute to the 3 pn equations of motion . the important thing here is that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\epsilon ^4}q_a^{{k_l}i}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\epsilon ^4}r_a^{{k_l}ij}$\end{document } are at most \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^4})$\end{document } in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{b\;n = 0}^{\mu \nu}$\end{document}. finally , since the order of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{bn}^{\mu \nu}(n \geq 1)$\end{document } is higher than that of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{b\;n = 0}^{\mu \nu}$\end{document } , we conclude that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_b^{\mu \nu } = { \mathcal o}({\epsilon ^4})$\end{document}. as far as the n / b contribution is concerned , since the integrand \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\lambda _ n^{\mu \nu } = - gt_{{\rm{ll}}}^{\mu \nu } + { \chi ^{\mu \nu \alpha \beta}}_{,\alpha \beta}$\end{document } is at least quadratic in the small deviation field h , we make the post - newtonian expansion in the integrand . then , basically , with the help of ( super-)potentials \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$g(\vec x)$\end{document } which satisfy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta g(\vec x ) = f(\vec x)$\end{document } , denoting the laplacian , we have for each integral ( see e.g. the n = 0 term in equation ( 77 ) ) 101\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_{n / b } { { d^3 } } y{{f(\vec y ) } \over { \vert \vec x - \vec y\vert } } = - 4\pi g(\vec x ) + \oint\nolimits_{\partial ( n / b ) } d { s_k}\,\left [ { { 1 \over { \vert \vec x - \vec y\vert}}{{\partial g(\vec y ) } \over { \partial { y^k } } } - g(\vec y){\partial \over { \partial { y^k}}}\left({{1 \over { \vert \vec x - \vec y\vert } } } \right ) } \right].$$\end{document } equation ( 101 ) can be derived without using dirac delta distributions ( see appendix b of ) . for the n 1 terms in equation ( 77 ) , we use potentials many times to convert all the volume integrals into surface integrals and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- 4\pi g(\vec x)$\end{document } terms8 . in fact finding the super - potentials is one of the most formidable tasks especially when we proceed to high post - newtonian orders . fortunately , up to 2.5 pn order , all the required super - potentials are available . at 3 pn order , there appear many integrands for which we could not find the required super - potentials . to obtain the 3 pn equations of motion , we devise an alternative method similar to the method employed by blanchet and faye . now the lowest order integrands can be evaluated with the body zone contribution \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_b^{\mu \nu}$\end{document } , and since \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_b^{\mu \nu}$\end{document } is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^4})$\end{document } , we find 102\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda _ n^{\tau \tau } = { \mathcal o}({\epsilon ^6}),$$\end{document } 103\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda _ n^{\tau i } = { \mathcal o}({\epsilon ^6}),$$\end{document } 104\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda _ n^{ij } = ( - g)t_{{\rm{ll}}}^{ij } + { \mathcal o}({\epsilon ^8 } ) = { \epsilon ^4}{1 \over { 64\pi}}\left({{\delta ^i}_k{\delta ^j}_l - { 1 \over 2}{\delta ^{ij}}{\delta _ { kl } } } \right){\,_4}h_b^{\tau \tau , k}\,{}_4h_b^{\tau \tau , l } + { \mathcal o}({\epsilon ^5}),$$\end{document } where we expanded \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_b^{\mu \nu}$\end{document } in an series , 105\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_b^{\mu \nu } = \sum\limits_{n = 0 } { { \epsilon ^{4 + n}}_n } h_b^{\mu \nu}.$$\end{document } similarly , in the following we expand h in an series . from these equations we find that the deviation field in n / b , h , is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^4})$\end{document}. ( it should be noted that in the body zone h is assumed to be of order unity and within our method we can not calculate h there explicitly . to obtain in the body zone , we have to know the internal structure of the star . ) in equations ( 81 , 82 , 83 ) , we have defined multipole moments of a star . the definition of those multipole moments are operational ones and are not necessarily equal to intrinsic multipole moments of the star . this is clear , for example , if we remember that a moving ball has spurious multipole moments due to lorentz contraction . we have adopted the generalized fermi normal coordinates ( gfc ) as the star s reference coordinates to address this problem . a question specific to our formalism is whether the differences between the multipole moments defined in equations ( 81 , 82 , 83 ) and the multipole moments in gfc give purely monopole terms . if so , we of course have to take into account such terms in the field to compute the equations of motion for two intrinsically spherical star binaries . this problem is addressed in the appendix c of and the differences are mainly attributed to the shape of the body zone . the body zone ba which is spherical in the near zone coordinates ( nzc ) is not spherical in the gfc mainly because of a kinematic effect ( lorentz contraction ) . to derive the 3 pn equations of motion , it turns out that it is sufficient to compute the difference in the stf quadrupole moment up to 1 pn order . the result is 106\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \delta i_a^{\langle ij\rangle } \equiv i_{a,{\rm{nzc}}}^{\langle ij\rangle } - i_{a,{\rm{gfc}}}^{\langle ij\rangle}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { = { \epsilon ^{- 6}}{1 \over 2}v_a^kv_a^l\oint\nolimits_{\partial { b_a } } d { s_k}y_a^l\,y_a^{\langle i}y_a^{j\rangle}{\lambda ^{\tau \tau } } + { \mathcal o}({\epsilon ^3 } ) } \\ { = - { \epsilon ^2}{{4m_a^3 } \over 5}v_a^{\langle i}v_a^{j\rangle } + { \mathcal o}({\epsilon ^3}),\quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$i_{a,{\rm{nzc}}}^{ij } \equiv i_a^{ij } \cdot i_{a,{\rm{gfc}}}^{ij}$\end{document } are the quadrupole moments defined in the generalized fermi normal coordinates . note that the difference \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta i_a^{\langle ij\rangle}$\end{document } is expressed in a surface integral form . as is obvious from equation ( 106 ) , this difference appears even if the companion star does not exist . we note that we could derive the 3 pn metric for an isolated star a moving at a constant velocity using our method explained in this section by simply letting the mass of the companion star be zero . actually , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta i_a^{\langle ij\rangle}$\end{document } above is a necessary term which makes the so - obtained 3 pn metric equal to the schwarzschild metric boosted at the constant velocity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec v_a}$\end{document } in harmonic coordinates . the reason why the influence of a body s lorentz contraction appears starting from 3 pn order is as follows . the body s lorentz contraction appears as an apparent deformation of the body , namely , apparent quadrupole moment as the leading order in the frame where the body is moving . the body s ( real ) quadrupole affects the field from 2 pn order through ( see equation ( 78 ) ) 107\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{{\rm{quad}}}^{\tau \tau } = { \epsilon ^8}\sum\limits_a { { { 3i_a^{\langle ij\rangle}r_a^ir_a^j } \over { 2r_a^5}}}.$$\end{document } the radius of a compact body a is of order of its mass ma , and the lorentz contraction deforms the body so that the radius of the body changes by the amount \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\epsilon ^2}{m_a}v_a^2 + \mathcal o({\epsilon ^4})$\end{document}. so the apparent quadrupole moments due to lorentz contraction is of order of 108\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i_{a\,{\rm{apparent}}}^{\langle ij\rangle}\sim{\epsilon ^2}m_a^3v_a^2.$$\end{document } this field is the 3 pn field and thus the lorentz contraction of a body affects the equations of motion starting from 3 pn order . from the definition of the four - momentum , 109\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_a^\mu ( \tau ) = { \epsilon ^2}\int\nolimits_{{b_a } } { { d^3 } } { \alpha _ a}\lambda _ n^{\tau \mu},$$\end{document } and the conservation law ( 67 ) , we have the evolution equations for the four - momentum : 110\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{dp_a^\mu } \over { d\tau } } = - { \epsilon ^{- 4}}\oint\nolimits_{\partial { b_a } } d { s_k}\,\lambda _ n^{k\mu } + { \epsilon ^{- 4}}v_a^k\oint\nolimits_{\partial { b_a } } d { s_k}\,\lambda _ n^{\tau \mu}.$$\end{document } here we used the fact that the size and the shape of the body zone are defined to be fixed ( in the near zone coordinates ) , while the center of the body zone moves at the velocity of the star s representative point . substituting the momentum - velocity relation ( 86 ) into the spatial components of equation ( 110 ) , we obtain the general form of equations of motion for star a , 111\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { p_a^\tau { { dv_a^i } \over { d\tau } } = - { \epsilon ^{- 4}}\oint\nolimits_{\partial { b_a } } d { s_k}\,\lambda _ n^{ki } + { \epsilon ^{- 4}}v_a^k\oint\nolimits_{\partial { b_a } } d { s_k}\,\lambda _ n^{\tau i}\quad \quad \quad } \\ { + { \epsilon ^{- 4}}v_a^i\left({\oint\nolimits_{\partial { b_a } } d { s_k}\,\lambda _ n^{k\tau } - v_a^k\oint\nolimits_{\partial { b_a } } d { s_k}\,\lambda _ n^{\tau \tau } } \right ) } \\ { - { { dq_a^i } \over { d\tau } } - { \epsilon ^2}{{{d^2}d_a^i } \over { d{\tau ^2}}}.\quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } all the right hand side terms in equation ( 111 ) except the dipole moment are expressed as surface integrals . we can specify the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } freely to determine the representative point \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i(\tau)$\end{document } of star a. up to 2.5 pn order we take \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i = 0$\end{document } and simply call \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } the center of mass of star a. note that in order to obtain the spin - orbit coupling force in the same form as in previous works [ 46 , 108 , 152 ] , we have to make another choice for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } ( see and appendix b.1 ) . at 3 pn order , yet another choice of the value of the dipole moment \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } shall be examined ( see sections 7 and 8.2 ) . in equation ( 111 ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document } rather than the mass of star a appears . hence we have to derive a relation between the mass and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document}. we shall derive that relation by solving the temporal component of the evolution equations ( 110 ) functionally . then , since all the equations are expressed with surface integrals except \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } to be specified , we can derive the equations of motion for a strongly self - gravitating star using the post - newtonian approximation . since we have introduced the body zone by hand , the arbitrary body zone size ra seems to appear in the metric , the multipole moments of the stars , and the equations of motion . more specifically ra appears in them because of ( i ) the splitting of the deviation field into two parts ( i.e. b and n / b contributions ) , the definition of the moments , and ( ii ) the surface integrals that we evaluate to derive the equations of motion . b and n / b contributions to the field depend on the body zone boundary ra . but thus it is natural to expect that there are renormalized multipole moments which are independent of ra since we use nonsingular matter sources . this renormalization would absorb the ra dependence occuring in the computation of the n / b field ( see section 4.8 for an example of such a renormalization ) . one possible practical obstacle for this expectation might be the ln(ra ) dependence of multipole moments . although at 3 pn order there appear such logarithmic terms , it is found that we could remove them by rechoosing the value of the dipole moment \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } of the star . though we use the same symbol for the moments henceforth as before for notational simplicity , it should be understood that they are the renormalized ones . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\mu$\end{document } for the renormalized \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\mu$\end{document}. since we compute integrals over the body zone boundary , in general the resulting equations of motion seem to depend on the size of the body zone boundary , ra . actually this is not the case . in the derivation of equation ( 111 ) , if we did not use the conservation law ( 67 ) until the final step , we have 112\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { p_a^\tau { { dv_a^i } \over { d\tau } } + { \epsilon ^{- 4}}\oint\nolimits_{\partial { b_a } } d { s_k}\,\lambda _ n^{ki } - { \epsilon ^{- 4}}v_a^k\oint\nolimits_{\partial { b_a } } d { s_k}\,\lambda _ n^{\tau i } - { \epsilon ^{- 4}}v_a^i\left({\oint\nolimits_{\partial { b_a } } d { s_k}\,\lambda _ n^{k\tau } - v_a^k\oint\nolimits_{\partial { b_a } } d { s_k}\,\lambda _ n^{\tau \tau } } \right ) + } \\ { { { dq_a^i } \over { d\tau } } + { \epsilon ^2}{{{d^2}d_a^i } \over { d{\tau ^2 } } } } \\ { = { \epsilon ^{- 4}}\int\nolimits_{{b_a } } { { d^3 } } y\,\lambda _ { n,\,\nu}^{i\nu } - { \epsilon ^{- 4}}v_a^i\int\nolimits_{{b_a } } { { d^3 } } y\,\lambda _ { n,\,\nu}^{\tau \nu } + { \epsilon ^{- 4}}{d \over { d\tau}}\left({\int\nolimits_{{b_a } } { { d^3 } } y\,\lambda _ { n,\,\nu}^{\tau \nu}y_a^i } \right ) . } \\ \end{array}$$\end{document } now the conservation law is satisfied for whatever value we take for ra , then the right hand side of the above equation is zero for any ra . hence the equations of motion ( 111 ) do not depend on ra ( a similar argument can be found in ) . along the same line , the momentum - velocity relation ( 86 ) does not depend on ra . in section 4.8 we shall explicitly show the irrelevance of the field and the equations of motion to ra by checking the cancellation among the ra dependent terms up to 0.5 pn order . we first derive the newtonian equations of motion and the 1 pn correction to the acceleration . the derivation of the 1 pn equations of motion includes some essences of our formalism and shows how it works properly . thus we shall give a detailed explanation about the derivation , though the calculation is elementary and the resulting equations of motion are well - known . now let us derive the newtonian mass - energy relation first . from equations ( 102 , 103 ) and the time component of equation ( 110 ) , we have 113\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{dp_a^\tau } \over { d\tau } } = { \mathcal o}({\epsilon ^2}).$$\end{document } then we define the mass of star a as the integrating constant of this equation , 114\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_a } \equiv \underset{\epsilon \rightarrow 0 } { \lim } p_a^\tau.$$\end{document } here ma is the adm mass that star a had if it were isolated . ( we took zero limit in equation ( 114 ) to ensure that the mass defined above does not include the effect of the companion star and the orbital motion of the star itself . by this limit we ensure that this mass is the integrating constant of equation ( 113 ) . ) by definition ma is constant . then we obtain the lowest order h : 115\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_4{h^{\tau \tau}}{= _ 4}h_b^{\tau \tau } = 4 \sum\limits_{a = 1,2 } { { { { m_a } } \over { { r_a}}}}.$$\end{document } second , from equation ( 91 ) with equations ( 102 ) and ( 103 ) we obtain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i = 0$\end{document } at the lowest order . thus we have the newtonian momentum - velocity relation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^i = { m_a}v_a^i + \mathcal o({\epsilon ^2})$\end{document } from equation ( 86 ) ( we set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i = 0$\end{document } ) . substituting equation ( 104 ) with the lowest order h into the general form of equations of motion ( 111 ) and using the newtonian momentum - velocity relation , we have for star 1 : 116\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { m_1}{{dv_1^i } \over { d\tau } } = - \oint\nolimits_{\partial { b_1 } } d { s_{k(4)}}\,[(- g)t_{{\rm{ll}}}^{ik}]\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { = - { 1 \over { 4\pi}}\left({\delta _ l^i\delta _ m^k - { 1 \over 2}{\delta ^{ik}}{\delta _ { lm } } } \right)\sum\limits_{a = 1,2 } { \sum\limits_{b = 1,2 } { \oint\nolimits_{\partial { b_1 } } d } } { s_k}\,{{{m_a}{m_b}y_a^ly_b^m } \over { y_a^3y_b^3}},\quad \quad \quad \quad \quad \quad } \\ { = - { 1 \over { 4\pi}}\left({\delta _ l^i\delta _ m^k - { 1 \over 2}{\delta ^{ik}}{\delta _ { lm } } } \right)\oint d { \omega _ { { { \bf{n}}_{\bf{1}}}}}\,n_1^k({{m_1 ^ 2n_1^ln_1^m } \over { { { ( \epsilon { r_1})}^2 } } } + { { 2{m_1}{m_2}n_a^{(l}(r_{12}^{m ) } + \epsilon { r_1}n_1^{m ) } ) } \over { \vert { { \vec r}_{12 } } + \epsilon { r_1}{{\vec n}_1}{\vert ^3 } } } } \\ { \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad + { { { { ( \epsilon { r_1})}^2}m_2 ^ 2(r_{12}^l + \epsilon { r_1}n_1^l)(r_{12}^m + \epsilon { r_1}n_1^m ) } \over { \vert { { \vec r}_{12 } } + \epsilon { r_1}{{\vec n}_1}{\vert ^3 } } } ) , } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$y_a^i$\end{document } is the integral variable and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec y_a } = \vec y - { \vec z_a}$\end{document}. we defined \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\vec n}_1}$\end{document } as the spatial unit vector9 emanating from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\vec z}_1}$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\vec r}_{12 } } \equiv { { \vec z}_1 } - { { \vec z}_2}$\end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$n_{12}^i \equiv r_{12}^i/{r_{12}}$\end{document}. we used \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec r_1 } = \epsilon { r_1}{\vec n_1}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec r_2 } = { \vec r_{12 } } + \epsilon { r_1}{\vec n_1}$\end{document}. for details see figure 3 ) . figure 3the vectors used in the surface integral over the boundary of the body zone 1 . the vectors used in the surface integral over the boundary of the body zone 1 . in the above equation , by virtue of the angular integral the first term ( which is singular when the zero limit is taken ) vanishes . the third term vanishes by letting go to zero . only the second term survives and the n = 1 term in the retardation expansion series of h , equation ( 76 ) , gives no contribution at the 1 pn order by the constancy of the mass ma , i.e. 5h = 0 . now we obtain 4h from the newtonian momentum - velocity relation : 117\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_4{h^{\tau i } } = 4\sum\limits_{a = 1,2 } { { { { m_a}v_a^i } \over { { r_a}}}}.$$\end{document } we evaluate the surface integrals in the evolution equation for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document } at 1 pn order . the result for star 1 is 118\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{dp_1^\tau } \over { d\tau } } = { \epsilon ^2}{m_1}{d \over { d\tau}}\left({{1 \over 2}v_1 ^ 2 + { { 3{m_2 } } \over { { r_{12 } } } } } \right ) + { \mathcal o}({\epsilon ^3}),$$\end{document } where we used the newtonian equations of motion . from this equation we have the mass - energy relation at 1 pn order , 119\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_1^\tau = { m_1}\left [ { 1 + { \epsilon ^2}\left({{1 \over 2}v_1 ^ 2 + { { 3{m_2 } } \over { { r_{12 } } } } } \right ) } \right ] + { \mathcal o}({\epsilon ^3}).$$\end{document } then we have to calculate the 1 pn order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i$\end{document}. the result is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i = { \epsilon ^2}m_a^2v_a^i/(6\epsilon { r_a})$\end{document}. as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i$\end{document } depends on ra , we ignore it ( see section 4.5 ) . as a result we obtain the momentum - velocity relation at 1 pn order , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^i = p_a^\tau v_a^i + \mathcal o({\epsilon ^3})$\end{document } from equation ( 86 ) . now as for 4h , we first calculate the surface integrals \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^{ij}$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_a^{ij}$\end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_a^{kij}$\end{document } from equations ( 91 ) and ( 92 ) . we then find that they depend on ra , hence we ignore them and obtain 120\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_b^{ij } = 4{\epsilon ^4}\sum\limits_{a = 1,2 } { { { { m_a}v_a^iv_a^j } \over { { r_a } } } } + { \mathcal o}({\epsilon ^5}).$$\end{document } to derive 6h and 4h , we have to evaluate non - compact support integrals for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$_6h_{n / b}^{\tau \tau}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$_4h_{n / b}^{ij}$\end{document } , and the n = 2 term in equation ( 76 ) for h : 121\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{\tau \tau } } = 4{\epsilon ^4}\sum\limits_{a = 1,2 } { { { p_a^\tau } \over { { r_a } } } } + 4{\epsilon ^6}\int\nolimits_{n / b } { { { { d^3}y } \over { \vert \vec x - \vec y{\vert ^6 } } } } [ ( - g)t_{{\rm{ll}}}^{\tau \tau } ] + 2{\epsilon ^6}{\partial \over { \partial { \tau ^2}}}\sum\limits_{a = 1,2 } { p_a^\tau } { r_a},$$\end{document } 122\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{ij } } = 4{\epsilon^4}\sum\limits_{a = 1,2 } { { { { m_a}\upsilon _ a^i\upsilon _ a^j } \over { { r_a } } } } + 4{\epsilon^4}\int\nolimits_{n / b } { { { { d^3}y } \over { \vert \vec x - \vec y\vert}}\,{}_4 } [ ( - g)t_{{\rm{ll}}}^{ij}],$$\end{document } with 123\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_6[(- 16\pi g)t_{{\rm{ll}}}^{\tau \tau } ] = - { 7 \over 8}{\,_4}{h^{\tau \tau , k}}_4{h^{\tau \tau}}_{,k},$$\end{document } 124\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_4[(- 16\pi g)t_{{\rm{ll}}}^{ij } ] = { 1 \over 4}\left({{\delta ^i}_k{\delta ^i}_k - { 1 \over 2}{\delta ^{ij}}{\delta _ { kl } } } \right){\,_4}{h^{\tau \tau , k}}_4{h^{\tau \tau , l}}.$$\end{document } the evaluation of the twice retardation expansion term ( the last term in equation ( 121 ) ) is straightforward . for the non - compact support integrals , it is sufficient to consider the following integral : 125\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_{n / b } { { { { d^3}y } \over { 16\pi |\vec x - \vec y|}}\,{}_4 } { h^{\tau \tau , i}}_4{h^{\tau \tau , j } } = { 1 \over \pi}\sum\limits_{a = 1,2 } { p_a^{\tau 2 } } \int\nolimits_{n / b } { { { { d^3}y } \over { |\vec x - \vec y| } } } { { r_a^ir_a^j } \over { r_a^6 } } + { 2 \over \pi}p_1^\tau p_2^\tau \int\nolimits_{n / b } { { { { d^3}y } \over { |\vec x - \vec y| } } } { { r_1^{(i}r_2^{j ) } } \over { r_{\,\,1}^3r_{\,\,2}^3}}.$$\end{document } to evaluate the above poisson integrals , we use equation ( 101 ) , thus we need to find superpotentials for the integrands . for this purpose , it is convenient to transform the tensorial integrands into scalar integrands with differentiation operators , 126\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{r_a^ir_a^j } \over { r_a^6 } } = { 1 \over 8}\left({{\delta ^i}_k{\delta ^j}_l + { \delta ^{ij}}{\delta _ { kl } } } \right){{{\partial ^2 } } \over { \partial { y^k}{y^l}}}\left({{1 \over { r_a^2 } } } \right),$$\end{document } 127\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{r_1^ir_2^j } \over { r_1 ^ 3r_2 ^ 3 } } = { \partial \over { \partial z_1^i}}{\partial \over { \partial z_2^i}}\left({{1 \over { { r_1}{r_2 } } } } \right).$$\end{document } then it is relatively easy to find the super - potentials for these scalars . the results are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta \ln { r_1 } = 1/r_1 ^ 2$\end{document } and ln s = 1/(r1r2 ) where s = r1 + r2 + r12 . equation ( 101 ) for each integrand then becomes 128\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_{n / b } { { { { d^3}y } \over { \vert \vec x - \vec y\vert } } } { { r_a^ir_a^j } \over { r_a^6 } } = - 4\pi \left({{{{\delta ^{ij } } } \over { 4r_a^2 } } - { { r_a^ir_a^j } \over { 4r_a^4 } } } \right ) + { { 4\pi { \delta ^{ij } } } \over { 3\epsilon { r_1}{r_1 } } } + { \mathcal o}(\epsilon { r_a}),$$\end{document } 129\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \int\nolimits_{n / b } { { { { d^3}y } \over { \vert \vec x - \vec y\vert } } } { { r_1^ir_2^j } \over { r_1 ^ 3r_2 ^ 3 } } = - 4\pi \left({- { { { \delta ^{ij } } } \over { { r_{12}}s } } - { { r_1^ir_{12}^j } \over { { r_1}{r_{12}}{s^2 } } } + { { r_{12}^ir_{12}^j } \over { r_{12}^2{s^2 } } } + { { r_{12}^ir_{12}^j } \over { r_{12}^3s } } - { { r_1^ir_{12}^j } \over { { r_1}{r_2}{s^2 } } } + { { r_{12}^ir_2^j } \over { { r_{12}}{r_2}{s^2 } } } } \right ) } \\ { + { \mathcal o}(\epsilon { r_a}){.}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } the second last term of equation ( 128 ) and the terms abbreviated as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}(\epsilon { r_a})$\end{document } in the above two equations arise from the surface integrals in equation ( 101 ) . since they depend on ra , we ignore it ( see section 4.8 ) . substituting equations ( 128 ) and ( 129 ) back into equation ( 125 ) , we can compute the non - compact support integrals . using the above results and the newtonian equations of motion for the twice retardation expansion term , we finally obtain 130\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { h^{\tau \tau } } = 4{\epsilon ^4}\sum\limits_{a = 1,2 } { { { p_a^\tau } \over { { r_a}}}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } } \\ { + \,{\epsilon ^6 } \left [ { - 2\sum\limits_{a = 1,2 } { { { { m_a } } \over { { r_a}}}\{{{({{\vec n}_a}\cdot{{\vec v}_a})}^2 } - v_a^2\ } + 2{{{m_1}{m_2 } } \over { r_{12}^2}}{{\vec n}_{12}}\cdot({{\vec n}_1 } - { { \vec n}_2 } ) } } \right . } \\ { \quad \quad \quad + \left . { 7\sum\limits_{a = 1,2 } { { { m_a^2 } \over { r_a^2 } } } + 14{{{m_1}{m_2 } } \over { { r_1}{r_2 } } } - 14{{{m_1}{m_2 } } \over { { r_{12}}}}\sum\limits_{a = 1,2 } { { 1 \over { { r_a } } } } } \right ] + { \mathcal o}({\epsilon ^7 } ) , } \\ \end{array}$$\end{document } 131\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { h^{ij } } = 4{\epsilon ^4}\sum\limits_{a = 1,2 } { { { { m_a}v_a^iv_a^j } \over { { r_a } } } } \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + \,{\epsilon ^4}\left [ { \sum\limits_{a = 1,2 } { { { m_a^2 } \over { r_a^2 } } } n_a^in_a^j - { { 8{m_1}{m_2 } } \over { { r_{12}}s}}n_{12}^in_{12}^j\quad \quad \quad \quad \quad \quad } \right.\quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { - \left . { 8\left({{\delta ^i}_k{\delta ^j}_l - { 1 \over 2}{\delta ^{ij}}{\delta _ { kl } } } \right){{{m_1}{m_2 } } \over { { s^2}}}{{({{\vec n}_{12 } } - { { \vec n}_1})}^{(k}}{{({{\vec n}_{12 } } + { { \vec n}_2})}^{l ) } } } \right ] + { \mathcal o}({\epsilon ^5 } ) , } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec n_a } \equiv { \vec r_a}/{r_a}$\end{document}. evaluating the surface integrals in equation ( 111 ) as in the newtonian case , we obtain the 1 pn equations of motion , 132\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { m_1}{{dv_1^i } \over { d\tau } } = - { { { m_1}{m_2 } } \over { r_{12}^2}}n_{12}^i\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + \,{\epsilon ^2}{{{m_1}{m_2 } } \over { r_{12}^2}}\left [ { n_{12}^i\left({- v_1 ^ 2 - 2v_2 ^ 2 + { 3 \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } + 4({{\vec v}_1}\cdot{{\vec v}_2 } ) + { { 5{m_1 } } \over { { r_{12 } } } } + { { 4{m_2 } } \over { { r_{12 } } } } } \right ) } \right . } \\ { \left . { + { v^i}\left({4({{\vec n}_{12}}\cdot{{\vec v}_1 } ) - 3({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right ) } \right],\quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } where we defined the relative velocity as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vec v \equiv { \vec v_1 } - { \vec v_2}$\end{document } and we used the newtonian equations of motion as well as equation ( 119 ) . with the n pn order equations of motion and h in hand , we first derive the n + 1 pn evolution equation for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document}. then we solve it functionally and obtain the mass - energy relation at n + 1 pn order . next we calculate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i$\end{document } at n + 1 pn order and derive the momentum - velocity relation at n + 1 pn order . then we calculate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^{{k_l}i}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_a^{{k_l}ij}$\end{document}. with the n + 1 pn mass - energy relation , the n + 1 pn momentum - velocity relation , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^{{k_l}i}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_a^{{k_l}ij}$\end{document } we next derive the n + 1 pn deviation field h. finally we evaluate the surface integrals which appear in the right hand side of equation ( 111 ) and obtain the n + 1 pn equations of motion . in the above calculations we use the n pn order equations of motion to reduce the order of the equations of motion whenever an acceleration appears in the right hand of the resulting equations of motion . for instance , when we meet \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\epsilon ^2}dv_1^i / d\tau$\end{document } in the right hand side of the equations motion and we have to evaluate this up to , then using the newtonian equations of motion , we replace it by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- { \epsilon ^2}{m_2}r_{12}^i / r_{12}^3$\end{document}. basically we shall derive the 3 pn equations of motion with the procedure as described above . as explained in section 4.5 we discard the body zone boundary dependent terms in the field , since we expect that they cancel out between the body zone contribution and the n / b contribution . before moving on to the higher order calculations , however , it is instructive to see that such a cancellation really occurs in the field and consequently the equations of motion up to 0.5 pn order . first , we show the independence of the body zone radius ra in the 0.5 pn field . returning back to the derivation of the 1 pn h with care for the ra dependence ( see equation ( 128 ) ) , we get 133\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { h^{\tau \tau } } = 4{\epsilon ^4}\sum\limits_{a = 1,2 } { { { p_a^\tau } \over { { r_a } } } } + 4{\epsilon ^6}\int\nolimits_{c / b } { { d^3 } } y\,{{{}_6\lambda _ n^{\tau \tau}(\tau , { y^k } ) } \over { \vert \vec x - \vec y\vert } } + 2{\epsilon ^6}{{{\partial ^2 } } \over { \partial { \tau ^2}}}\sum\limits_{a = 1,2 } { p_a^\tau } { r_a } + { \mathcal o}({\epsilon ^7 } ) } \\ { = 4{\epsilon ^4}\sum\limits_{a = 1,2 } { { 1 \over { { r_a } } } } \left({p_a^\tau - { \epsilon ^2}{{7m_a^2 } \over { 2\epsilon { r_a } } } } \right ) + { \mathcal o}({\epsilon ^6}).\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } now we split \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document } as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau = \bar p_a^\tau + \tilde p_a^\tau$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar p_a^\tau$\end{document } does not depend on ra while \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde p_a^\tau$\end{document } does . in order to evaluate the ra dependent part of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde p_a^\tau$\end{document } we use the fact that the integral of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\lambda _ n^{\tau \tau}$\end{document } over the near zone does not depend on the size of the body zone . ( notice that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document } is defined as the volume integral of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\lambda _ n^{\tau \tau}$\end{document } over ba . ) in fact , from the relevant expression of the pseudotensor , we obtain at the lowest order 134\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \epsilon ^2}\int\nolimits_{n / b } { { d^3 } } \alpha { \epsilon ^6}_6\lambda _ n^{\tau \tau } = { \epsilon ^2}\int\nolimits_{n / b } { { d^3 } } { y_6}\lambda _ n^{\tau \tau}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { = - { \epsilon ^2}{7 \over 2}\sum\limits_{a = 1,2 } { { { m_a^2 } \over { \epsilon { r_a}}}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \quad \quad } \\ { \quad + ( { \rm{terms}}\,{\rm{independent}}\,{\rm{of}}\,{r_a},\,{\rm{or}}\,{\rm{terms}}\,{\rm{having}}\,{\rm{positive}}\,{\rm{power}}({\rm{s}})\,{\rm{of}}\,{r_a}){. } } \\ \end{array}$$\end{document } hence we find \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde p_a^\tau = { \epsilon ^2}7m_a^2/(2\epsilon { r_a } ) + { \mathcal o}({\epsilon ^2})$\end{document}. the above equation shows us that the 0.5 pn field is independent of ra and fully expressed by the ra independent energy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar p_a^\tau$\end{document } , or mass up to 0.5 pn order . in the similar manner we split \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^i$\end{document } as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^i = \bar p_a^i + \tilde p_a^i$\end{document } and obtain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde p_a^i = { \epsilon ^2}11m_a^2v_a^i/(3\epsilon { r_a } ) + { \mathcal o}({\epsilon ^2})$\end{document } from the definition of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^i$\end{document}. thus from the fact that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i = { \epsilon ^2}m_a^2v_a^i/(6\epsilon { r_a } ) + { \mathcal o}({\epsilon ^2})$\end{document } , we find that the 0.5 pn momentum - velocity relation does not depend on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_a}:\;\bar p_a^i = \bar p_a^\tau v_a^i + { \mathcal o}({\epsilon ^2})$\end{document}. finally evaluating the surface integrals in the general form of equations of motion using the renormalized ( barred ) moments , we find that the equations of motion are independent of ra as was expected . as remarked in section 4.5 from now on we use the same symbol for the renormalized moments as for the bare moments henceforth as before for notational simplicity . in the post - newtonian approximation , we need to solve poisson equations to find the metric . up however , it seems impossible to derive the 3 pn accurate gravitational field in harmonic coordinates in a closed form completely . the problem is that it seems difficult ( if at all possible ) to find a particular solution of the poisson equations for non - compact sources . the works so far overcome this problem by not solving the poisson equations but keeping the poisson integral unevaluated . then to derive the equations of motion , we basically interchange the order of operations ; first we evaluate surface integrals with the poisson integrals as integrands ( in the surface integral approach ) or compute derivatives of the poisson integrals ( when one adopts the geodesic equation ) , and then we evaluate the remaining volume integrals . we first explain the usual method and a method to derive a field just around the star , and then explain the method mentioned above . up to 2.5 pn order , we have solved all the poisson equations necessary to derive the 2.5 pn gravitational field . at 3 pn order , we have found a part of the solutions of the poisson equations , which we call ( super-)potentials10 . for example , 135\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{r_1^i\,r_1^j\,r_1^k\,r_2^l } \over { r_1 ^ 5\,r_2 ^ 3 } } = \delta \left [ { - { 1 \over 3}{{{\partial ^4 } } \over { \partial z_1^i\,\partial z_1^j\,\partial z_1^k\,\partial z_2^l}}{f^{(1 , - 1 ) } } + { 1 \over 3}({\delta ^{ij}}{\partial _ { z_1^k } } + { \delta ^{ik}}{\partial _ { z_1^j } } + { \delta ^{jk}}{\partial _ { z_1^i}}){\partial _ { z_2^l}}\ln s } \right],$$\end{document } where s = r1 + r2 + r12 , and f which satisfies f = r1/r2 is given in as 136\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f^{(1 , - 1 ) } } = { 1 \over { 18}}(- r_1 ^ 2 - 3{r_1}{r_{12 } } - r_{12}^2 + 3{r_1}{r_2 } + 3{r_{12}}{r_2 } + r_2 ^ 2 ) + { 1 \over 6}(- r_1 ^ 2 + r_{12}^2 + r_2 ^ 2)\ln s.$$\end{document } it is possible to add any homogeneous solution to super - potentials . in our formalism , the only place where we use super - potentials is equation ( 101 ) . in the case above , we could add , say , 1/r1 to f. ( note that to evaluate the surface integrals in the general form of equations of motion ( 111 ) we need super - potentials in the spatial region n / b which do not include any singularity due to the point particle limit . ) it is easy to see that contribution from a possible additional homogeneous solution cancels out between the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- 4\pi g(\vec x)$\end{document } term and the surface integral in equation ( 101 ) . useful super - potentials for derivation of the 3 pn field are given in [ 30 , 27 , 91 , 99 ] . as all what we need to do is to evaluate the surface integrals in the general form of equations of motion ( 111 ) , we need an expression for the gravitational field only around the star . in fact , we have developed such a method in for the source term of the following form 137\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ { z_a^i}}{\partial _ { z_{a^{\prime}}^j}}g(\vec x ) \equiv { \partial _ { z_a^i}}{\partial _ { z_{a^{\prime}}^j}}\left({{{{{(\ln { r_1})}^p}{{(\ln { r_2})}^q } } \over { r_1^ar_2^b } } } \right),$$\end{document } where a and b are integers and p = 0 , 1 , q = 0 , 1 . then , we take spatial derivatives out of the poisson integral , 138\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_{n / b } { { { { d^3}y } \over { \vert \vec x - \vec y\vert } } } { \partial _ { z_a^i}}{\partial _ { z_{a^{\prime}}^j}}g(\vec y ) = { \partial _ { z_a^i}}{\partial _ { z_{a^{\prime}}^j}}\int\nolimits_{n / b } { d^3}y\,{{g(\vec y ) } \over { \vert \vec x - \vec y\vert } } + { \partial _ { z_a^i}}\oint\nolimits_{\partial { b_{a^{\prime } } } } d{s_j}\,{{g(\vec y ) } \over { \vert \vec x - \vec y\vert } } + \oint\nolimits_{\partial { b_a } } d{s_i}\,{{{\partial _ { z_{a^{\prime}}^j}}g(\vec y ) } \over { \vert \vec x - \vec y\vert}}.$$\end{document } note that the integration region is n / b and therefore \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$g(\vec x)$\end{document } is nonsingular in n / b . for this kind of source term , we have given a method in to find a field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f_{[a , c]}^{(m , n)}$\end{document } in the neighborhood of star a in the following sense : 139\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta f_{[a , c]}^{(p , m , q , n ) } - { ( \ln { r_1})^p}r_1^m{(\ln { r_2})^q}r_2^n = { \mathcal o}\left({{{r_a^{c + 1 } } \over { r_{12}^{c + 1 } } } } \right)\qquad { \rm{as}}\;{r_a } \rightarrow 0.$$\end{document } we have checked at 3 pn order that the resulting field from this method is equal to the field obtained from the usual ( super - potential ) method whenever the super - potentials are available . unfortunately , however , this method is not perfect and we need another method to derive the equations of motion which we explain now . in the surface integral approach , we need to evaluate a surface integral of the landau - lifshitz pseudotensor which has a form h,h,. from an order counting , it would be clear that we need to evaluate a surface integral of the form 3 pn potential to find the 3 pn equations of motion , where it seems impossible to find the 3 pn potential in a closed form , as mentioned above . the surface integral mentioned here thus has the form 140\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\oint\nolimits_{\partial { b_1 } } d { s_k}{{r_a^l } \over { r_a^3}}\underset { \epsilon { r_a } } { { \rm{disc } } } \int\nolimits_{n / b } { { d^3 } } y\,{{f(\vec y ) } \over { \vert \vec x - \vec y\vert}}$$ \end{document } for star 1 . here the operator \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{dis}}{{\rm{c}}_{\epsilon { r_a}}}$\end{document } means to discard all the ra dependent terms other than logarithms of ra . the method to evaluate this type of integral is to exchange the order of integrals , first calculate the surface integral , and then calculate the volume integral . one caveat is that we can not simply exchange the order of integrals , and we put an operation discra in front of the poisson integral as in equation ( 140 ) above . we first exchange the order of integrals , 141\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\oint\nolimits_{\partial { b_1 } } d { s_k}\,{{r_a^l } \over { r_a^3}}\underset { \epsilon { r_a } } { { \rm{disc } } } \int\nolimits_{n / b } { { d^3 } } y\,{{f({{\vec y}_1 } ) } \over { \vert { { \vec r}_1 } - { { \vec y}_1}\vert } } = \underset { { r_1}\prime \to \epsilon { r_1 } } { \lim } \underset { \epsilon { r_a } } { { \rm{disc } } } \int\nolimits_{n / b } { { d^3 } } { y_1}\,f({\vec y_1})\,\oint\nolimits_{\partial { { b\prime}_1 } } d { s_k}\,{1 \over { \vert { { \vec y}_1 } - r_1\prime { { \vec n}_1}\vert}}{\partial _ { z_a^l}}{1 \over { { r_a}}},$$\end{document } where we defined a sphere \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${b_1^\prime}$\end{document } whose center is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec z_1}$\end{document } and that has a radius \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_1^\prime}$\end{document } which is a constant slightly larger than ra for any ( small ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\epsilon \ ; ( \epsilon { r_1 } < r_1^{\prime } \ll { r_{12}})$\end{document}. the reason we introduced \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_1^\prime}$\end{document } is as follows . suppose that we treat an integrand for which the super - potential is available . by calculating the poisson integral the piece generally depends on ra , however we reasonably discard such ra - dependent terms ( other than logarithmic dependence ) as explained in section 4.5 . using the so - obtained ra - independent field , we evaluate the surface integrals in the general form of the 3 pn equations of motion by discarding the ra dependence emerging from the surface integrals , and obtain the equations of motion . thus the discarding-ra procedure must be employed each time when the field is derived and also when equations of motion are derived , not just once . thus \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_1^\prime}$\end{document } was introduced to distinguish the two kinds of ra dependence and to discard the ra dependence in the right order . let us consider the following integral : 142\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\oint\nolimits_{\partial { b_1 } } d { s_k}\,{{r_1^k } \over { r_1 ^ 3}}\int\nolimits_{n / b } { { { { d^3}y } \over { \vert \vec x - \vec y\vert } } } { 1 \over { y_1 ^ 2}}.$$\end{document } using \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta \ln { r_1 } = 1/r_1 ^ 2$\end{document } , we can integrate the poisson integral and obtain the field , 143\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_{n / b } { { { { d^3}y } \over { \vert \vec x - \vec y\vert } } } { 1 \over { y_1 ^ 2 } } = - 4\pi \ln \left({{{{r_1 } } \over { r/\epsilon } } } \right ) + 4\pi - 4\pi { { \epsilon { r_1 } } \over { { r_1 } } } + { \mathcal o}\left({{{\left({\epsilon { r_a } } \right)}^2 } } \right).$$\end{document } since the body zone contribution must have an r1 dependence hidden in the moments as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$4\pi \epsilon { r_1}/{r_1 } [ + { \mathcal o}({(\epsilon { r_a})^2})]$\end{document } ( see section 4.5 ) , the last term should be discarded before we evaluate the the surface integral in equation ( 142 ) using this field . , 144\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$16{\pi ^2}\left({\ln \left({{{r/\epsilon } \over { \epsilon { r_1 } } } } \right ) + 1 } \right).$$\end{document } on the other hand , we can derive the equations of motion by first evaluating the surface integral over \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\partial b_1^{\prime}$\end{document } , 145\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \oint\nolimits_{\partial { b_1 } } d { s_k}\,{{r_1^k } \over { r_1 ^ 3}}\int\nolimits_{n / b } { { { { d^3}y } \over { \vert \vec x - \vec y\vert } } } { 1 \over { y_1 ^ 2 } } = \int\nolimits_{n / b } { { { { d^3}y } \over { y_1 ^ 2 } } } \oint\nolimits_{\partial b_1^\prime } d { s_k}{{r_1^k } \over { r_1 ^ 3}}{1 \over { \vert { { \vec r}_1 } - { { \vec y}_1}\vert } } } \\ { = 16{\pi ^2}\left [ { \int\nolimits_{\epsilon { r_1}}^{r_1^\prime } { { { dy } \over { r_1^\prime } } } + \int\nolimits_{r_1^\prime}^{r/\epsilon } { { { dy } \over { { y_1 } } } } } \right ] } \\ { = 16{\pi ^2}\left({\ln \left({{{{\mathcal r}/\epsilon } \over { r_1^\prime } } } \right ) + 1 - { { \epsilon { r_1 } } \over { r_1^\prime } } } \right ) . } \\ \end{array}$$\end{document } thus , if we take b1 as the integral region instead of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\partial b_1^{\prime}$\end{document } in the first equality in equation ( 145 ) , or if we take \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\lim \nolimits _ { r_1^{\prime}\rightarrow \epsilon r_{1}}$\end{document } without employing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm{dis}}{{\rm{c}}_{\epsilon { r_a}}}$\end{document } beforehand , we will obtain an incorrect result , 146\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$16{\pi ^2}\ln \left({{{{\mathcal r}/\epsilon } \over { \epsilon { r_1 } } } } \right),$$\end{document } which disagrees with equation ( 144 ) . with this caution in mind , it is straightforward ( though tedious ) to evaluate the surface integrals and then the volume integrals , and thus evaluate all the necessary integrals for our derivation of the 3 pn equations of motion . when we have derived the 3 pn equations of motion , we have used the super - potential method whenever possible , and used a combination of the above three methods when necessary . in fact , for a computational check , we have used the direct - integration method to evaluate the contributions to the equations of motion from all of the 3 pn n / b nonretarded field , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$_8h_{n / b\;n = 0}^{\tau i}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$_{10}h_{n / b\;n = 0}^{\tau \tau}{+ _ 8}h_{n / b\;n = { 0^l}}^l$\end{document}. as expected , we obtain the same result from two computations ; the result from the direct - integration method agrees with that from the combination of the three methods : the direct - integration method , the super - potential method , and the super - potential - in - series method . it was found that the direct - integration part does not play any role in the evaluation of the evolution equation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document } at 3 pn order . thus we use the same method as in the evaluation of the 2.5 pn equations of motion . evaluating the surface integrals in equation ( 110 ) , we obtain the evolution equation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document } as 147\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { dp_{1\theta}^\tau } \over { d\tau } } = - { \epsilon ^2}{{{m_1}{m_2 } } \over { r_{12}^2}}\left [ { 4({{\vec n}_{12}}\cdot{{\vec v}_1 } ) - 3({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right]\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + { \epsilon ^4}{{{m_1}{m_2 } } \over { r_{12}^2}}\left [ { - { 9 \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3 } + { 1 \over 2}v_1 ^ 2({{\vec n}_{12}}\cdot{{\vec v}_2 } ) + 6({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}\quad } \right . } \\ { \quad \quad \quad \quad \quad \quad \quad \quad - 2v_1 ^ 2({{\vec n}_{12}}\cdot{{\vec v}_1 } ) + 4({{\vec v}_1}\cdot{{\vec v}_2})({{\vec n}_{12}}\cdot\vec v ) + 5v_2 ^ 2({{\vec n}_{12}}\cdot{{\vec v}_2 } ) - 4v_2 ^ 2({{\vec n}_{12}}\cdot{{\vec v}_1})\quad } \quad \quad \quad \quad + \left . { { { { m_1 } } \over { { r_{12}}}}(- 4({{\vec n}_{12}}\cdot{{\vec v}_2 } ) + 6({{\vec n}_{12}}\cdot{{\vec v}_1 } ) ) + { { { m_2 } } \over { { r_{12}}}}(- 10({{\vec n}_{12}}\cdot{{\vec v}_1 } ) + 11({{\vec n}_{12}}\cdot{{\vec v}_2 } ) ) } \right ] } \\ { \quad \quad \ , + { \epsilon ^6}{{{m_1}{m_2 } } \over { { r_{12}}}}\left [ { - \left({{3 \over 2}v_1 ^ 4 + 2v_1 ^ 2v_2 ^ 2 + 4v_2 ^ 4 } \right)({{\vec n}_{12}}\cdot{{\vec v}_1 } ) + \left({{5 \over 8}v_1 ^ 4 + { 3 \over 2}v_1 ^ 2v_2 ^ 2 + 7v_2 ^ 4 } \right)({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right . } \\ { \quad \quad \quad \quad + \left({2v_1 ^ 2 + 4v_2 ^ 2 } \right)({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec v}_1}\cdot{{\vec v}_2 } ) - \left({2v_1 ^ 2 + 8v_2 ^ 2 } \right)({{\vec n}_{12}}\cdot{{\vec v}_2})({{\vec v}_1}\cdot{{\vec v}_2 } ) } \\ { \quad \quad \quad \quad + \left({3v_1 ^ 2 + 12v_2 ^ 2 } \right)({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } - \left({{3 \over 4}v_1 ^ 2 + 12v_2 ^ 2 } \right){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3 } } \\ { \quad \quad \quad \quad \quad \quad \quad + 2({{\vec n}_{12}}\cdot{{\vec v}_2}){{({{\vec v}_1}\cdot{{\vec v}_2})}^2 } - 6({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}({{\vec v}_1}\cdot{{\vec v}_2 } ) + 6{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3}({{\vec v}_1}\cdot{{\vec v}_2 } ) } \\ { - { { 15 } \over 2}({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^4 } - { { 45 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^5}\quad \quad \quad \quad } \\ { + { { { m_1 } } \over { { r_{12}}}}\left\{{\left({- 42v_1 ^ 2 - { { 117 } \over 4}v_2 ^ 2 } \right)({{\vec n}_{12}}\cdot{{\vec v}_1 } ) + 60{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^3 } } \right . } \\ { \quad \quad \quad \quad \quad \quad + \left({{{137 } \over 4}v_1 ^ 2 + { { 37 } \over 2}v_2 ^ 2 } \right)({{\vec n}_{12}}\cdot{{\vec v}_2 } ) + { { 297 } \over 4}({{\vec n}_{12 } } \cdot { { \vec v}_1})(\vec v \cdot { { \vec v}_2 } ) } \\ { \quad \quad \quad \quad - { { 219 } \over 4}({{\vec n}_{12 } } \cdot { { \vec v}_2})(\vec v \cdot { { \vec v}_2 } ) - 151{{({{\vec n}_{12 } } \cdot { { \vec v}_1})}^2}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \\ { \left . { \quad + 109({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } - 23{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3 } } \right\ } } \\ { \quad \quad \quad \quad + { { { m_2 } } \over { { r_{12}}}}\left\{{- \left({13v_1 ^ 2 + 18v_2 ^ 2 } \right)({{\vec n}_{12}}\cdot{{\vec v}_1 } ) + \left({17v_1 ^ 2 + 25v_2 ^ 2 } \right)({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right . } \\ { \quad \quad \quad \quad + 26({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec v}_1}\cdot{{\vec v}_2 } ) - 28({{\vec n}_{12}}\cdot{{\vec v}_2})({{\vec v}_1}\cdot{{\vec v}_2 } ) + 2{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \\ { \quad + \left . { 16({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } - 20{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3 } } \right\ } } \\ { \qquad \qquad \quad \,\quad \quad \quad + { { m_1 ^ 2 } \over { r_{12}^2}}\left({{{33 } \over 4}({{\vec n}_{12}}\cdot{{\vec v}_1 } ) - { { 13 } \over 2}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right ) - { { { m_1}{m_2 } } \over { r_{12}^2}}\left({{{35 } \over 4}({{\vec n}_{12}}\cdot{{\vec v}_1 } ) + { { 17 } \over 4}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right ) } \\ { \ , + \left . { { { m_2 ^ 2 } \over { r_{12}^2}}\left({- 12({{\vec n}_{12}}\cdot{{\vec v}_1 } ) + { { 23 } \over 2}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right ) } \right]\quad \quad \quad \quad } \\ { + { \mathcal o}({\epsilon ^7}).\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } remarkably , we can integrate equation ( 147 ) functionally : 148\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{1\theta}^\tau = { m_1}\left({1 + { \epsilon ^2}_2{\gamma _ 1 } + { \epsilon ^4}_4{\gamma _ 1 } + { \epsilon ^6}_6{\gamma _ 1 } } \right ) + { \mathcal o}({\epsilon ^7}),$$\end{document } with 149\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_2{\gamma _ 1 } = { 1 \over 2}v_1 ^ 2 + { { 3{m_2 } } \over { { r_{12}}}},$$\end{document } 150\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$_4{\gamma _ 1 } = - { { 3{m_2 } } \over { 2{r_{12}}}}{({\vec n_{12}}\cdot{\vec v_2})^2 } + { { 2{m_2 } } \over { { r_{12}}}}v_2 ^ 2 + { { 7{m_2 } } \over { 2{r_{12}}}}v_1 ^ 2 - { { 4{m_2 } } \over { { r_{12}}}}({\vec v_1}\cdot{\vec v_2 } ) + { 3 \over 8}v_1 ^ 4 + { { 7m_2 ^ 2 } \over { 2r_{12}^2 } } - { { 5{m_1}{m_2 } } \over { 2r_{12}^2}},$$\end{document } 151\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { _ 6{\gamma _ 1 } = { { m_1 ^ 2{m_2 } } \over { 2r_{12}^3 } } + { { 21{m_1}m_2 ^ 2 } \over { 4r_{12}^3 } } + { { 5m_2 ^ 3 } \over { 2r_{12}^3 } } + { 5 \over { 16}}v_1 ^ 6\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { \,\ , + { { m_2 ^ 2 } \over { r_{12}^2}}\left({{{45 } \over 4}v_1 ^ 2 + { { 19 } \over 2}v_2 ^ 2 + { 1 \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2 } - 19({{\vec v}_1}\cdot{{\vec v}_2 } ) - ( { { \vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2 } ) - 3{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } } \right)\quad \quad } \\ { \quad \quad + { { { m_1}{m_2 } } \over { r_{12}^2}}\left({{{43 } \over 8}v_1 ^ 2 + { { 53 } \over 8}v_2 ^ 2 - { { 69 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2 } - { { 53 } \over 4}({{\vec v}_1}\cdot{{\vec v}_2 } ) + { { 85 } \over 4}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2 } ) - { { 69 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } } \right ) } \\ { + { { { m_2 } } \over { { r_{12}}}}\left({{{33 } \over 8}v_1 ^ 4 + { 3 \over 2}v_1 ^ 2v_2 ^ 2 + v_2 ^ 4 - 6v_1 ^ 2({{\vec v}_1}\cdot{{\vec v}_2 } ) - 4v_2 ^ 2({{\vec v}_1}\cdot{{\vec v}_2 } ) - { 7 \over 4}v_1 ^ 2{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}\quad \quad \quad \quad \quad } \right . } \\ { \quad - \left . { { 5 \over 2}v_2 ^ 2{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } + 2{{({{\vec v}_1}\cdot{{\vec v}_2})}^2 } + 2{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}({{\vec v}_1}\cdot{{\vec v}_2 } ) + { 9 \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^4 } } \right).\quad \quad \quad } \\ \end{array}$$\end{document } equation ( 148 ) together with equations ( 149 , 150 , 151 ) gives the 3 pn order mass - energy relation . in this section we explain the meaning of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_{a\theta}^\tau$\end{document}. first of all , we expand in an series the four - velocity of star a normalized as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${g_{\mu \nu}}u_a^\mu u_a^\nu = - { \epsilon ^{- 2}}$\end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$u_a^i = u_a^\tau v_a^i$\end{document}. the result is 152\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { u_a^\tau = 1 + { \varepsilon ^2}\left [ { { 1 \over 2}v_a^2 + { 1 \over 4}\,{}_4{h^{\tau \tau } } } \right]\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + { \varepsilon ^4}\left [ { { 1 \over 4}\,{}_6{h^{\tau \tau } } + { 1 \over 4}\,{}_4{h^k}_k - { 3 \over { 32}}{{{(_4}{h^{\tau \tau}})}^2 } + { 5 \over 8}\,{}_4{h^{\tau \tau}}v_a^2 - \,{}_4{h^\tau}_kv_a^k + { 3 \over 8}v_a^4 } \right]\quad \quad \quad } \\ { + { \varepsilon ^5}{1 \over 4}\left [ { { } _ 7{h^{\tau \tau } } + \,{}_5{h^k}_k } \right]\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { \quad \quad + { \varepsilon ^6}\left [ { { 1 \over 4}\,{}_8{h^{\tau \tau } } + { 1 \over 4}\,{}_6{h^k}_k + { 1 \over { 16}}\,{}_4{h^{\tau \tau}}_4{h^k}_k - { 3 \over { 16}}\,{}_4{h^{\tau \tau}}_6{h^{\tau \tau } } + { 7 \over { 128}}{{{(_4}{h^{\tau \tau}})}^3 } + { 1 \over 4}\,{}_4{h^{\tau k}}_4{h^\tau}_k } \right . } \\ { \quad \quad { - _ 6}{h^\tau}_kv_a^k - { 1 \over 4}\,{}_4{h^{\tau \tau}}_4{h^\tau}_kv_a^k + { 1 \over 2}\,{}_4{h_{kl}}v_a^kv_a^l + { 1 \over 8}\,{}_4{h^k}_kv_a^2 + { 5 \over { 64}}{{{(_4}{h^{\tau \tau}})}^2}v_a^2 } \\ 2}\,{}_4{h^\tau}_kv_a^kv_a^2 + { { 27 } \over { 32}}\,{}_4{h^{\tau \tau}}v_a^4 + { 5 \over { 16}}v_a^6 } \right]\quad \quad \quad \quad \quad } \\ { \quad + { \mathcal o}({\varepsilon ^7}).\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } the field should be evaluated somehow at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec z_a}$\end{document}. this is a formal series since the metric derived via the point particle description diverges at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec z_a}$\end{document}. now let us regularize this equation with the hadamard partie finie regularization ( see [ 88 , 143 ] and for example , [ 26 , 30 ] in the literature of the post - newtonian approximation ) . consider a function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f(\vec x)$\end{document } which can be expanded around \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec z_1}$\end{document } in the form 153\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f({\vec r_1 } ) = \sum\limits_{p = { p_0 } } { { 1 \over { r_1^p } } } \underset { p}{\hat f } ( { \vec n_1}).$$\end{document } then the hadamard partie finie at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec z_1}$\end{document } of the function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f(\vec x)$\end{document } is defined by 154\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\oint { { { d{\omega _ { { { \bf{n}}_{\bf{1 } } } } } } \over { 4\pi } } } \underset { 0}{\hat f}({\vec n_1}).$$\end{document } for example , by this procedure h becomes ( see equation ( 130 ) ) 155\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[{h^{\tau \tau}}]_1^h = 4{\epsilon ^4}{{p_2^\tau } \over { { r_{12 } } } } + { \epsilon ^6}\left [ { - 2{{{m_2 } } \over { { r_{12}}}}\{{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } - v_2 ^ 2\ } - 16{{{m_1}{m_2 } } \over { r_{12}^2 } } + 7{{m_2 ^ 2 } \over { r_{12}^2 } } } \right ] + { \mathcal o}({\epsilon ^7})$$\end{document } for star 1 . in the above equation , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$[f]_a^{\rm{h}}$\end{document } means that we regularize the quantity f at star a by the hadamard partie finie . evaluating equation ( 152 ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$[\sqrt { - g } ] _ a^{\rm{h}}$\end{document } by this procedure , then comparing the result to equation ( 148 ) combined with equations ( 149 , 150 , 151 ) , we find at least up to 3 pn order : 156\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{a\theta}^\tau = { m_a}[\sqrt { - g } u_a^\tau ] _ a^{\rm{h}}.$$\end{document } it is important that even after regularizing all the divergent terms , there remains a nonlinear effect . equation ( 156 ) is natural . and note that we have never assumed this relation in advance . this relation has been derived by solving the evolution equation for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_{a\theta}^\tau$\end{document } functionally . in this regard , it is worth mentioning that the naturality of equation ( 156 ) supports the use of the hadamard partie finie regularization ( or any regularization procedures if we can reproduce equation ( 156 ) with them ) to derive the 3 pn mass - energy relation to deal with divergences when one uses dirac delta distributions11 . finally , we note that up to 3 pn order 157\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\sqrt { - g } u_a^\tau ] _ a^{\rm{h } } + { \mathcal o}({\epsilon ^7})$$\end{document } is satisfied if we use the hadamard partie finie regularization explained above . we now derive the 3 pn momentum - velocity relation by calculating the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i$\end{document } integral at 3 pn order . from the definition of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i$\end{document } integral , equation ( 91 ) , 158\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q_a^i = { \epsilon ^6}\oint\nolimits_{\partial { b_a } } { d{s_k}({}_{10}\lambda _ n^{\tau k } - v_a^k{}_{10}\lambda _ n^{\tau \tau})y_a^i,}$$\end{document } we find that the calculation required is almost the same as that in the equation for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$dp_a^\tau / d\tau$\end{document}. namely , it turns out that we do not need to use the direct - integration method to compute the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i$\end{document } integral . therefore it is straightforward to evaluate the surface integrals in the definition of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i$\end{document}. here we split \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i$\end{document } into \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_{a\theta}^i$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_{a\chi}^i$\end{document } as 159\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q_a^i = q_{a\theta}^i + q_{a\chi}^i,$$\end{document } with 160\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q_{a\theta}^i = { \epsilon ^{- 4}}\oint\nolimits_{\partial { b_a } } d { s_k}\left({\theta _ n^{\tau k } - v_a^k\theta _ n^{\tau \tau } } \right)y_a^i,$$\end{document } 161\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q_{a\chi}^i = { \epsilon ^{- 4}}\oint\nolimits_{\partial { b_a } } d { s_k}\left({\chi _ n^{\tau k\alpha \beta}{}_{,\alpha \beta } - v_a^k\chi _ n^{\tau \tau \alpha \beta}{}_{,\alpha \beta } } \right)y_a^i,$$\end{document } and we show only \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_{a\theta}^i$\end{document } : 162\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${}_{\leq 6}q_{1\theta}^i = - { \epsilon ^6}{{m_1 ^ 3\,{m_2}\,n_{12}^{\langle ij\rangle}v_{12}^j } \over { 2r_{12}^3 } } = - { \epsilon ^6}{d \over { d\tau}}\left({{{m_1 ^ 3{m_2 } } \over { 6r_{12}^3}}r_{12}^i } \right ) = { \epsilon ^6}{d \over { d\tau}}\left({{1 \over 6}m_1 ^ 3a_1^i } \right),$$\end{document } where nf is the quantity f up to order n inclusively . here it should be understood that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a_a^i$\end{document } in the last expression is evaluated with the newtonian acceleration . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i$\end{document } of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^6})$\end{document } appears at the 4 pn or higher order field . thus up to 3 pn order , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$_6q_a^i$\end{document } affects the equations of motion only through the 3 pn momentum - velocity relation . for this reason , not \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_{a\chi}^i$\end{document } but \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_{a\theta}^i$\end{document } is necessary to derive the 3 pn equations of motion . the explicit expression for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_{a\chi}^i$\end{document } is given in . now with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_{a\theta}^i$\end{document } in hand , we obtain the momentum - velocity relation . it turns out that the part of the momentum velocity relation is a trivial identity12 . thus , defining the parts of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\mu$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } in the same way as for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i$\end{document } , we obtain 163\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{1\theta}^i = p_{1\theta}^\tau v_1^i + { \epsilon ^6}{d \over { d\tau}}\left({{1 \over 6}m_1 ^ 3a_1^i } \right ) + { \epsilon ^2}{{dd_{1\theta}^i } \over { d\tau}}.$$\end{document } as explained in the previous sections 3.4 and 4.4 , we define the representative point \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } of star a by choosing the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document}. in other words , one can freely choose \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } in principle13 . one may set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } equal to zero up to 2.5 pn order . natural to see a three - momentum proportial to a three - velocity and take another choice , 164\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d_{a\theta}^i = - { \epsilon ^4}{1 \over 6}m_a^3a_a^i = { \epsilon ^4}\delta _ { a\theta}^i.$$\end{document } henceforth , we shall define \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } by this equation . finally , it is important to realize that the nonzero dipole moment \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } of order affects the 3 pn field and the 3 pn equations of motion in essentially the same manner as the newtonian dipole moment affects the newtonian field and equations of motion . from equations ( 78 , 79 , 80 ) we see that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta _ { a\theta}^i$\end{document } appears only at 10h as 165\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{\tau \tau}}{\vert _ { { \delta _ { a\theta } } } } = 4{\epsilon ^{10}}\sum\limits_{a = 1,2 } { { { \delta _ { a\theta}^kr_a^k } \over { r_a^3 } } } + { \mathcal o}({\epsilon ^{11}}){.}$$\end{document } then the corresponding acceleration becomes 166\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_1}a_1^i{\vert _ { { \delta _ { a\theta } } } } = - { \epsilon ^6}{{3{m_1}\delta _ { 2\theta}^i } \over { r_{12}^3}}n_{12}^{\langle ik\rangle } + { \epsilon ^6}{{3{m_2}\delta _ { 1\theta}^i } \over { r_{12}^3}}n_{12}^{\langle ik\rangle } - { \epsilon ^6}{{{d^2}\delta _ { 1\theta}^i } \over { d{\tau ^2}}}.$$\end{document } the last term compensates the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^i$\end{document } integral contribution appearing through the momentum - velocity relation ( 163 ) . note that this change of the acceleration does not affect the existence of the conservation of the ( newtonian - sense ) energy , 167\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_1}a_1^i{\vert _ { { \delta _ { a\theta}}}}v_1^i + { m_2}a_2^i{\vert _ { { \delta _ { a\theta}}}}v_2^i = { \epsilon ^6}{d \over { d\tau}}\sum\limits_{a = 1,2 } { \left [ { \delta _ { a\theta}^k{{dv_a^k } \over { d\tau } } - v_a^k{d \over { d\tau}}\delta _ { a\theta}^k } \right]}.$$\end{document } to derive the 3 pn equations of motion , we evaluate the surface integrals in the general form of the equations of motion ( 111 ) using the field 8h , the field 6h , the 3 pn body zone contributions , and the 3 pn n / b contributions corresponding to the results from the super - potential method and the super - potential - in - series method . we then combine the result with the terms from the direct - integration method . from 3 pn order , the effects of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$q_a^{{k_l}i}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_a^{{k_l}ij}$\end{document } integrals appearing in the 3 pn field in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_b^{\mu \nu}$\end{document } contribute to the 3 pn equations of motion . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$_6q_{a\theta}^i$\end{document } given in equation ( 162 ) affects the 3 pn equations of motion through the 3 pn momentum - velocity relation . since we define the representative points of the stars via equation ( 164 ) , we add the corresponding acceleration given by equation ( 166 ) . furthermore , our choice of the representative points of the stars makes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_{a\chi}^i$\end{document } appear independently of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_{a\theta}^i$\end{document } in the field , and hence \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$_4d_{a\chi}^i$\end{document } affects the 3 pn equations of motion . in summary , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$_{\leq 4}q_a^{{k_l}i}{,_{\ ; \leq 4}}r_a^{{k_l}ij},{\;_6}q_{a\theta}^i,\;\delta _ { a\theta}^i$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$_4d_{a\chi}^i$\end{document } contributions to the 3 pn field can be written as 168\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${}_{10}{h^{\tau \tau } } + { } _ 8{h^k}_k = 4\sum\limits_{a = 1,2 } { { { r_a^k } \over { r_a^3 } } } \left({\delta _ { a\theta}^k + { } _ 4d_{a\chi}^k + { } _ 4r_a^{kll } - { 1 \over 2}{}_4r_a^{llk } } \right ) + \ldots,$$\end{document } where are other contributions . on the other hand , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$_6q_{a\theta}^i$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta _ { a\theta}^i$\end{document } affect the equations of motion through the momentum - velocity relation in equation ( 111 ) , 169\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_1}{\left({{{dv_1^i } \over { d\tau } } } \right)_{\leq 3{\rm{pn } } } } = - { \epsilon ^6}{{{d_6}q_{1\theta}^i } \over { d\tau } } - { \epsilon ^6}{{{d^2}\delta _ { 1\theta}^i } \over { d{\tau ^2 } } } + \ldots,$$\end{document } but they cancel each other out , since we choose equation ( 164 ) . then these contributions to a 3 pn acceleration can be summarized into 170\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left({\begin{array}{*{20}c } { { \rm{the}}\;{\rm{contribution}}\;{\rm{to}}\;{m_1}a_1^i\;{\rm{from}}\;}\\ { { } _ { \leq 4}q_a^{{k_l}i},{}_{\leq 4}r_a^{{k_l}ij},{}_6q_{a\theta}^i,\delta _ { a\theta}^i,{}_4d_{a\chi}^i}\\ \end{array } } \right ) = { \epsilon ^6}{{118 } \over 9}{{m_1 ^ 3m_2 ^ 2 } \over { r_{12}^6}}r_{12}^i + { \epsilon ^6}{{118 } \over 9}{{m_1 ^ 2m_2 ^ 3 } \over { r_{12}^6}}r_{12}^i.$$\end{document } collecting these contributions mentioned above , we obtain the 3 pn equations of motion . however , we found that logarithmic terms having the arbitrary constants ra in their arguments survive , 171\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { m_1}{{\left({{{dv_1^i } \over { d\tau } } } \right)}^{{\rm{with}}\,{\rm{log } } } } = { m_1}{{\left({{{dv_1^i } \over { d\tau } } } \right)}_{\le 2.5{\rm{pn}}}}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + { \epsilon ^6}{{m_1 ^ 2{m_2 } } \over { r_{12}^3}}\left [ { { { 44m_1 ^ 2 } \over { 3r_{12}^2}}n_{12}^i\ln \left({{{{r_{12 } } } \over { \epsilon { r_1 } } } } \right ) - { { 44m_2 ^ 2 } \over { 3r_{12}^2}}n_{12}^i\ln \left({{{{r_{12 } } } \over { \epsilon { r_2 } } } } \right)\quad \quad \quad \quad \quad \quad } \right.\quad \quad \quad \quad \quad } \\ { + \left . { { { 22{m_1 } } \over { { r_{12}}}}\left({5{{({{\vec n}_{12}}\cdot\vec v)}^2}n_{12}^i - { v^2}n_{12}^i - 2({{\vec n}_{12}}\cdot\vec v){v^i } } \right)\ln \left({{{{r_{12 } } } \over { \epsilon { r_1 } } } } \right ) } \right ] } \\ { + \ldots + { \mathcal o}({\epsilon ^7}),\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \end{array}$$\end{document } where the acceleration through 2.5 pn order , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${(dv_1^i / d\tau)_{\leq 2.5{\rm{pn}}}}$\end{document } , is the damour and deruelle 2.5 pn acceleration . in our formalism , we have computed it in . since this equation contains two arbitrary constants , the body zone radii ra , at first sight its predictive power on the orbital motion of the binary seems to be limited . in the next section 8.2 , we shall show that by a reasonable redefinition of the representative points of the stars , we can remove ra from our equations of motion . there , we show the explicit form of the 3 pn equations of motion we obtained . the reason logarithms appear in the 3 pn equations of motion ( 171 ) is in a sense easy to understand . since the post - newtonian approximation is a weak field expansion , at some level of iteration in the evaluation of field , we have inevitably logarithms in the field through volume integrals such as 172\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_{n / b } { { { { d^3}y } \over { \vert \vec x - \vec y\vert } } } { \left({{m \over y } } \right)^n},$$\end{document } where m and y are typical the mass and length scale in the orbital motion , respectively , and n is a positive integer . for instance consider \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_1 ^ 3({\vec r_1 } \cdot { \vec a_1})/r_1 ^ 5$\end{document } as an integrand . then we find 173\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_{n / b } { { { { d^3}y } \over { \vert \vec x - \vec y\vert } } } { { m_1 ^ 3({{\vec y}_1}\cdot{{\vec a}_1 } ) } \over { y_1 ^ 5 } } = { { m_1 ^ 3({{\vec r}_1}\cdot{{\vec a}_1 } ) } \over { 3r_1 ^ 3}}\ln \left({{{{r_1 } } \over { \epsilon { r_1 } } } } \right ) + \ldots.$$\end{document } actually , we could remove the ra dependence in our 3 pn equations of motion via an alternative choice of the center of mass . the following alternative choice of the representative point of star a removes the ra dependence in equation ( 171 ) : 174\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d_{a\theta , { \rm{new}}}^i(\tau ) = { \epsilon ^4}\delta _ { a\theta}^i(\tau ) - { \epsilon ^4}{{22 } \over 3}m_a^3a_a^i\ln \left({{{{r_{12 } } } \over { \epsilon { r_a } } } } \right ) \equiv { \epsilon ^4}\delta _ { a\theta}^i(\tau ) + { \epsilon ^4}\delta _ { a\ln}^i(\tau ) \equiv { \epsilon ^4}\delta _ a^i(\tau){.}$$\end{document } note that this redefinition of the center of mass does not affect the existence of the energy conservation as was shown by equation ( 167 ) . we can examine the effect of this redefinition on the equations of motion using equation ( 166 ) ( using \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta _ { a\;\ln}^i$\end{document } instead of a ) . then we have 175\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { m_1}a_1^i{\vert _ { { \delta _ { a\ln } } } } = - { \epsilon ^6}{{3{m_1}\delta _ { 2\ln}^i } \over { r_{12}^3}}n_{12}^{\langle ik\rangle } + { \epsilon ^6}{{3{m_2}\delta _ { 1\ln}^i } \over { r_{12}^3}}n_{12}^{\langle ik\rangle } - { \epsilon ^6}{{{d^2}\delta _ { 1\ln}^i } \over { d{\tau ^2}}}\quad \quad \quad \quad \quad}\\ { = - { { 44m_1 ^ 3m_2 ^ 2 } \over { 3r_{12}^5}}n_{12}^i\ln \left({{{{r_{12 } } } \over { \epsilon { r_1 } } } } \right ) + { { 44m_1 ^ 2m_2 ^ 3 } \over { 3r_{12}^5}}n_{12}^i\ln \left({{{{r_{12 } } } \over { \epsilon { r_2 } } } } \right)\quad \quad \quad \quad \quad \quad \quad \quad}\\ { - { { 22m_1 ^ 3{m_2 } } \over { r_{12}^4}}\left({5{{({{\vec n}_{12}}\cdot\vec v)}^2}n_{12}^i - { v^2}n_{12}^i - 2({{\vec n}_{12}}\cdot\vec v){v^i } } \right)\ln \left({{{{r_{12 } } } \over { \epsilon { r_1 } } } } \right)\quad \quad \quad \quad}\\ { + { { 22m_1 ^ 3{m_2 } } \over { 3r_{12}^4}}\left({{{{m_1 } } \over { { r_{12}}}}n_{12}^i + { { { m_2 } } \over { { r_{12}}}}n_{12}^i - { v^2}n_{12}^i + 8{{({{\vec n}_{12}}\cdot\vec v)}^2}n_{12}^i - 2{{({{\vec n}_{12}}\cdot\vec v)}^2}{v^i } } \right).}\\ \end{array}$$\end{document } comparing the above equations with equation ( 171 ) , we easily conclude that the representative point \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } of star a defined by 176\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d_{a\theta , { \rm{new}}}^i(\tau ) = { \epsilon ^{- 6}}\int\nolimits_{{b_a } } { { d^3 } } y\,({y^i } - z_a^i(\tau))\,\theta _ n^{\tau \tau}(\tau , { y^k } ) = { \epsilon ^4}\delta _ a^i(\tau)$$\end{document } obeys the equations of motion free from any logarithmic term and hence free from any ambiguity up to 3 pn order inclusively . we note that in our formalism \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } is defined by the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } and in turn we have a freedom to assign to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } any value as we like ( though it may be natural to set the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } such that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } resides inside star a ) . we also note that we define \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } order by order . we mention here that blanchet and faye have already noticed that in their 3 pn equations of motion a suitable coordinate transformation removes ( parts of ) the logarithmic dependence of arbitrary parameters corresponding ( roughly ) to our body zone radii14 . it is well - known that choosing different values of dipole moments corresponds to a coordinate transformation . our new choice of the dipole moments of the stars is in a sense natural . to see this , let us consider the harmonic condition : 177\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 = { h^{\tau \mu}}_{,\mu } = 4{\epsilon ^4}\sum\limits_{a = 1,2 } { \left [ { { { \dot p_a^\tau } \over { { r_a } } } + { { r_a^i } \over { r_a^3}}\left({p_a^\tau v_a^i + { \epsilon ^2}\dot d_a^i - p_a^i } \right ) } \right ] + \sum\limits_{a = 1,2 } { \oint\nolimits_{\partial { b_a } } { { { d{s_i } } \over { \vert \vec x - \vec y\vert}}(\lambda _ n^{\tau i } - v_a^i\lambda _ n^{\tau \tau } ) \ldots,}}}$$\end{document } 178\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 = { h^{i\mu}}_{,\mu } = 4{\epsilon ^4}\sum\limits_{a = 1,2 } { { { \dot p_a^i } \over { { r_a } } } + \sum\limits_{a = 1,2 } { \oint\nolimits_{\partial { b_a } } { { { d{s_j } } \over { \vert \vec x - \vec y\vert}}\left(\lambda _ n^{ij } - v_a^j\lambda _ n^{\tau i}\right ) + \ldots,}}}$$\end{document } where are irrelevant terms . these equations are a manifestation of the fact that the harmonic condition is consistent with the evolution equation for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document } , the momentum - velocity relation , and the equations of motion ( and relations among higher multipole moments , hidden in ) . thus if the logarithmic dependence of ra arises from the second term of equation ( 178 ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^i$\end{document } must have the same logarithmic dependence ( times minus sign ) to ensure harmonicity . this and the momentum velocity relation in turn mean \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$v_a^i = \dot z_a^i$\end{document } or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } have corresponding logarithmic dependence . since we already know that the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_a^\tau$\end{document } have no logarithm up to 3 pn order , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } should have logarithms . this is consistent with the fact that a choice of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } determines \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document}. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } depends on logarithms if the old choice is taken , while it does not if our new choice is taken . let us retain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i \neq 0$\end{document } for a while . we then find that the near zone dipole moment \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_n^i$\end{document } defined by a volume integral of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\lambda _ n^{\tau \tau}{y^i}$\end{document } becomes 179\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\epsilon ^2}d_n^i \equiv { \epsilon ^{- 4}}\int\nolimits_n { { d^3 } } y\,\lambda _ n^{\tau \tau}{y^i } = \sum\limits_{a = 1,2 } { p_a^\tau } z_a^i + { \epsilon ^2}\sum\limits_{a = 1,2 } { d_a^i } + { \epsilon ^{- 4}}\int\nolimits_{n / b } { { d^3 } } y\,\lambda _ n^{\tau \tau}{y^i}.$$\end{document } then if we take the old choice of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } , the volume integral becomes 180\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_{n / b } { { d^3 } } y\,\lambda _ n^{\tau \tau}{y^i } = { \epsilon ^4}{{22 } \over 3}\sum\limits_{a = 1,2 } m_a^3a_a^i\ln \left({{{{r_{12 } } } \over { \epsilon { r_a } } } } \right ) + \ldots,$$\end{document } where terms denoted by notice that the near zone dipole moment can be freely determined , say , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_n^i = 0$\end{document } because we can define the origin of the near zone freely . by taking temporal derivatives twice of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_n^i$\end{document } we see that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_{a,{\rm{new}}}^i$\end{document } gives a natural definition of the center of the mass in terms of which the 3 pn equations of motion are independent of ra . by adding \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${m_1}a_1^i{\vert _ { { \delta _ { a\;\ln}}}}$\end{document } to equation ( 171 ) , we obtain our 3 pn equations of motion for two spherical compact stars whose representative points are defined by equation ( 176 ) , 181\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { m_1}{{dv_1^i } \over { d\tau } } = - { { { m_1}{m_2 } } \over { r_{12}^2}}n_{12}^i\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + { \epsilon ^2}{{{m_1}{m_2 } } \over { r_{12}^2}}n_{12}^i\left [ { - v_1 ^ 2 - 2v_2 ^ 2 + 4({{\vec v}_1}\cdot{{\vec v}_2 } ) + { 3 \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } + { { 5{m_1 } } \over { { r_{12 } } } } + { { 4{m_2 } } \over { { r_{12 } } } } } \right]\quad } \\ { + { \epsilon ^2}{{{m_1}{m_2 } } \over { r_{12}^2}}{v^i}\left [ { 4({{\vec n}_{12}}\cdot{{\vec v}_1 } ) - 3({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right]\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { \quad \quad \quad \quad + { \epsilon ^4}{{{m_1}{m_2 } } \over { r_{12}^2}}n_{12}^i\left [ { - 2v_2 ^ 4 + 4v_2 ^ 2({{\vec v}_1}\cdot{{\vec v}_2 } ) - 2{{({{\vec v}_1}\cdot{{\vec v}_2})}^2 } + { 3 \over 2}v_1 ^ 2{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } + { 9 \over 2}v_2 ^ 2{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } } \right . } \\ \quad \quad \quad \quad \quad \quad \quad - 6({{\vec v}_1}\cdot{{\vec v}_2}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } - { { 15 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^4 } - { { 57 } \over 4}{{m_1 ^ 2 } \over { r_{12}^2 } } - 9{{m_2 ^ 2 } \over { r_{12}^2 } } - { { 69 } \over 2}{{{m_1}{m_2 } } \over { r_{12}^2 } } } \\ { \quad \quad \quad + { { { m_1 } } \over { { r_{12}}}}\left({- { { 15 } \over 4}v_1 ^ 2 + { 5 \over 4}v_2 ^ 2 - { 5 \over 2}({{\vec v}_1}\cdot{{\vec v}_2 } ) + { { 39 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2 } } \right . } \\ { \quad \quad \quad \quad - \left . { - 39({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2 } ) + { { 17 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } } \right ) } \\ { \left . { \quad \quad \quad \quad \quad \quad \quad + { { { m_2 } } \over { { r_{12}}}}\left({4v_2 ^ 2 - 8({{\vec v}_1}\cdot{{\vec v}_2 } ) + 2{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2 } - 4({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2 } ) - 6{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } } \right ) } \right ] } \\ { + { \epsilon ^4}{{{m_1}{m_2 } } \over { r_{12}^2}}{v^i}\left [ { { { { m_1 } } \over { { r_{12}}}}\left({- { { 63 } \over 4}({{\vec n}_{12}}\cdot{{\vec v}_1 } ) + { { 55 } \over 4}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right ) + { { { m_2 } } \over { { r_{12}}}}\left({- 2({{\vec n}_{12}}\cdot{{\vec v}_1 } ) - 2({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right ) } \right . } \\ { \quad \quad \quad \quad + v_1 ^ 2({{\vec n}_{12}}\cdot{{\vec v}_2 } ) + 4v_2 ^ 2({{\vec n}_{12}}\cdot{{\vec v}_1 } ) - 5v_2 ^ 2({{\vec n}_{12}}\cdot{{\vec v}_2 } ) - 4({{\vec v}_1}\cdot{{\vec v}_2})({{\vec n}_{12}}\cdot\vec v ) } \\ { - \left . { 6({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } + { 9 \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3 } } \right]\quad \quad \quad \quad \quad } \\ { + { \epsilon ^5}{{4m_1 ^ 2{m_2 } } \over { 5r_{12}^3}}\left [ { n_{12}^i({{\vec n}_{12}}\cdot\vec v)\left({- 6{{{m_1 } } \over { { r_{12 } } } } + { { 52 } \over 3}{{{m_2 } } \over { { r_{12 } } } } + 3{v^2 } } \right ) + { v^i}\left({2{{{m_1 } } \over { { r_{12 } } } } - 8{{{m_2 } } \over { { r_{12 } } } } - { v^2 } } \right ) } \right ] } \\ { + { \epsilon ^6}{{{m_1}{m_2 } } \over { r_{12}^2}}n_{12}^i\left [ { { { 35 } \over { 16}}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^6 } - { { 15 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^4}v_1 ^ 2 + { { 15 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^4}({{\vec v}_1}\cdot{{\vec v}_2 } ) } \right.\quad \quad \quad } \\ { + 3{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}{{({{\vec v}_1}\cdot{{\vec v}_2})}^2 } - { { 15 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^4}v_2 ^ 2 + { 3 \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}v_1 ^ 2v_2 ^ 2 } \\ { - 12{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}({{\vec v}_1}\cdot{{\vec v}_2})v_2 ^ 2 - 2{{({{\vec v}_1}\cdot{{\vec v}_2})}^2}v_2 ^ 2 + { { 15 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}v_2 ^ 4 } \\ { + 4({{\vec v}_1}\cdot{{\vec v}_2})v_2 ^ 4 - 2v_2 ^ 6\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + { { { m_1 } } \over { { r_{12}}}}\left({- { { 171 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^4 } + { { 171 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^3}({{\vec n}_{12}}\cdot{{\vec v}_2})\quad \quad \quad } \right . } \\ { \quad \quad \quad - { { 723 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } + { { 383 } \over 2}({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3 } } \\ { \quad \quad \quad \quad \quad \quad - { { 455 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^4 } + { { 229 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}v_1 ^ 2 - { { 205 } \over 2}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2})v_1 ^ 2 } \\ { \,\quad \quad + { { 191 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}v_1 ^ 2 - { { 91 } \over 8}v_1 ^ 4 - { { 229 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}({{\vec v}_1}\cdot{{\vec v}_2 } ) } \\ { \quad \quad \quad + 244({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2})({{\vec v}_1}\cdot{{\vec v}_2 } ) - { { 225 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}({{\vec v}_1}\cdot{{\vec v}_2 } ) } \\ { \,\quad \quad + { { 91 } \over 2}v_1 ^ 2({{\vec v}_1}\cdot{{\vec v}_2 } ) - { { 177 } \over 4}{{({{\vec v}_1}\cdot{{\vec v}_2})}^2 } + { { 229 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}v_2 ^ 2 } \\ { \quad \quad \quad \quad - { { 283 } \over 2}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2})v_2 ^ 2 + { { 259 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}v_2 ^ 2 - { { 91 } \over 4}v_1 ^ 2v_2 ^ 2 } \\ { \ , + \left . { 43({{\vec v}_1}\cdot{{\vec v}_2})v_2 ^ 2 - { { 81 } \over 8}v_2 ^ 4 } \right)\quad \quad \quad \quad \quad \quad \quad \quad } \\ { + { { { m_2 } } \over { { r_{12}}}}\left({- 6{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } + 12({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3}\quad \quad \quad \quad } \right . } \\ { + 6{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^4 } + 4({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2})({{\vec v}_1}\cdot{{\vec v}_2})\quad \quad \quad } \\ { + 12{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}({{\vec v}_1}\cdot{{\vec v}_2 } ) + 4{{({{\vec v}_1}\cdot{{\vec v}_2})}^2}\quad \quad \quad \quad \quad } \\ { \quad \quad \quad \quad \quad - \left . { 4({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2})v_2 ^ 2 - 12{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}v_2 ^ 2 - 8({{\vec v}_1}\cdot{{\vec v}_2})v_2 ^ 2 + 4v_2 ^ 4 } \right ) } \\ { + { { m_2 ^ 2 } \over { r_{12}^2}}\left({- { { ( { { \vec n}_{12}}\cdot{{\vec v}_1})}^2 } + 2({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2 } ) + { { 43 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } } \right.\quad \quad \quad } \\ { \left . { + 18({{\vec v}_1}\cdot{{\vec v}_2 } ) - 9v_2 ^ 2 } \right ) } \\ { + { { { m_1}{m_2 } } \over { r_{12}^2}}\left({{{415 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2 } - { { 375 } \over 4}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2 } ) + { { 1113 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } } \right . } \\ { \quad \quad \quad \quad - \left . { { { 615{\pi ^2 } } \over { 64}}{{({{\vec n}_{12}}\cdot\vec v)}^2 } + 18v_1 ^ 2 + { { 123{\pi ^2 } } \over { 64}}{v^2 } + 33({{\vec v}_1}\cdot{{\vec v}_2 } ) - { { 33 } \over 2}v_2 ^ 2 } \right ) } \\ { \quad + { { m_1 ^ 2 } \over { r_{12}^2}}\left({- { { 2069 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2 } + 543({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2 } ) - { { 939 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } } \right . } \\ { \quad + \left . { { { 471 } \over 8}v_1 ^ 2 - { { 357 } \over 4}({{\vec v}_1}\cdot{{\vec v}_2 } ) + { { 357 } \over 8}v_2 ^ 2 } \right)\quad \quad \quad \quad \quad } \\ { \quad + \left . { { { 16m_2 ^ 3 } \over { r_{12}^3 } } + { { m_1 ^ 2{m_2 } } \over { r_{12}^3}}\left({{{547 } \over 3 } - { { 41{\pi ^2 } } \over { 16 } } } \right ) - { { 13m_1 ^ 3 } \over { 12r_{12}^3 } } + { { { m_1}m_2 ^ 2 } \over { r_{12}^3}}\left({{{545 } \over 3 } - { { 41{\pi ^2 } } \over { 16 } } } \right ) } \right ] } \\ { + { \epsilon ^6}{{{m_1}{m_2 } } \over { r_{12}^2}}{v^i}\left [ { { { 15 } \over 2}({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^4 } - { { 45 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^5 } - { 3 \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3}v_1 ^ 2\quad \quad \quad \quad \quad \quad \quad \quad \quad } \right . } \\ { + 6({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}({{\vec v}_1}\cdot{{\vec v}_2 } ) - 6{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3}({{\vec v}_1}\cdot{{\vec v}_2})\quad \quad \quad \quad \quad } \\ { - 2({{\vec n}_{12}}\cdot{{\vec v}_2}){{({{\vec v}_1}\cdot{{\vec v}_2})}^2 } - 12({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}v_2 ^ 2 + 12{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3}v_2 ^ 2 } \\ { + ( { { \vec n}_{12}}\cdot{{\vec v}_2})v_1 ^ 2v_2 ^ 2 - 4({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec v}_1}\cdot{{\vec v}_2})v_2 ^ 2 + 8({{\vec n}_{12}}\cdot{{\vec v}_2})({{\vec v}_1}\cdot{{\vec v}_2})v_2 ^ 2\quad } \\ { + 4({{\vec n}_{12}}\cdot{{\vec v}_1})v_2 ^ 4 - 7({{\vec n}_{12}}\cdot{{\vec v}_2})v_2 ^ 4\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + { { { m_2 } } \over { { r_{12}}}}\left({- 2{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) + 8({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } + 2{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3 } } \right . } \\ { + 2({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec v}_1}\cdot{{\vec v}_2 } ) + 4({{\vec n}_{12}}\cdot{{\vec v}_2})({{\vec v}_1}\cdot{{\vec v}_2})\quad \quad \quad \quad } \\ { - \left . { 2({{\vec n}_{12}}\cdot{{\vec v}_1})v_2 ^ 2 - 4({{\vec n}_{12}}\cdot{{\vec v}_2})v_2 ^ 2 } \right)\quad \quad \quad \quad \quad \quad \quad } \\ { + { { { m_1 } } \over { { r_{12}}}}\left({- { { 243 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^3 } + { { 565 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right.\quad \quad \quad \quad \quad \quad } \\ { \quad \quad \quad - { { 269 } \over 4}({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } - { { 95 } \over { 12}}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3 } + { { 207 } \over 8}({{\vec n}_{12}}\cdot{{\vec v}_1})v_1 ^ 2 } \\ { \quad \quad \quad \quad - { { 137 } \over 8}({{\vec n}_{12}}\cdot{{\vec v}_2})v_1 ^ 2 - 36({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec v}_1}\cdot{{\vec v}_2 } ) + { { 27 } \over 4}({{\vec n}_{12}}\cdot{{\vec v}_2})({{\vec v}_1}\cdot{{\vec v}_2 } ) } \\ { + \left . { { { 81 } \over 8}({{\vec n}_{12}}\cdot{{\vec v}_1})v_2 ^ 2 + { { 83 } \over 8}({{\vec n}_{12}}\cdot{{\vec v}_2})v_2 \quad \quad \quad } \\ { + { { m_2 ^ 2 } \over { r_{12}^2}}\left({4({{\vec n}_{12}}\cdot{{\vec v}_1 } ) + 5({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right)\quad \quad \quad \quad \quad \quad } \\ { \quad \quad \quad \quad + { { { m_1}{m_2 } } \over { r_{12}^2}}\left({- { { 307 } \over 8}({{\vec n}_{12}}\cdot{{\vec v}_1 } ) + { { 479 } \over 8}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) + { { 123{\pi ^2 } } \over { 32}}({{\vec n}_{12}}\cdot\vec v ) } \right ) } \\ { + \left . { { { m_1 ^ 2 } \over { r_{12}^2}}\left({{{311 } \over 4}({{\vec n}_{12}}\cdot{{\vec v}_1 } ) - { { 357 } \over 4}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right ) } \right]\quad \quad \quad } \\ { + { \mathcal o}({\epsilon ^7}),\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } in the harmonic gauge . now we list some features of our 3 pn equations of motion . in the test - particle limit , our 3 pn equations of motion coincide with a geodesic equation for a test - particle in the schwarzschild metric in harmonic coordinates ( up to 3 pn order ) . suppose that star 1 is a test particle , star 2 is represented by the schwarzschild metric , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec v_2 } = 0$\end{document}. then the geodesic equation for star 1 becomes 182\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { a_1^i = - { { { m_2 } } \over { r_{12}^2}}n_{12}^i + { \epsilon^2}{{{m_2 } } \over { r_{12}^2}}\left({- v_1 ^ 2n_{12}^i + { { 4{m_2 } } \over { { r_{12}}}}n_{12}^i + 4({{\vec n}_{12}}\cdot{{\vec v}_1})v_1^i } \right)\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad}\\ { + { \epsilon^4}{{m_2 ^ 2 } \over { r_{12}^3}}\left({2{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}n_{12}^i - 9{{{m_2 } } \over { { r_{12}}}}n_{12}^i - 2({{\vec n}_{12}}\cdot{{\vec v}_1})v_1^i } \right)\quad \quad \quad \quad}\\ { + { \epsilon^6}{{m_2 ^ 3 } \over { r_{12}^4}}\left({- { { ( { { \vec n}_{12}}\cdot{{\vec v}_1})}^3}n_{12}^i + { { 16{m_2 } } \over { { r_{12}}}}n_{12}^i + 4({{\vec n}_{12}}\cdot{{\vec v}_1})v_1^i } \right ) + { \mathcal o}({\epsilon^7})}\\ \end{array}$$\end{document } in the harmonic gauge . thus , in the test particle limit equation ( 181 ) coincides with the geodesic equation for a test particle in the schwarzschild metric up to 3 pn order . with the help of the formulas developed in , we have checked the lorentz invariance of equation ( 181 ) ( in the post - newtonian perturbative sense ) . also , we have checked that our 3 pn acceleration admits a conserved energy of the binary orbital motion ( modulo the 2.5 pn radiation reaction effect ) . in fact , the energy e of the binary associated with equation ( 181 ) is 183\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { e = { 1 \over 2}{m_1}v_1 ^ 2 - { { { m_1}{m_2 } } \over { 2{r_{12}}}}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { \quad \quad \quad \quad + { \epsilon ^2}\left [ { { 3 \over 8}{m_1}v_1 ^ 4 + { { m_1 ^ 2{m_2 } } \over { 2r_{12}^2 } } + { { { m_1}{m_2 } } \over { 2{r_{12}}}}\left({3v_1 ^ 2 - { 7 \over 2}({{\vec v}_1}\cdot{{\vec v}_2 } ) - { 1 \over 2}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2 } ) } \right ) } \right]\quad \quad \quad \quad } \\ { + { \epsilon ^4}\left [ { { 5 \over { 16}}{m_1}v_1 ^ 6 - { { m_1 ^ 3{m_2 } } \over { 2r_{12}^3 } } - { { 19m_1 ^ 2m_2 ^ 2 } \over { 8r_{12}^3}}\quad } \right.\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { \quad \quad \quad \quad \quad + { { m_1 ^ 2{m_2 } } \over { 2r_{12}^2}}\left({- 3v_1 ^ 2 + { 7 \over 2}v_2 ^ 2 + { { 29 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2 } - { { 13 } \over 2}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2 } ) + { { ( { { \vec n}_{12}}\cdot{{\vec v}_2})}^2 } } \right ) } \\ { \quad \quad \quad \quad \quad \quad + { { { m_1}{m_2 } } \over { 4{r_{12}}}}\left({{3 \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^3}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) + { 3 \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } - { 9 \over 2}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2})v_1 ^ 2 } \right . } \\ { \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad - { { 13 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}v_1 ^ 2 + { { 21 } \over 2}v_1 ^ 4 + { { 13 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}({{\vec v}_1}\cdot{{\vec v}_2})\quad \quad \quad \quad \quad \quad \quad \quad } \\ { \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \,\,\,+ \left . { \left . { 3({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2})({{\vec v}_1}\cdot{{\vec v}_2 } ) - { { 55 } \over 2}v_1 ^ 2({{\vec v}_1}\cdot{{\vec v}_2 } ) + { { 17 } \over 2}{{({{\vec v}_1}\cdot{{\vec v}_2})}^2 } + { { 31 } \over 4}v_1 ^ 2v_2 ^ 2 } \right ) } \right ] } \\ { + { \epsilon ^6}\left [ { { { 35 } \over { 128}}{m_1}v_1 ^ 8 + { { 3m_1 ^ 4{m_2 } } \over { 8r_{12}^4 } } + { { 469m_1 ^ 3m_2 ^ 2 } \over { 18r_{12}^4}}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad { \quad \quad \quad \quad + { { m_1 ^ 2m_2 ^ 2 } \over { 2r_{12}^3}}\left({{{547 } \over 6}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2 } - { { 3115 } \over { 24}}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2 } ) - { { 123{\pi ^2 } } \over { 32}}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot\vec v ) } \right . } \\ { - \left . { { { 575 } \over 9}v_1 ^ 2 + { { 41{\pi ^2 } } \over { 32}}(\vec v\cdot{{\vec v}_2 } ) + { { 4429 } \over { 72}}({{\vec v}_1}\cdot{{\vec v}_2 } ) } \right)\quad } \\ { \quad \quad + { { m_1 ^ 3{m_2 } } \over { 2r_{12}^3}}\left({- { { 437 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2 } + { { 317 } \over 4}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2 } ) + 3{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } + { { 301 } \over { 12}}v_1 ^ 2 } \right . } \\ { - \left . { { { 337 } \over { 12}}({{\vec v}_1}\cdot{{\vec v}_2 } ) + { 5 \over 2}v_2 ^ 2 } \right)\quad \quad \quad \quad \quad \quad \quad \quad } \\ { \quad \quad \quad \quad \quad + { { { m_1}{m_2 } } \over { { r_{12}}}}\left({- { 5 \over { 16}}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^5}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) - { 5 \over { 16}}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^4}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } - { 5 \over { 32}}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^3}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3 } } \right . } \\ { \quad + { { 19 } \over { 16}}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^3}({{\vec n}_{12}}\cdot{{\vec v}_2})v_1 ^ 2 + { { 15 } \over { 16}}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}v_1 ^ 2 } \\ { \quad \quad \quad \quad \quad + { 3 \over 4}({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3}v_1 ^ 2{{19 } \over { 16}}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^4}v_1 ^ 2 - { { 21 } \over { 16}}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2})v_1 ^ 4 } \\ { \quad \quad \quad \quad \quad \quad \quad \quad - 2{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}v_1 ^ 4 + { { 55 } \over { 16}}v_1 ^ 6 - { { 19 } \over { 16}}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^4}({{\vec v}_1}\cdot{{\vec v}_2 } ) - { { ( { { \vec n}_{12}}\cdot{{\vec v}_1})}^3}({{\vec n}_{12}}\cdot{{\vec v}_2})({{\vec v}_1}\cdot{{\vec v}_2 } ) } \\ { \quad \quad - { { 15 } \over { 32}}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}({{\vec v}_1}\cdot{{\vec v}_2 } ) + { { 45 } \over { 16}}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}v_1 ^ 2({{\vec v}_1}\cdot{{\vec v}_2 } ) } \\ { \quad \quad \quad \quad \quad \quad \quad + { 5 \over 4}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2})v_1 ^ 2({{\vec v}_1}\cdot{{\vec v}_2 } ) + { { 11 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}v_1 ^ 2({{\vec v}_1}\cdot{{\vec v}_2 } ) - { { 139 } \over { 16}}v_1 ^ 4({{\vec v}_1}\cdot{{\vec v}_2 } ) } \\ { \quad \quad \quad \quad \quad \quad - { 3 \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}{{({{\vec v}_1}\cdot{{\vec v}_2})}^2 } + { 5 \over { 16}}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2}){{({{\vec v}_1}\cdot{{\vec v}_2})}^2 } + { { 41 } \over 8}v_1 ^ 2{{({{\vec v}_1}\cdot{{\vec v}_2})}^2 } } \\ { \quad \quad \quad \quad \quad \quad \quad + { 1 \over { 16}}{{({{\vec v}_1}\cdot{{\vec v}_2})}^3 } - { { 45 } \over { 16}}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}v_1 ^ 2v_2 ^ 2 - { { 23 } \over { 32}}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2})v_1 ^ 2v_2 ^ 2 + { { 79 } \over { 16}}v_1 ^ 4v_2 ^ 2 } \\ { \qquad \qquad \qquad \quad \ , - \left . { { { 161 } \over { 32}}v_1 ^ 2v_2 ^ 2({{\vec v}_1}\cdot{{\vec v}_2 } ) } \right)\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { \quad \quad \quad + { { m_1 ^ 2{m_2 } } \over { r_{12}^2}}\left({- { { 49 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^4 } + { { 75 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^3}({{\vec n}_{12}}\cdot{{\vec v}_2 } ) - { { 187 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2 } + { { 11 } \over 2}v_1 ^ 4 } \right . } \\ { \quad \quad \quad \quad + { { 247}\over { 24}}({{\vec n}_{12}}\cdot{{\vec v}_1}){{({{\vec n}_{12}}\cdot{{\vec v}_2})}^3 } + { { 49 } \over 8}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}v_1 ^ 2 + { { 81 } \over { \quad \quad \quad \quad - { { 21 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}v_1 ^ 2 - { { 15 } \over 2}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}({{\vec v}_1}\cdot{{\vec v}_2 } ) - { 3 \over 2}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2})({{\vec v}_1}\cdot{{\vec v}_2 } ) } \\ { \quad \quad \quad \quad + { { 21 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}({{\vec v}_1}\cdot{{\vec v}_2 } ) - 27v_1 ^ 2({{\vec v}_1}\cdot{{\vec v}_2 } ) + { { 55 } \over 2}{{({{\vec v}_1}\cdot{{\vec v}_2})}^2 } + { { 49 } \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_1})}^2}v_2 ^ 2 } \\ { \quad \quad \quad - { { 27 } \over 2}({{\vec n}_{12}}\cdot{{\vec v}_1})({{\vec n}_{12}}\cdot{{\vec v}_2})v_2 ^ 2 + { 3 \over 4}{{({{\vec n}_{12}}\cdot{{\vec v}_2})}^2}v_2 ^ 2 + { { 55 } \over 4}v_1 ^ 2v_2 ^ 2 - 28({{\vec v}_1}\cdot{{\vec v}_2})v_2 ^ 2 } \\ { + \left . { \left . { { { 135 } \over { 16}}v_2 ^ 4 } \right ) } \right]\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + ( 1 \leftrightarrow 2 ) + { \mathcal o}({\epsilon ^7}).\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } this orbital energy of the binary is computed based on that one found in blanchet and faye , the relation between their 3 pn equations of motion and our result described in section 8.5 below , and equation ( 167 ) . ( after constructing e given as in equation ( 183 ) , we have checked that our 3 pn equations of motion make e to be conserved . ) we note that equation ( 171 ) as well gives a correct geodesic equation in the test - particle limit , is lorentz invariant , and admits the conserved energy . these facts can be seen by the form of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a_1^i{\vert _ { { \delta _ { a\;\ln}}}}$\end{document } equation ( 175 ) ; it is zero when m1 0 , is lorentz invariant up to 3 pn order , and is the effect of the mere redefinition of the dipole moments which does not break energy conservation . we have retained during our calculation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document}-dependent terms with positive powers of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document } or logarithms of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document}. as stated below equation ( 76 ) , it is a good computational check to show that our equations of motion do not depend on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document } physically . in fact , we found that the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document}-dependent terms cancel each other out in the final result . there is no need to employ a gauge transformation to remove such an \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document } dependence . as for terms with negative powers of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document } , we simply assume that those terms cancel out the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document } dependent terms from the far zone contribution . indeed , pati and will [ 129 , 130 ] , whose method we have adopted to compute the far zone contribution , have proved that all the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal r}$\end{document}-dependent terms cancel out between the far zone and the near zone contributions through all post - newtonian orders . by comparing equation ( 181 ) with the blanchet and faye 3 pn equations of motion , we find the following relation : 184\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_1}\vec a_1^{\,{\rm{this}}\;{\rm{work } } } = { m_1}{(\vec a_1^{{\rm{bf}}})_{\lambda = - { { 1987 } \over { 3080 } } } } + { m_1}{\vec a_1}{\vert _ { { \delta _ { a\ln } } } } + \,{m_1}{\vec a_1}{\vert _ { { \delta _ { a,{\rm{bf}}}}}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${m_1}\vec a_1^{{\rm{this}}\;{\rm{work}}}$\end{document } is the 3 pn acceleration given in equation ( 181 ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${(\vec a_1^{{\rm{bf}}})_{\lambda = - 1987/3080}}$\end{document } is the blanchet and faye 3 pn acceleration with = 1987/3080 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${m_1}{\vec a_1}{\vert _ { { \delta _ { a\;\ln}}}}$\end{document } is given in equation ( 175 ) with ra replaced by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_a^{\prime}$\end{document } for notational consistency with the blanchet and faye 3 pn equations of motion shown in . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${m_1}{\vec a_1}{\vert _ { { \delta _ { a,{\rm{bf}}}}}}$\end{document } is an acceleration induced by the following dipole moments of the stars : 185\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ { a,{\rm{bf}}}^i = - { { 3709 } \over { 1260}}m_a^3a_a^i.$$\end{document } we can compute \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${m_1}a_1^i{\vert _ { { \delta _ { a,{\rm{bf}}}}}}$\end{document } by substituting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta _ { a,{\rm{bf}}}^i$\end{document } instead of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta _ { a\theta}^i$\end{document } equation ( 166 ) . thus , by choosing the dipole moments , 186\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d_{a\theta , { \rm{bf}}}^i = { \epsilon ^4}\delta _ { a\theta}^i - { \epsilon ^4}\delta _ { a,{\rm{bf}}}^i,$$\end{document } we have the 3 pn equations of motion in completely the same form as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${(\vec a_1^{{\rm{bf}}})_{\lambda = - 1987/3080}}$\end{document}. in other words , our 3 pn equations of motion physically agree with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${(\vec a_1^{{\rm{bf}}})_{\lambda = - 1987/3080}}$\end{document } modulo the definition of the dipole moments ( or equivalently , the coordinate transformation under the harmonic coordinates condition ) . in the value of that we found , = 1987/3080 , is perfectly consistent with the relation ( 1 ) and the result of ( static = 0 ) . finally , let us discuss the ambiguity in the 3 pn equations of motion previously derived by blanchet and faye in [ 25 , 27 ] . in their formalism , dirac delta distributions are used to achieve the point particle limit . the ( lorentz invariant generalized ) hadamard partie finie regularization has been extensively employed to regularize divergences caused by their use of a singular source . in fact , unless regularization is employed , divergences occur both in the evaluation of the 3 pn field where ( poisson ) integrals diverge at the location of the stars and in their derivation of the equations of motion where a substitution of the metric into a geodesic equation causes divergences . when we regularize some terms at a point , say , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } , where the terms are singular , using the hadamard partie finie regularization , roughly speaking we take an angular average of the finite part of the terms in the neighborhood of the singular point . then if there are logarithmic terms such as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\ln ( \vert \vec x - { { \vec z}_a}\vert/{r_{12}})$\end{document } , we should take an angular average over some sphere centered on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } with a finite radius . the radius of the sphere is arbitrary but we do not ignore it because we should ensure the argument of the logarithms to be dimensionless . the problem here is that there is a priori no reason to expect that the radius for each star introduced to regularize the field and another radius for that star introduced to regularize the geodesic equation coincide with each other . thus the blanchet and faye 3 pn equations of motion have four arbitrary constants instead of two in our equations of motion . in our framework , we can see the origin of the number if we assume that we have defined a different body zone \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b_a^{\prime}$\end{document } in the derivation of the equations of motion from ba used in the derivation of the 3 pn field . however , in reality , we have only one body zone for each star . in our formalism the field is expressed in terms of the four - momentum ( and multipole moments ) which are defined as volume integrals over the body zone . on the other hand our general form of the equations of motion has been derived based on the conservation law of the four - momentum , and thus we evaluate the surface integrals in the general form of the equations of motion over the boundary of the body zone . in fact , [ 25 , 27 ] have shown that two of the four arbitrary constants can be removed by using a gauge freedom remaining in the harmonic gauge condition ; the two places where the singular points exist are in some sense ambiguous . the remaining two turn out to appear as the ratios \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\zeta _ a } = r_a^\prime/{s_a}(a = 1,2)$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_a^\prime$\end{document } and sa are the four regularization parameters ( roughly speaking , the radii of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b_a^\prime$\end{document } and ba in the terminology in the previous paragraph ) . blanchet and faye [ 25 , 27 ] then proved that assuming the equations of motion are polynomials of the two masses of the stars , those two ratios should satisfy ln a = + (m1 + m2)ma where and are pure numbers . then they showed that in order for their equations of motion to admit conserved energy , then = 159/308 , while no argument was found to fix . the above argument in turn means that the blanchet and faye 3 pn equations of motion do not give a conserved energy unless \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b_a^\prime$\end{document } is different from ba . damour , jaranowski , and schfer pointed out that there is an unsatisfactory feature in the generalized partie finie regularization which contradicts with the mathematical structure of general relativity . indeed , by using dimensional regularization which is pointed out by them to be more satisfactory in this regard , derived an unambigous adm hamiltonian in the adm transeverse traceless gauge . later , blanchet et al . used dimensional regularization and found that their new equations of motion physically agree with ours and admit a conserved energy . to deal with strongly self - gravitating objects such as neutron stars , we have used the surface integral approach with the strong field point particle limit . the surface integral approach is achieved by using the local conservation of the energy momentum , which led us to the general form of the equations of motion that are expressed entirely in terms of surface integrals . the use of the strong field point particle limit and the surface integral approach makes our 3 pn equations of motion applicable to inspiraling compact binaries which consist of strongly self - gravitating regular stars ( modulo the scalings imposed on the initial hypersurface ) . our 3 pn equations of motion depend only on the masses of the stars and are independent of their internal structure such as their density profiles or radii . thus our result supports the strong equivalence principle up to 3 pn order . at 3 pn order , it does not seem possible to derive the field in a closed form . this is because not all the super - potentials required are available , and thus we could not evaluate all the poisson - type n / b integrals . some of the integrands allow us to derive super - potentials in a series form in the neighborhood of the stars . for others , we have adopted an idea that blanchet and faye have used in [ 25 , 26 , 27 ] . the idea is that while abandoning the complete derivation of the 3 pn gravitational field valid throughout n / b , one exchanges the order of integrals15 . we first evaluate the surface integrals in the evolution equation for the energy of a star and the general form of equations of motion , and then we evaluate the remaining volume integrals . using these methods , we first derived the 3 pn mass - energy relation and the momentum - velocity relation . the 3 pn mass - energy relation admits a natural interpretation . we then evaluated the surface integrals in the general form of equations of motion , and obtained the equations of motion up to 3 pn order of accuracy . at 3 pn order , our equations of motion contain logarithms of the body zone radii ra . we showed that we could remove the logarithmic terms by a suitable redefinition of the representative points of the stars . thus we could transform our 3 pn equations of motion into unambiguous equations which do not contain any arbitrarily introduced free parameters . our so - obtained 3 pn equations of motion agree physically ( modulo a definition of the representative points of the stars ) with the result derived by blanchet and faye with = 1987/3080 , which is consistent with equation ( 1 ) and static = 0 reported by damour , jaranowski , and schfer . this result indirectly supports the validity of the dimensional regularization in the adm canonical approach in the admtt gauge . blanchet and faye [ 25 , 27 ] introduced four arbitrary parameters . in the hadamard partie finie regularization , one has to introduce a sphere around each singular point ( representing a point mass ) whose radius is a free parameter . in their framework , regularizations are employed in the evaluation of both the gravitational field having two singular points and the two equations of motion . since , in their formalism , there is a priori no reason to expect that the spheres introduced for the evaluation of the field and the equations of motion coincide , there arise four arbitrary parameters . this is in contrast to our formalism where each body zone introduced in the evaluation of the field is inevitably the same as the body zone with which we defined the energy and the three - momentum of each star for which we derived our equations of motion . actually , the redefinition of the representative points in our formalism corresponds to the gauge transformation in , and only two of the four parameters remain in . then they have used one of the remaining two free parameters to ensure the energy conservation , and there remains only one arbitrary parameter which they could not fix in their formalism . on the other hand , our 3 pn equations of motion have no ambiguous parameter , admit conservation of an orbital energy of the binary system ( when we neglect the 2.5 pn radiation reaction effect ) , and respect lorentz invariance in the post - newtonian perturbative sense . we emphasize that we do not need to a posteriori adjust some parameters to make our 3 pn equations of motion to satisfy the above three physical features . we here note that blanchet et al . , who computed the 3 pn equations of motion in the harmonic gauge using the dimensional regularization , have recently obtained the same value for . the gauge condition in a harmonic gauge is related to the equations of motion . one may ask if the 3 pn equations of motion that have been derived so far guarantee the harmonic gauge condition through the corresponding post - newtonian accuracy . let us call the n pn accurate metric components to be the components that are needed to compute the n pn equations of motion . then the harmonic condition for the n pn field requires that matter obeys the n 1 pn equations of motion . thus , we need the 4 pn field to check if our resulting 3 pn equations of motion are a necessary condition to fullfil the harmonic gauge condition . the 4 pn templates may be required to detect gravitational wave directly with high signal to noise ratio and extract astrophysical information from the wave . we have to derive the 4 pn equations of motion to derive the 4 pn templates if we use the energy balance . at this moment , we should say that it is difficult to derive the 4 pn equations of motion . the technical obstacles regarding the derivation of the 4 pn equations of motion are the following . first , to derive the 4 pn equations of motion , we have to derive the 4 pn gravitational field at least in the neighborhood of a star . this requires the 3 pn gravitational field valid throughout the near zone . as seen in this article , however , it seems impossible to derive the 3 pn accurate gravitational field in harmonic coordinates in a closed form completely . we have not yet found the super - potentials necessary for us to evaluate the 3 pn poisson integrals ( or , retarded integrals ) . second , the amounts of calculations required would be too large to derive the 4 pn equations of motion successfully . the gravitational field at 1 pn order consists of about 10 terms , while the landau - lifshitz pseudotensor has about 5 terms and thus we have to handle 50 terms to derive the 1 pn equations of motion . the landau - lifshitz pseudotensor in terms of h consists of about 50 terms and thus 10 terms must be treated to derive the 2 pn equations of motion . the number of terms in the 3 pn field are of order 10 10 , while landau - lifshitz pseudotensor has about 100 terms . thus we may encounter 10 terms to derive the 3 pn equations of motion . then at the 4 pn order , we may expect 10 10 terms for the field , 500 terms for the integrand , and 10 10 terms for the equations of motion . thus the number of terms in the intermediate expressions to be dealt with are about ten - fold the number quoted above at each order16 . such an enormous number of terms forces us to use algebraic computing softwares to deal with them . in this work , we have extensively used the algebraic computing software maple , mathematica , and mathtensor to deal with tensors . however , quantity changes quality . when some equations are produced through a sequence of black - boxes ( that is , calculations done by computers ) we could not check the equations term by term even if the calculations follow trivial procedures such as taking a taylor expansion of equations around a star and extracting off some coefficients from the taylor - expanded equations . then , how could one confirm one s result ? some possible tests include the following : do the resulting equations of motion respect lorentz invariance ? do the equations of motion admit the existence of a conserved energy ? does the gravitational field ( if obtainable ) satisfy the harmonic condition ? do the results obtained by more than two groups agree with each other ? then do all these consist of a set of necessary and sufficient criteria for confirmation ? one way to derive the 4 pn equations of motion is to use brute force ( if the first difficulty how to derive the required super - potentials at 3 pn order was successfully dealt with ) . on the other hand , one may find a scaling appropriate to the late inspiralling phase and construct an approximation scheme based on such a scaling , as the post - newtonian approximation is based on the newtonian scaling . in the post - newtonian approximation , the lowest order term is just the newtonian term , and the first order term is the 1 pn correction . in the post - minkowskian approximation , ( the lowest order is just a straight line and ) the first order correction is valid for any velocity but only for a weak field . likewise , a new approximation scheme ( if any ) would give ( the lowest order of the conservative dynamics and ) the first order correction to the radiation reaction effect . such an approximation may produce a smaller number of terms than the post - newtonian approximation and thus give easier - to - treat equations of motion . also the relatively lower order equations of motion obtained by such an approximation are expected to give templates with the same accuracy as the accuracy achieved by the relatively higher order post - newtonian equations of motion . thus , such an approximation scheme is an attractive alternative to the post - newtonian approximation .

we discuss various aspects of the post - newtonian approximation in general relativity . after presenting the foundation based on the newtonian limit , we show a method to derive post - newtonian equations of motion for relativistic compact binaries based on a surface integral approach and the strong field point particle limit . as an application we derive third post - newtonian equations of motion for relativistic compact binaries which respect the lorentz invariance in the post - newtonian perturbative sense , admit a conserved energy , and are free from any ambiguity .

Introduction Foundation of the Post-Newtonian Approximation Post-Newtonian Equations of Motion for Compact Binaries Formulation Third Post-Newtonian Gravitational Field Third Post-Newtonian Mass-Energy Relation Third Post-Newtonian Momentum-Velocity Relation Third Post-Newtonian Equations of Motion

studies of the post - newtonian approximation were made by lorentz and droste , einstein , infeld , and hoffmann , fock , plebansky and bazanski , and chandrasekhar and associates [ 40 , 41 , 42 , 43 ] . now it is widely known that the post - newtonian approximation is important in analyzing a number of relativistic problems , such as the equations of motion of binary pulsars [ 34 , 61 , 74 , 89 ] , solar - system tests of general relativity [ 159 , 160 , 161 , 164 ] , and gravitational radiation reaction [ 39 , 42 ] . in the case of the post - newtonian approximation the background spacetime is also minkowski spacetime , but there is another small parameter , that is , the typical velocity of the system divided by the speed of light . in the post - newtonian approximation , the equations of general relativity take the form of newton s equations in an appropriate limit as 0 . such a limit is called the newtonian limit and it will be the basis of constructing the post - newtonian approximation . before going into the details , we mention the reason for the growth of interest in the post - newtonian approximation in recent years . certainly the discovery of the binary neutron star system psr 1913 + 16 was a strong reason to have renewed interest in the post - newtonian approximation , since it is the first system found in which general relativistic gravity plays a fundamental role in its evolution . the most systematic among those works that have succeeded in achieving higher order iteration are the ones by blanchet , damour , and iyer who have developed a scheme to calculate the waveform at a higher order , where the post - minkowskian approximation is used to construct the external field and the post - newtonian approximation is used to construct the field near the material source . in this paper , we mainly discuss the foundation of the newtonian limit and the post - newtonian equations of motion for relativistic compact binaries in an inspiralling phase . the post - newtonian equations of motion are now available in harmonic coordinates up to 3.5 pn order inclusively . the equations of motion for a two point mass binary in harmonic coordinates up to 2.5 pn order , at which the radiation reaction effect first appears , were derived by damour and deruelle [ 52 , 46 ] based on the post - minkowskian approach . the present authors and asada on the other hand derived the 2.5 pn equations of motion for point particles with arbitrarily strong internal gravity using a regular point particle limit called the strong field point particle limit . these authors also used the local conservation law but adopted a surface integral approach , and they did not specify an explicit form of the stress - energy tensor but assumed that it satisfies some scaling on the initial hypersurface . currently we have the equations of motion for relativistic compact binaries through the 3.5 pn approximation of general relativity in hand . on the other hand , blanchet and faye have tackled the derivation of the 3 pn equations of motion for two point masses expressed as two dirac delta distributions in harmonic coordinates [ 25 , 27 ] based on their previous work . their equations of motion respect the lorentz invariance in the post - newtonian pertubative sense and admits a conserved energy of orbital motion modulo the 2.5 pn radiation reaction effect . the present authors have derived the 3 pn equations of motion for relativistic compact binaries [ 91 , 92 , 93 ] in harmonic coordinates based on their previous work . this article is organized as follows : in section 2 we introduce the newtonian limit in general relativity and present how to construct the post - newtonian hierarchy . in section 3 , we discuss the basics of our derivation of the post - newtonian equations of motion for relativistic compact binaries . there we discuss how to incorporate strong internal gravity in the post - newtonian approximation . in section 4 , , we derive the newtonian and the 1 pn equations of motion in our formalism . in sections 6 and 7 , we derive the mass - energy relation and the momentum - velocity relation up to 3 pn order , and finally in section 8 , we derive the 3 pn equations of motion . the resulting equations of motion respect lorentz invariance , admit a conserved energy ( modulo the 2.5 pn radiation reaction effect ) , and are free from any ambiguity . although in the main body of this article we focus our attention on spherical massive bodies ( or point particles ) , we shall apply our formalism to extended bodies in appendix b. in appendix c , using the strong field point particle limit and the surface integral approach , we discuss that a particle with strong internal gravity moves on a geodesic of some smooth metric part which is produced by the particle itself . throughout this article since the newtonian limit is the basis of the post - newtonian approximation , we shall first formulate the newtonian limit . here we first give a general discussion on the formulation of the post - newtonian approximation independent of any initial value formalism and then present the concrete treatment in harmonic coordinates . without this linkage of the region with the expansion parameter , the post - newtonian approximation leads to divergences in the higher orders . in this paper , for our computation of the 3 pn equations of motion , we will use the will and wiseman method to solve the problem of the breakdown of the post - newtonian approximation in the near zone . at the next derivative , in this section we review briefly the strong field point particle limit , the surface integral approach for the evaluation of the gravitational force , and scalings of matter and field variables on an initial hypersurface . also we revisit some elementary but useful equations , the newtonian velocity momentum relation and the newtonian equations of motion for extended bodies . following the basic idea explained in the previous section 3 , we develop now our formalism for the derivation of the post - newtonian equations of motion suitable for relativistic compact binaries . as we have assumed in the previous section 3.3 that the magnitude of the free data of the gravitational field on the initial hypersurface is 2.5 pn order , we expect that the homogeneous solution does not affect the equations of motion up to 3 pn order . then , since all the equations are expressed with surface integrals except \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d_a^i$\end{document } to be specified , we can derive the equations of motion for a strongly self - gravitating star using the post - newtonian approximation . since we have introduced the body zone by hand , the arbitrary body zone size ra seems to appear in the metric , the multipole moments of the stars , and the equations of motion . using the above results and the newtonian equations of motion for the twice retardation expansion term , we finally obtain 130\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { h^{\tau \tau } } = 4{\epsilon ^4}\sum\limits_{a = 1,2 } { { { p_a^\tau } \over { { r_a}}}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } } \\ { + \,{\epsilon ^6 } \left [ { - 2\sum\limits_{a = 1,2 } { { { { m_a } } \over { { r_a}}}\{{{({{\vec n}_a}\cdot{{\vec v}_a})}^2 } - v_a^2\ } + 2{{{m_1}{m_2 } } \over { r_{12}^2}}{{\vec n}_{12}}\cdot({{\vec n}_1 } - { { \vec n}_2 } ) } } \right . } for instance , when we meet \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\epsilon ^2}dv_1^i / d\tau$\end{document } in the right hand side of the equations motion and we have to evaluate this up to , then using the newtonian equations of motion , we replace it by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- { \epsilon ^2}{m_2}r_{12}^i / r_{12}^3$\end{document}. in the post - newtonian approximation , we need to solve poisson equations to find the metric . then to derive the equations of motion , we basically interchange the order of operations ; first we evaluate surface integrals with the poisson integrals as integrands ( in the surface integral approach ) or compute derivatives of the poisson integrals ( when one adopts the geodesic equation ) , and then we evaluate the remaining volume integrals . in the case above , we could add , say , 1/r1 to f. ( note that to evaluate the surface integrals in the general form of equations of motion ( 111 ) we need super - potentials in the spatial region n / b which do not include any singularity due to the point particle limit . ) in the surface integral approach , we need to evaluate a surface integral of the landau - lifshitz pseudotensor which has a form h,h,. using the so - obtained ra - independent field , we evaluate the surface integrals in the general form of the 3 pn equations of motion by discarding the ra dependence emerging from the surface integrals , and obtain the equations of motion . now let us regularize this equation with the hadamard partie finie regularization ( see [ 88 , 143 ] and for example , [ 26 , 30 ] in the literature of the post - newtonian approximation ) . note that this change of the acceleration does not affect the existence of the conservation of the ( newtonian - sense ) energy , 167\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_1}a_1^i{\vert _ { { \delta _ { a\theta}}}}v_1^i + { m_2}a_2^i{\vert _ { { \delta _ { a\theta}}}}v_2^i = { \epsilon ^6}{d \over { d\tau}}\sum\limits_{a = 1,2 } { \left [ { \delta _ { a\theta}^k{{dv_a^k } \over { d\tau } } - v_a^k{d \over { d\tau}}\delta _ { a\theta}^k } \right]}.$$\end{document } to derive the 3 pn equations of motion , we evaluate the surface integrals in the general form of the equations of motion ( 111 ) using the field 8h , the field 6h , the 3 pn body zone contributions , and the 3 pn n / b contributions corresponding to the results from the super - potential method and the super - potential - in - series method . since the post - newtonian approximation is a weak field expansion , at some level of iteration in the evaluation of field , we have inevitably logarithms in the field through volume integrals such as 172\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_{n / b } { { { { d^3}y } \over { \vert \vec x - \vec y\vert } } } { \left({{m \over y } } \right)^n},$$\end{document } where m and y are typical the mass and length scale in the orbital motion , respectively , and n is a positive integer . }\\ \end{array}$$\end{document } comparing the above equations with equation ( 171 ) , we easily conclude that the representative point \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$z_a^i$\end{document } of star a defined by 176\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d_{a\theta , { \rm{new}}}^i(\tau ) = { \epsilon ^{- 6}}\int\nolimits_{{b_a } } { { d^3 } } y\,({y^i } - z_a^i(\tau))\,\theta _ n^{\tau \tau}(\tau , { y^k } ) = { \epsilon ^4}\delta _ a^i(\tau)$$\end{document } obeys the equations of motion free from any logarithmic term and hence free from any ambiguity up to 3 pn order inclusively . with the help of the formulas developed in , we have checked the lorentz invariance of equation ( 181 ) ( in the post - newtonian perturbative sense ) . on the other hand our general form of the equations of motion has been derived based on the conservation law of the four - momentum , and thus we evaluate the surface integrals in the general form of the equations of motion over the boundary of the body zone . to deal with strongly self - gravitating objects such as neutron stars , we have used the surface integral approach with the strong field point particle limit . the use of the strong field point particle limit and the surface integral approach makes our 3 pn equations of motion applicable to inspiraling compact binaries which consist of strongly self - gravitating regular stars ( modulo the scalings imposed on the initial hypersurface ) . on the other hand , our 3 pn equations of motion have no ambiguous parameter , admit conservation of an orbital energy of the binary system ( when we neglect the 2.5 pn radiation reaction effect ) , and respect lorentz invariance in the post - newtonian perturbative sense . on the other hand , one may find a scaling appropriate to the late inspiralling phase and construct an approximation scheme based on such a scaling , as the post - newtonian approximation is based on the newtonian scaling . in the post - newtonian approximation , the lowest order term is just the newtonian term , and the first order term is the 1 pn correction . such an approximation may produce a smaller number of terms than the post - newtonian approximation and thus give easier - to - treat equations of motion .

use of human donor eyes was approved by the mayo clinic ( rochester , mn , usa ) institutional review board and conformed to the ethical guidelines defined by the declaration of helsinki . ten archived normal human eyes stored in different fixative solutions were used for this study ( table ) . four eyes , mean age 58 ( range , 3373 ) , were immersion fixed in either 2% or 4% paraformaldehyde in 0.1 m phosphate buffer . six additional eyes , mean age 73 ( range , 4988 ) were perfusion fixed at 10 mm hg in either 4% paraformaldehyde , 4% paraformaldehyde/2% glutaraldehyde , or in a light fixation regimen in which 3% paraformaldehyde/0.1% triton x-100 ( 5 minutes ) was followed by 3% paraformaldehyde ( 30 minutes ) with storage in 1.5% paraformaldehyde . all perfusion fixations were preceded by two hours of initial perfusion with dulbecco 's modified eagle 's media ( dmem ; mediatech , inc . , herndon , va , usa ) at 10 mm hg to assure the anterior segment outflow channels remained open . demographics of normal human eyes anterior segments were removed from all specimens and dissected into 1-mm radial wedges preserving the complex flaps and adjacent tissue . three to seven wedges were preferentially selected from each eye by laying the 1-mm radial sections flat on a glass slide in a drop of pbs and using transillumination on a dissecting scope to visualize collector channels , septa , and other prominent outer wall features ( n = 41 ) . briefly , with the radial wedge lying flat on a wax dissecting surface , a vertical cut was made at the anterior limit of schlemm 's canal . an angular cut was made from the posterior aspect of schlemm 's canal extending to the edge of the ciliary body remnant . any connections between the inner wall and outer wall were gently severed with a beaver blade to allow complete separation of the canal . the remnant of the ciliary body was then used to transfer the specimens so the endothelial surface was left undisturbed . tissue corresponding to inner and outer walls of each wedge were rinsed in 0.1 m phosphate buffer ( ph 7.2 ) and post - fixed in a phosphate buffered 1% osmium tetroxide solution for 1 hour . the specimens were dehydrated in a graded series of acetone solutions ( 50%100% ) , critical point dried , mounted , and sputter coated with gold . specimens were examined in a jeol 6510 scanning electron microscope ( peabody , ma , usa ) at 10 to 15 kv at magnifications from 200 to 10,000 . this included orifices , which were transected in preparation or partially obstructed from view by flaps . for example , the diameter of orifices with oval shapes was measured on the long axis . a total of 52 visible orifices were examined and categorized as either simple or complex . for each orifice , the largest measureable diameter was measured using a linear distance application that is included in analyze software ( mayo clinic , rochester , mn , usa ) . the measurements of diameter were carried out on four separate occasions by one of the authors ( mb ) and on one occasion by another of the authors ( ch ) . the intraclass correlation coefficient for the intraobserver measurements was 0.96 ( 95% coefficient intervals ( ci ) : 0.940.98 ) and for interobserver measurements was 0.98 ( 95% cis : 0.960.99 ) . unpaired t - tests were performed to examine whether fixation ( immersion versus perfusion ) or type of collector channel ( simple versus complex ) were statistically different . the reproducibility of the collector channel diameter measurements between intraobserver and interobserver was investigated using intraclass correlation coefficients . this was completed both within the four measurements from the first observer , and between the observers . the range of this statistical summary is from zero to one with a zero implying no reproducibility and 1 being perfect reproducibility . use of human donor eyes was approved by the mayo clinic ( rochester , mn , usa ) institutional review board and conformed to the ethical guidelines defined by the declaration of helsinki . ten archived normal human eyes stored in different fixative solutions were used for this study ( table ) . four eyes , mean age 58 ( range , 3373 ) , were immersion fixed in either 2% or 4% paraformaldehyde in 0.1 m phosphate buffer . six additional eyes , mean age 73 ( range , 4988 ) were perfusion fixed at 10 mm hg in either 4% paraformaldehyde , 4% paraformaldehyde/2% glutaraldehyde , or in a light fixation regimen in which 3% paraformaldehyde/0.1% triton x-100 ( 5 minutes ) was followed by 3% paraformaldehyde ( 30 minutes ) with storage in 1.5% paraformaldehyde . all perfusion fixations were preceded by two hours of initial perfusion with dulbecco 's modified eagle 's media ( dmem ; mediatech , inc . , herndon , va , usa ) at 10 mm hg to assure the anterior segment outflow channels remained open . anterior segments were removed from all specimens and dissected into 1-mm radial wedges preserving the complex flaps and adjacent tissue . three to seven wedges were preferentially selected from each eye by laying the 1-mm radial sections flat on a glass slide in a drop of pbs and using transillumination on a dissecting scope to visualize collector channels , septa , and other prominent outer wall features ( n = 41 ) . briefly , with the radial wedge lying flat on a wax dissecting surface , a vertical cut was made at the anterior limit of schlemm 's canal . an angular cut was made from the posterior aspect of schlemm 's canal extending to the edge of the ciliary body remnant . any connections between the inner wall and outer wall were gently severed with a beaver blade to allow complete separation of the canal . the remnant of the ciliary body was then used to transfer the specimens so the endothelial surface was left undisturbed . tissue corresponding to inner and outer walls of each wedge were rinsed in 0.1 m phosphate buffer ( ph 7.2 ) and post - fixed in a phosphate buffered 1% osmium tetroxide solution for 1 hour . the specimens were dehydrated in a graded series of acetone solutions ( 50%100% ) , critical point dried , mounted , and sputter coated with gold . specimens were examined in a jeol 6510 scanning electron microscope ( peabody , ma , usa ) at 10 to 15 kv at magnifications from 200 to 10,000 . this included orifices , which were transected in preparation or partially obstructed from view by flaps . for example , the diameter of orifices with oval shapes was measured on the long axis . a total of 52 visible orifices were examined and categorized as either simple or complex . for each orifice , the largest measureable diameter was measured using a linear distance application that is included in analyze software ( mayo clinic , rochester , mn , usa ) . the measurements of diameter were carried out on four separate occasions by one of the authors ( mb ) and on one occasion by another of the authors ( ch ) . the intraclass correlation coefficient for the intraobserver measurements was 0.96 ( 95% coefficient intervals ( ci ) : 0.940.98 ) and for interobserver measurements was 0.98 ( 95% cis : 0.960.99 ) . unpaired t - tests were performed to examine whether fixation ( immersion versus perfusion ) or type of collector channel ( simple versus complex ) were statistically different . the reproducibility of the collector channel diameter measurements between intraobserver and interobserver was investigated using intraclass correlation coefficients . this was completed both within the four measurements from the first observer , and between the observers . the range of this statistical summary is from zero to one with a zero implying no reproducibility and 1 being perfect reproducibility . collector channel orifices were evaluated and split into two groups : simple or complex . of the 52 orifices examined , 19 ( 37% ) had simple oval openings in the outer wall of schlemm 's canal while 33 ( 63% ) were complex due to bridge - like coverings , flap - like structures , or association with septal columns . the long axis of the oval orifices corresponded to the long axis of schlemm 's canal . simple orifices had an oval appearance with a 54.7 4.6 m diameter opening ( n = 19 , range , 20232 m ) . no difference in diameter size was identified between immersion ( n = 12 ) or perfusion fixed tissue ( n = 7 ; p = 0.25 ) . in regions that were torn or cut during preparation , the orifices were lined with a single thin layer of outer wall endothelial cells that appeared narrow in width ( 510 m ) and elongated ( 50100 m ) , with slightly raised edges at the junction of adjacent cells ( fig . simple orifices were most often seen in planar regions in the middle or anterior portion of schlemm 's canal . six orifices had a thickened endothelial rim that surrounded the entire circumference at the junction with schlemm 's canal ( fig . seven orifices had a partial lip - like rim that was located opposite to a smooth sloping entrance and appeared to funnel fluid downward into the collector channel ( fig . a partially denuded simple orifice revealed an extensive branching of extracellular matrix with a dense circumferential fibrous appearance at the edge of the orifice ( fig . four of 19 simple orifices were located within 100 to 300 m of a complex orifice . various types of simple collector channel ( cc ) orifices in the outer wall of schlemm 's canal . ( a ) representative image showing a simple orifice with thickened rim surrounding entire cc opening . ( b ) transected orifice showing lip - like rim ( white arrow ) in association with a cc . complex orifices were similar in structure and size to the simple orifice with an average mean diameter of 62.7 3.4 m ( n = 33 , range , 20236 m ; p = 0.16 ) with no size difference noted between immersion ( n = 10 ) or perfusion fixed tissue ( n = 23 ; p = 0.82 ) . the complex orifices were most often located in the middle and posterior portions of schlemm 's canal . during gross dissection , large intrascleral vessels were observed posterior to schlemm 's canal within the sclera adjacent to the scleral spur . these vessels in some cases extended further posteriorly terminating on the surface of the sclera beneath the posterior portion of the ciliary body . sixteen of 33 complex collector channel orifices were elongated and were spanned by a wide bridge - like structure dividing the orifice . the bridge - like structures appeared to be covering either one or in some cases two collector channel orifices ( fig . the bridge structures were lined with outer wall endothelial cells oriented parallel to the span of the bridge crossing the orifice . the bridges had an underlying fibrous matrix core that was continuous with the underlying connective tissue . outer wall ( ow , top ) and inner wall ( iw , bottom ) are joined at corresponding oval adhesion sites ( asterisks ) . inner wall cells appeared rounded and elevated in contrast to ow cells that appeared elongated and flat . in addition to bridge - like structures , twelve complex orifices had flap - like structures ( figs . 3 , 4 ) . these flap - like structures extended between the outer and inner walls with a luminal surface lined by endothelial cells that were oriented in relation to the shape of the particular structure . various configurations of endothelial lined extensions with diameters of 10 to 20 m were present that fanned over the orifice and anchored the flaps to the inner and outer wall ( figs . the extensions and tubular - like structures could also be observed in gross dissections ( fig . 5b ) facing the transected area shown in figure 4b confirmed the presence and complexity of the flap - like structures associated with the orifices . in these sections , the connective tissue core in the flaps and extensions was continuous with the juxtacanalicular tissue . complex cc showing relationship to adjacent simple cc , flap covering orifice and intrascleral cc . ( a ) low power of ow showing complex orifice with flap ( asterisk ) and simple orifice ( arrow ) . ( b ) higher magnification of complex flap with endothelial cells covering its surface ( asterisk ) and two flap anchors extending from its edge ( arrowheads ) . flap covers entrance to cc in the sclera ( red arrow head ) and distally extends from the orifice 175 m . ( c ) side view showing the orifice neck ( red arrowheads ) and cc proper ( black arrows ) . complex flap is visible over the cc orifice in the ow with extension into the cc proper in the sclera . complex cc orifice associated with flaps . ( a ) corresponding ow ( top ) and iw ( bottom ) showing flap - structure on ow overlying orifice at the junctional region of the iws and ows ( red arrow ) . lip of the orifice ( iw panel , middle ) is delineated by three red dashes on the top and a red arrow , which points to the darkened orifice region . extensions and corresponding anchor sites on iws and ows are labeled a and b. anterior and posterior are labeled in ow and iw wedges . b showing underlying fibrous structure of anchoring tether ( t ) and tubule - like structure ( arrow ) . ( a ) gross dissection of radial wedge of anterior segment showing trabecular meshwork , iw , ow , and schlemm 's canal ( sc ) . anterior ( ant ) and posterior ( post ) are noted in the overview of the gross dissection . inset ( black square ) shows complex u - shaped flap ( cflp , black arrows ) spanning iw and ow with septal columns ( sepc , yellow arrows ) and tubule - like structure ( t , red arrow ) in the posterior portion of schlemm 's canal . ( b ) correlative plastic section of sc in same orientation as figure 5a with transected portions of complex flap ( cflp , black arrows ) on iw and ow , sepc ( yellow arrows ) on the iw and funnel - shaped attachment of tubule - like structure ( t , red arrow ) on the ow . septal columns in association with collector channels were visible in gross dissections before the separation of the inner and outer wall ( fig . the endothelial cells of the outer wall were mostly flat and elongated with the longitudinal axes of the outer wall endothelial cells frequently oriented toward the orifices , giving an impression of streaming in both simple and complex collector channels ( figs . 1b , 6a , 6b ) . in contrast , the endothelial cells of the inner wall were more rounded , slightly narrower ( 4- to 6-m wide ) , shorter in length ( 4060 m ) , and often bulging toward the lumen of schlemm 's canal ( figs . 2 , 6c ) . the junction between cells varied from slightly raised edges to grooves or fissures between the cells . the elevated cells were more numerous in areas of the inner wall directly underneath both simple and complex collector channel orifices , giving the inner wall a cobblestone appearance . inner wall endothelium formed complex folds , pockets , and clefts in areas opposite the collector channel orifices . plaque - like areas ( 50200 m ) containing fibrous material were found on corresponding inner and outer walls , most likely representing areas that were in close apposition . small delicate tubular - shaped structures were often seen in the gross dissections in the anterior and middle portion of schlemm 's canal ( fig . ( c ) inner wall cells with prominent bulging nuclei surround remnant of septal column and show cell alignment near orifice . simple orifices had an oval appearance with a 54.7 4.6 m diameter opening ( n = 19 , range , 20232 m ) . no difference in diameter size was identified between immersion ( n = 12 ) or perfusion fixed tissue ( n = 7 ; p = 0.25 ) . in regions that were torn or cut during preparation , the orifices were lined with a single thin layer of outer wall endothelial cells that appeared narrow in width ( 510 m ) and elongated ( 50100 m ) , with slightly raised edges at the junction of adjacent cells ( fig . simple orifices were most often seen in planar regions in the middle or anterior portion of schlemm 's canal . six orifices had a thickened endothelial rim that surrounded the entire circumference at the junction with schlemm 's canal ( fig . seven orifices had a partial lip - like rim that was located opposite to a smooth sloping entrance and appeared to funnel fluid downward into the collector channel ( fig . a partially denuded simple orifice revealed an extensive branching of extracellular matrix with a dense circumferential fibrous appearance at the edge of the orifice ( fig . . four of 19 simple orifices were located within 100 to 300 m of a complex orifice . various types of simple collector channel ( cc ) orifices in the outer wall of schlemm 's canal . ( a ) representative image showing a simple orifice with thickened rim surrounding entire cc opening . ( b ) transected orifice showing lip - like rim ( white arrow ) in association with a cc . complex orifices were similar in structure and size to the simple orifice with an average mean diameter of 62.7 3.4 m ( n = 33 , range , 20236 m ; p = 0.16 ) with no size difference noted between immersion ( n = 10 ) or perfusion fixed tissue ( n = 23 ; p = 0.82 ) . the complex orifices were most often located in the middle and posterior portions of schlemm 's canal . during gross dissection , large intrascleral vessels were observed posterior to schlemm 's canal within the sclera adjacent to the scleral spur . these vessels in some cases extended further posteriorly terminating on the surface of the sclera beneath the posterior portion of the ciliary body . sixteen of 33 complex collector channel orifices were elongated and were spanned by a wide bridge - like structure dividing the orifice . the bridge - like structures appeared to be covering either one or in some cases two collector channel orifices ( fig . the bridge structures were lined with outer wall endothelial cells oriented parallel to the span of the bridge crossing the orifice . the bridges had an underlying fibrous matrix core that was continuous with the underlying connective tissue . outer wall ( ow , top ) and inner wall ( iw , bottom ) are joined at corresponding oval adhesion sites ( asterisks ) . inner wall cells appeared rounded and elevated in contrast to ow cells that appeared elongated and flat . in addition to bridge - like structures , twelve complex orifices had flap - like structures ( figs . 3 , 4 ) . these flap - like structures extended between the outer and inner walls with a luminal surface lined by endothelial cells that were oriented in relation to the shape of the particular structure . various configurations of endothelial lined extensions with diameters of 10 to 20 m were present that fanned over the orifice and anchored the flaps to the inner and outer wall ( figs . 3 , 4 ) . the flap with its extensions covered not only the orifice proper ( fig . 5b ) facing the transected area shown in figure 4b confirmed the presence and complexity of the flap - like structures associated with the orifices . in these sections , the connective tissue core in the flaps and extensions was continuous with the juxtacanalicular tissue . complex cc showing relationship to adjacent simple cc , flap covering orifice and intrascleral cc . ( a ) low power of ow showing complex orifice with flap ( asterisk ) and simple orifice ( arrow ) . ( b ) higher magnification of complex flap with endothelial cells covering its surface ( asterisk ) and two flap anchors extending from its edge ( arrowheads ) . flap covers entrance to cc in the sclera ( red arrow head ) and distally extends from the orifice 175 m . ( c ) side view showing the orifice neck ( red arrowheads ) and cc proper ( black arrows ) . complex flap is visible over the cc orifice in the ow with extension into the cc proper in the sclera . ( a ) corresponding ow ( top ) and iw ( bottom ) showing flap - structure on ow overlying orifice at the junctional region of the iws and ows ( red arrow ) . lip of the orifice ( iw panel , middle ) is delineated by three red dashes on the top and a red arrow , which points to the darkened orifice region . extensions and corresponding anchor sites on iws and ows are labeled a and b. anterior and posterior are labeled in ow and iw wedges . b showing underlying fibrous structure of anchoring tether ( t ) and tubule - like structure ( arrow ) . ( a ) gross dissection of radial wedge of anterior segment showing trabecular meshwork , iw , ow , and schlemm 's canal ( sc ) . anterior ( ant ) and posterior ( post ) are noted in the overview of the gross dissection . inset ( black square ) shows complex u - shaped flap ( cflp , black arrows ) spanning iw and ow with septal columns ( sepc , yellow arrows ) and tubule - like structure ( t , red arrow ) in the posterior portion of schlemm 's canal . ( b ) correlative plastic section of sc in same orientation as figure 5a with transected portions of complex flap ( cflp , black arrows ) on iw and ow , sepc ( yellow arrows ) on the iw and funnel - shaped attachment of tubule - like structure ( t , red arrow ) on the ow . septal columns in association with collector channels were visible in gross dissections before the separation of the inner and outer wall ( fig . the endothelial cells of the outer wall were mostly flat and elongated with the longitudinal axes of the outer wall endothelial cells frequently oriented toward the orifices , giving an impression of streaming in both simple and complex collector channels ( figs . 1b , 6a , 6b ) . in contrast , the endothelial cells of the inner wall were more rounded , slightly narrower ( 4- to 6-m wide ) , shorter in length ( 4060 m ) , and often bulging toward the lumen of schlemm 's canal ( figs . 2 , 6c ) . the junction between cells varied from slightly raised edges to grooves or fissures between the cells . the elevated cells were more numerous in areas of the inner wall directly underneath both simple and complex collector channel orifices , giving the inner wall a cobblestone appearance . inner wall endothelium formed complex folds , pockets , and clefts in areas opposite the collector channel orifices . plaque - like areas ( 50200 m ) containing fibrous material were found on corresponding inner and outer walls , most likely representing areas that were in close apposition . small delicate tubular - shaped structures were often seen in the gross dissections in the anterior and middle portion of schlemm 's canal ( fig . ( c ) inner wall cells with prominent bulging nuclei surround remnant of septal column and show cell alignment near orifice . the results of the present study indicate that collector channel orifices have diverse morphologic appearances ranging from simple oval openings to complex orifice structures . complex orifices were observed frequently with bridge - like structures , tethered flaps and in association with septal columns . in addition , complex orifice flap - like structures were connected to the inner and the outer wall of schlemm 's canal . it is unlikely that the complex orifices were a result of tissue preparation because they were observed in eyes that were immersion fixed and also in eyes that were perfusion fixed . there was considerable variation in orifice diameters , which ranged between 20 and 110 m . the orifice diameters were similar in both simple and complex orifices . while single flaps and bridging sieve - like structures have been described , multiple flap - like connections to the inner and outer wall of schlemm 's canal have not been previously reported . this could be due in part to the use of routine dissection and the representative sampling techniques used for light , transmission , and scanning microscopy . in our study , collector channel regions were preferentially selected by dissection and preserved with surrounding tissue to get a 3-dimensional analysis of collector channel regions . collector channels in radial wedges greater than 50 m in size were visible in the outer wall . it is interesting to note that many collector channels associate with large intrascleral vessels or connect to ciliary vessels . it is interesting to speculate that different orifice structures ( simple versus complex ) may relate to specific connections within the venous system and indicate a systematic hierarchy of moving aqueous fluid between different vascular beds . answers to the exact functions of these complex collector channels will rely on further studies of the outflow system to definitively establish the nature of downstream connections . complex collector channels with flap - like extensions connecting the inner and outer wall may also function similar to valves found in veins and lymph vessels by limiting fluid backflow . in this regard , the flaps could potentially prevent aqueous fluid that had passed into the collector channels from returning to schlemm 's canal when pressures in schlemm 's canal are similar to episcleral venous pressure . it is known that reflux of blood occurs when the iop is lower than episcleral venous pressure in the range of 8 to 9 mm hg . when episcleral venous pressure is higher than schlemm 's canal pressure , these flaps may leak , causing blood reflux into schlemm 's canal . ramos et al . suggested that schlemm 's canal endothelium share many morphologic and functional characteristics with endothelium of both blood and lymphatic vessels . work has shown the presence of prospero homeobox protein 1 ( prox1 ) , a protein responsible for regulating lymphatic development , and forkhead transcription factor foxc2 , a lymphatic valve marker protein , in schlemm 's canal endothelial cells of lymphatic and blood vasculature reporter mice . additionally , park et al . found that schlemm 's canal cells also expressed integrin 9 and had vascular endothelium - cadherin ( ve - cadherin ) containing junctions similar to collecting lymphatics . these findings along with new work demonstrating the responsiveness of schlemm 's canal development in mouse , zebrafish , and human eye tissue to vegf - c , a lymphangiogenic growth factor , suggests that schlemm 's canal has lymphatic characteristics and may function as a rudimentary lymphatic vessel . li et al . using a primate model system and phase sensitive optical coherence tomography ( phs - oct ) have observed that the trabecular meshwork and schlemm 's canal are sensitive to pulse - induced movement . using phs - oct with defined parameters of pressure and pulsed - flow demonstrated dilation of schlemm 's canal , distension of trabecular meshwork , and increased visibility of collagenous structures within the canal and at collector channel ostia . these observations suggest that a mechanism , similar to the valve - like function proposed here for complex flaps , may be necessary to keep orifices patent by being readily deformable to compensate for rapid pressure changes as found during head movement , blinking , and sneezing , and may also function in response to transient movement in normal iop . in the present study , we observed adhesion areas and septal columns where the inner and outer walls were in close contact . the septal columns maintained schlemm 's canal patency especially in the vicinity of collector channel orifices , preventing herniation of the inner wall into the collector channel . smit and johnstone suggested that extensions bridging the inner and outer wall may serve as tethers that maintain the shape of schlemm 's canal . the flap - like extensions of complex collector channels may relay extracellular matrix tension or stiffness through schlemm 's canal endothelial cells by coupling the matrix of the inner and outer wall . this could occur via the elastin fiber system , which connects to the actin cytoskeleton of schlemm 's canal cells through connecting fibrils . regions where the endothelium was disrupted or missing on flaps or orifices , revealed a matrix with a mesh pattern similar to that reported for the elastin fiber system of rat venules . the appearance of the extracellular mesh was also similar to that observed by scanning electron microscopy in preparations from our laboratory where the inner wall endothelium of schlemm 's canal was removed in anterior segment culture with varying concentrations of ethacrynic acid . additional hints of coupling that may relay matrix tension or stiffness was observed in the mirror alignment of outer wall cells in regions of collector channels , which are contiguous in flaps , septums , and tubule - like structures . the alignment of endothelial cells was apparent in our study with outer wall endothelial cells aligned with one another toward collector channel orifices . its associated pressure gradient and the alignment of f - actin in the endothelial cells appears to provide critical mechanical reinforcement as noted by ethier et al . notably the actin cytoskeleton is linked to the elastin system , which may act in concert to sense changes in the extracellular matrix environment along with integrins and other signaling proteins . variations in collector channel orifice structure imply a possible regulatory function through linkage to the distal outflow system . in addition , the presence of different types of collector channels may compensate for changing conditions in fluid flow and pressure dynamics adjacent to the orifices and in schlemm 's canal at large . in aging or disease , if one collector channel orifice becomes blocked or a region of schlemm 's canal becomes adherent , other collector channels would be available . taken together , the anatomical structures identified in this study indicate a possible regulatory role in fluid dynamics by schlemm 's canal by virtue of collector channels and their associated structural entities such as flap - like structures , septa , and tubule - like structures . further studies are warranted to explore the distal outflow pathway , its structures and its role in aqueous humor dynamics .

purposeto examine the anatomical variation of normal human collector channel orifices and their relationship with schlemm 's canal.methodsten human anterior segments fixed by immersion or perfusion were dissected radially and further divided by fine dissection into corresponding inner and outer wall segments . the tissues were dehydrated , critical - point dried , sputter coated , and examined by scanning electron microscopy . images were obtained at magnifications from 200 to 10,000 . selected radial collector channel regions were processed for plastic embedding.resultstwo classes of collector channel orifices were identified . simple oval orifices ( 54.7 4.6m diameter ) were lined with endothelial cells and most often occurred on a planar region of schlemm 's canal outer wall . complex orifices ( 62.7 3.4m diameter ) were often found associated with septal columns and bridges , and typically covered with flap - like structures ( 1040 m ) that extended between the inner and outer wall and over the collector channel orifices . both simple and complex orifices had complete or partial lip - like rims . in orifices with partial rims , a trough - like groove was often visible on the outer wall surface opposite the lip . transected septa and inner and outer wall adhesion sites were often found in association with complex collector channel orifices.conclusionscollector channel orifice structure varied from simple ovals to complex tethered flaps and bridges . collector channel orifices with complex flaps connect the inner and outer walls of schlemm 's canal , and may serve to enhance and regulate aqueous outflow in these regions .

Methods Human Eyes Tissue Dissection Scanning Electron Microscopy Orifice Analysis Correlative Light Microscopy Statistical Analysis Results Simple Collector Channel Orifices Complex Collector Channel Orifices Schlemm's Canal in Region of Collector Channel Orifices Discussion

demographics of normal human eyes anterior segments were removed from all specimens and dissected into 1-mm radial wedges preserving the complex flaps and adjacent tissue . three to seven wedges were preferentially selected from each eye by laying the 1-mm radial sections flat on a glass slide in a drop of pbs and using transillumination on a dissecting scope to visualize collector channels , septa , and other prominent outer wall features ( n = 41 ) . briefly , with the radial wedge lying flat on a wax dissecting surface , a vertical cut was made at the anterior limit of schlemm 's canal . an angular cut was made from the posterior aspect of schlemm 's canal extending to the edge of the ciliary body remnant . any connections between the inner wall and outer wall were gently severed with a beaver blade to allow complete separation of the canal . tissue corresponding to inner and outer walls of each wedge were rinsed in 0.1 m phosphate buffer ( ph 7.2 ) and post - fixed in a phosphate buffered 1% osmium tetroxide solution for 1 hour . the specimens were dehydrated in a graded series of acetone solutions ( 50%100% ) , critical point dried , mounted , and sputter coated with gold . specimens were examined in a jeol 6510 scanning electron microscope ( peabody , ma , usa ) at 10 to 15 kv at magnifications from 200 to 10,000 . for example , the diameter of orifices with oval shapes was measured on the long axis . unpaired t - tests were performed to examine whether fixation ( immersion versus perfusion ) or type of collector channel ( simple versus complex ) were statistically different . the reproducibility of the collector channel diameter measurements between intraobserver and interobserver was investigated using intraclass correlation coefficients . this was completed both within the four measurements from the first observer , and between the observers . anterior segments were removed from all specimens and dissected into 1-mm radial wedges preserving the complex flaps and adjacent tissue . three to seven wedges were preferentially selected from each eye by laying the 1-mm radial sections flat on a glass slide in a drop of pbs and using transillumination on a dissecting scope to visualize collector channels , septa , and other prominent outer wall features ( n = 41 ) . briefly , with the radial wedge lying flat on a wax dissecting surface , a vertical cut was made at the anterior limit of schlemm 's canal . an angular cut was made from the posterior aspect of schlemm 's canal extending to the edge of the ciliary body remnant . any connections between the inner wall and outer wall were gently severed with a beaver blade to allow complete separation of the canal . tissue corresponding to inner and outer walls of each wedge were rinsed in 0.1 m phosphate buffer ( ph 7.2 ) and post - fixed in a phosphate buffered 1% osmium tetroxide solution for 1 hour . the specimens were dehydrated in a graded series of acetone solutions ( 50%100% ) , critical point dried , mounted , and sputter coated with gold . specimens were examined in a jeol 6510 scanning electron microscope ( peabody , ma , usa ) at 10 to 15 kv at magnifications from 200 to 10,000 . for example , the diameter of orifices with oval shapes was measured on the long axis . unpaired t - tests were performed to examine whether fixation ( immersion versus perfusion ) or type of collector channel ( simple versus complex ) were statistically different . the reproducibility of the collector channel diameter measurements between intraobserver and interobserver was investigated using intraclass correlation coefficients . this was completed both within the four measurements from the first observer , and between the observers . collector channel orifices were evaluated and split into two groups : simple or complex . of the 52 orifices examined , 19 ( 37% ) had simple oval openings in the outer wall of schlemm 's canal while 33 ( 63% ) were complex due to bridge - like coverings , flap - like structures , or association with septal columns . the long axis of the oval orifices corresponded to the long axis of schlemm 's canal . simple orifices had an oval appearance with a 54.7 4.6 m diameter opening ( n = 19 , range , 20232 m ) . in regions that were torn or cut during preparation , the orifices were lined with a single thin layer of outer wall endothelial cells that appeared narrow in width ( 510 m ) and elongated ( 50100 m ) , with slightly raised edges at the junction of adjacent cells ( fig . simple orifices were most often seen in planar regions in the middle or anterior portion of schlemm 's canal . six orifices had a thickened endothelial rim that surrounded the entire circumference at the junction with schlemm 's canal ( fig . seven orifices had a partial lip - like rim that was located opposite to a smooth sloping entrance and appeared to funnel fluid downward into the collector channel ( fig . various types of simple collector channel ( cc ) orifices in the outer wall of schlemm 's canal . ( b ) transected orifice showing lip - like rim ( white arrow ) in association with a cc . complex orifices were similar in structure and size to the simple orifice with an average mean diameter of 62.7 3.4 m ( n = 33 , range , 20236 m ; p = 0.16 ) with no size difference noted between immersion ( n = 10 ) or perfusion fixed tissue ( n = 23 ; p = 0.82 ) . the complex orifices were most often located in the middle and posterior portions of schlemm 's canal . during gross dissection , large intrascleral vessels were observed posterior to schlemm 's canal within the sclera adjacent to the scleral spur . sixteen of 33 complex collector channel orifices were elongated and were spanned by a wide bridge - like structure dividing the orifice . the bridge - like structures appeared to be covering either one or in some cases two collector channel orifices ( fig . the bridge structures were lined with outer wall endothelial cells oriented parallel to the span of the bridge crossing the orifice . outer wall ( ow , top ) and inner wall ( iw , bottom ) are joined at corresponding oval adhesion sites ( asterisks ) . in addition to bridge - like structures , twelve complex orifices had flap - like structures ( figs . these flap - like structures extended between the outer and inner walls with a luminal surface lined by endothelial cells that were oriented in relation to the shape of the particular structure . various configurations of endothelial lined extensions with diameters of 10 to 20 m were present that fanned over the orifice and anchored the flaps to the inner and outer wall ( figs . the extensions and tubular - like structures could also be observed in gross dissections ( fig . 5b ) facing the transected area shown in figure 4b confirmed the presence and complexity of the flap - like structures associated with the orifices . in these sections , the connective tissue core in the flaps and extensions was continuous with the juxtacanalicular tissue . ( b ) higher magnification of complex flap with endothelial cells covering its surface ( asterisk ) and two flap anchors extending from its edge ( arrowheads ) . ( a ) corresponding ow ( top ) and iw ( bottom ) showing flap - structure on ow overlying orifice at the junctional region of the iws and ows ( red arrow ) . ( a ) gross dissection of radial wedge of anterior segment showing trabecular meshwork , iw , ow , and schlemm 's canal ( sc ) . inset ( black square ) shows complex u - shaped flap ( cflp , black arrows ) spanning iw and ow with septal columns ( sepc , yellow arrows ) and tubule - like structure ( t , red arrow ) in the posterior portion of schlemm 's canal . septal columns in association with collector channels were visible in gross dissections before the separation of the inner and outer wall ( fig . the endothelial cells of the outer wall were mostly flat and elongated with the longitudinal axes of the outer wall endothelial cells frequently oriented toward the orifices , giving an impression of streaming in both simple and complex collector channels ( figs . in contrast , the endothelial cells of the inner wall were more rounded , slightly narrower ( 4- to 6-m wide ) , shorter in length ( 4060 m ) , and often bulging toward the lumen of schlemm 's canal ( figs . the junction between cells varied from slightly raised edges to grooves or fissures between the cells . the elevated cells were more numerous in areas of the inner wall directly underneath both simple and complex collector channel orifices , giving the inner wall a cobblestone appearance . inner wall endothelium formed complex folds , pockets , and clefts in areas opposite the collector channel orifices . plaque - like areas ( 50200 m ) containing fibrous material were found on corresponding inner and outer walls , most likely representing areas that were in close apposition . small delicate tubular - shaped structures were often seen in the gross dissections in the anterior and middle portion of schlemm 's canal ( fig . simple orifices had an oval appearance with a 54.7 4.6 m diameter opening ( n = 19 , range , 20232 m ) . in regions that were torn or cut during preparation , the orifices were lined with a single thin layer of outer wall endothelial cells that appeared narrow in width ( 510 m ) and elongated ( 50100 m ) , with slightly raised edges at the junction of adjacent cells ( fig . simple orifices were most often seen in planar regions in the middle or anterior portion of schlemm 's canal . six orifices had a thickened endothelial rim that surrounded the entire circumference at the junction with schlemm 's canal ( fig . seven orifices had a partial lip - like rim that was located opposite to a smooth sloping entrance and appeared to funnel fluid downward into the collector channel ( fig . various types of simple collector channel ( cc ) orifices in the outer wall of schlemm 's canal . ( b ) transected orifice showing lip - like rim ( white arrow ) in association with a cc . complex orifices were similar in structure and size to the simple orifice with an average mean diameter of 62.7 3.4 m ( n = 33 , range , 20236 m ; p = 0.16 ) with no size difference noted between immersion ( n = 10 ) or perfusion fixed tissue ( n = 23 ; p = 0.82 ) . the complex orifices were most often located in the middle and posterior portions of schlemm 's canal . during gross dissection , large intrascleral vessels were observed posterior to schlemm 's canal within the sclera adjacent to the scleral spur . sixteen of 33 complex collector channel orifices were elongated and were spanned by a wide bridge - like structure dividing the orifice . the bridge - like structures appeared to be covering either one or in some cases two collector channel orifices ( fig . the bridge structures were lined with outer wall endothelial cells oriented parallel to the span of the bridge crossing the orifice . outer wall ( ow , top ) and inner wall ( iw , bottom ) are joined at corresponding oval adhesion sites ( asterisks ) . in addition to bridge - like structures , twelve complex orifices had flap - like structures ( figs . these flap - like structures extended between the outer and inner walls with a luminal surface lined by endothelial cells that were oriented in relation to the shape of the particular structure . various configurations of endothelial lined extensions with diameters of 10 to 20 m were present that fanned over the orifice and anchored the flaps to the inner and outer wall ( figs . 5b ) facing the transected area shown in figure 4b confirmed the presence and complexity of the flap - like structures associated with the orifices . in these sections , the connective tissue core in the flaps and extensions was continuous with the juxtacanalicular tissue . ( a ) corresponding ow ( top ) and iw ( bottom ) showing flap - structure on ow overlying orifice at the junctional region of the iws and ows ( red arrow ) . ( a ) gross dissection of radial wedge of anterior segment showing trabecular meshwork , iw , ow , and schlemm 's canal ( sc ) . inset ( black square ) shows complex u - shaped flap ( cflp , black arrows ) spanning iw and ow with septal columns ( sepc , yellow arrows ) and tubule - like structure ( t , red arrow ) in the posterior portion of schlemm 's canal . septal columns in association with collector channels were visible in gross dissections before the separation of the inner and outer wall ( fig . the endothelial cells of the outer wall were mostly flat and elongated with the longitudinal axes of the outer wall endothelial cells frequently oriented toward the orifices , giving an impression of streaming in both simple and complex collector channels ( figs . in contrast , the endothelial cells of the inner wall were more rounded , slightly narrower ( 4- to 6-m wide ) , shorter in length ( 4060 m ) , and often bulging toward the lumen of schlemm 's canal ( figs . the junction between cells varied from slightly raised edges to grooves or fissures between the cells . the elevated cells were more numerous in areas of the inner wall directly underneath both simple and complex collector channel orifices , giving the inner wall a cobblestone appearance . inner wall endothelium formed complex folds , pockets , and clefts in areas opposite the collector channel orifices . plaque - like areas ( 50200 m ) containing fibrous material were found on corresponding inner and outer walls , most likely representing areas that were in close apposition . small delicate tubular - shaped structures were often seen in the gross dissections in the anterior and middle portion of schlemm 's canal ( fig . the results of the present study indicate that collector channel orifices have diverse morphologic appearances ranging from simple oval openings to complex orifice structures . complex orifices were observed frequently with bridge - like structures , tethered flaps and in association with septal columns . in addition , complex orifice flap - like structures were connected to the inner and the outer wall of schlemm 's canal . it is unlikely that the complex orifices were a result of tissue preparation because they were observed in eyes that were immersion fixed and also in eyes that were perfusion fixed . the orifice diameters were similar in both simple and complex orifices . while single flaps and bridging sieve - like structures have been described , multiple flap - like connections to the inner and outer wall of schlemm 's canal have not been previously reported . in our study , collector channel regions were preferentially selected by dissection and preserved with surrounding tissue to get a 3-dimensional analysis of collector channel regions . collector channels in radial wedges greater than 50 m in size were visible in the outer wall . complex collector channels with flap - like extensions connecting the inner and outer wall may also function similar to valves found in veins and lymph vessels by limiting fluid backflow . in this regard , the flaps could potentially prevent aqueous fluid that had passed into the collector channels from returning to schlemm 's canal when pressures in schlemm 's canal are similar to episcleral venous pressure . work has shown the presence of prospero homeobox protein 1 ( prox1 ) , a protein responsible for regulating lymphatic development , and forkhead transcription factor foxc2 , a lymphatic valve marker protein , in schlemm 's canal endothelial cells of lymphatic and blood vasculature reporter mice . found that schlemm 's canal cells also expressed integrin 9 and had vascular endothelium - cadherin ( ve - cadherin ) containing junctions similar to collecting lymphatics . these findings along with new work demonstrating the responsiveness of schlemm 's canal development in mouse , zebrafish , and human eye tissue to vegf - c , a lymphangiogenic growth factor , suggests that schlemm 's canal has lymphatic characteristics and may function as a rudimentary lymphatic vessel . using a primate model system and phase sensitive optical coherence tomography ( phs - oct ) have observed that the trabecular meshwork and schlemm 's canal are sensitive to pulse - induced movement . using phs - oct with defined parameters of pressure and pulsed - flow demonstrated dilation of schlemm 's canal , distension of trabecular meshwork , and increased visibility of collagenous structures within the canal and at collector channel ostia . these observations suggest that a mechanism , similar to the valve - like function proposed here for complex flaps , may be necessary to keep orifices patent by being readily deformable to compensate for rapid pressure changes as found during head movement , blinking , and sneezing , and may also function in response to transient movement in normal iop . in the present study , we observed adhesion areas and septal columns where the inner and outer walls were in close contact . the septal columns maintained schlemm 's canal patency especially in the vicinity of collector channel orifices , preventing herniation of the inner wall into the collector channel . smit and johnstone suggested that extensions bridging the inner and outer wall may serve as tethers that maintain the shape of schlemm 's canal . the flap - like extensions of complex collector channels may relay extracellular matrix tension or stiffness through schlemm 's canal endothelial cells by coupling the matrix of the inner and outer wall . this could occur via the elastin fiber system , which connects to the actin cytoskeleton of schlemm 's canal cells through connecting fibrils . the appearance of the extracellular mesh was also similar to that observed by scanning electron microscopy in preparations from our laboratory where the inner wall endothelium of schlemm 's canal was removed in anterior segment culture with varying concentrations of ethacrynic acid . additional hints of coupling that may relay matrix tension or stiffness was observed in the mirror alignment of outer wall cells in regions of collector channels , which are contiguous in flaps , septums , and tubule - like structures . the alignment of endothelial cells was apparent in our study with outer wall endothelial cells aligned with one another toward collector channel orifices . variations in collector channel orifice structure imply a possible regulatory function through linkage to the distal outflow system . in addition , the presence of different types of collector channels may compensate for changing conditions in fluid flow and pressure dynamics adjacent to the orifices and in schlemm 's canal at large . in aging or disease , if one collector channel orifice becomes blocked or a region of schlemm 's canal becomes adherent , other collector channels would be available . taken together , the anatomical structures identified in this study indicate a possible regulatory role in fluid dynamics by schlemm 's canal by virtue of collector channels and their associated structural entities such as flap - like structures , septa , and tubule - like structures .

molecular oxygen is found only in those biotopes that harbor organisms carrying out oxygenic photosynthesis . in oxygen - deficient systems , the nature of the redox couple and concentrations of electron acceptor / donor determine the succession of dissimilatory metabolisms due to thermodynamic conditions . for a given substrate and under standard conditions , the aerobic dissimilatory metabolisms provide about one order of magnitude more energy than the anaerobic ones , for example , glucose respiration ( g = 2877 kj / mol ) versus glucose fermentation ( g = 197 kj / mol ) . therefore , in sedimentary environments oxygen is exhausted at deeper layers and the dissimilatory metabolisms are anaerobic as a result . anaerobic microorganisms are of interest in extreme environments because environmental parameters such as temperature and salinity regulate the rates of organic matter remineralization . interestingly , the low activity of water and the expense on biosynthesis only select heterotrophs and strict aerobes as extreme halophiles . however , some moderate halophilic and strict anaerobes have been described ; one example is the methanogen methanohalobium evestigatum , which uses methylated compounds ( e.g. , methylamine and methanol ) to generate methane . methylated substrates yield more energy ( g = 78.7 to 191.1 kj per mol substrate ) than h2/co2 ( g = 34 kj / mol substrate ) or acetate ( g = 31 kj mol substrate ) and allow that methylotrophic methanogens such as m. evestigatum can tolerate up to 29.2% nacl . differences in bioenergetic yield determine an exclusion of hydrogenotrophic methanogens such as methanocalculus halotolerans , which tolerates a lesser salinity : up to 12% nacl . a similar pattern has been described for sulfate - reducing prokaryotes : acetoclastic sulfate reducers ( g = 47.6 kj mol substrate ) , most of them belonging to desulfobacteraceae , cease to tolerate high osmolarity conditions , for example , desulfobacter halotolerans grows up to 13% nacl ; on the other hand , desulfohalobiaceae members have higher salt tolerances ( up to 25% nacl ) and grow with h2/co2 ( g = 152.2 kj mol ) or lactate ( g = 160 kj / mol ) . to define whether extremes of salinity are relevant in composition and persistence of anaerobic ecotypes , the ephemeral systems and spatial gradients constitute appropriate sites of study . even though there are some studies about microbial communities present along salt gradients , those approaches have been performed on thalassic microbial mats ; therefore , they are depleted in sulfate at deep layers , but most of them are also formed only on intertidal zones . sulfate is the second most abundant electron acceptor on earth and consequently the dominant electron acceptor for anaerobic metabolism in marine sediments . one interesting ephemeral and sulfate - rich system is tirez lagoon , or sabkha , because it is subjected to flooding / desiccation regimes , located in la mancha , an arid region in spain . tirez lagoon is athalassic since the ionic composition is far from seawater and it is characterized by a low cl : so4 ratio ( about 1.18 in flooded season and 0.35 in the dry season ) , whilst in the dead sea this ratio is above 10 . this system is maintained at a neutral ph due to a high mg and ca concentration in combination with a low co3 content at the saltern and sediment environments . the traces of co3 are removed as dolomite ( camg(co3)2 ) preventing alkalinization . given this scenario , the primary objective of this study was to characterize the composition of the anaerobic populations in the ephemeral and sulfate rich tirez lagoon . the identification of environmental sulfate reducing prokaryotes ( srp ) and sulfur oxidizing prokaryotes ( sop ) can be performed by enrichment culturing and molecular ecology fingerprinting ; however , a characterization of methanogenic archaea ( ma ) through isolation techniques is problematic given their slow growth rates . the use of molecular ecology fingerprinting techniques such as denaturing gradient gel electrophoresis ( dgge ) from pcr - amplified genes is informative to assess the temporal and spatial qualitative diversity in natural samples , and it also requires fewer sequencing resources in comparison to clone libraries and/or metagenomic analysis . instead of the 16s rrna gene , the use of dgge from pcr - amplified functional gene markers is profitable to elucidate the composition of the anaerobic pathways of sulfate respiration ( sr ) , sulfur oxidation ( so ) , and methanogenesis ( mt ) . the 16s rrna gene - based analysis can not provide an unambiguous link between gene sequences and its physiological or metabolic role . whereas the srp and sop organisms are phylogenetically and physiologically disperse along the bacteria and archaea domains , ma organisms are monophyletic restricted to archaea . in the dissimilatory pathways of sulfate reduction and sulfur oxidation , dissimilatory sulfite reductase ( dsr ) and adenosine-5-phosphosulfate ( aps ) reductase ( apr ) are considered as key enzymes . in the sulfate - reducing pathway , sulfate has to be activated to aps by atp - sulfurylase ( ec : 2.7.7.4 ) at the expense of atp ; apr ( ec : 1.8.99.2 ) converts the aps to sulfite and amp ; hereafter , sulfite is reduced to sulfide by dsr ( ec : 1.8.7.1 ) . for the sulfur - oxidizing pathway the alpha subunits of apr and dsr enzymes are found in all known srp and most of sop . regarding the methanogenesis pathway , the methyl coenzyme - m reductase ( mcr ) ( ec : 2.8.4.1 ) catalyses the reduction of a methyl group bound to coenzyme - m , with the concomitant release of methane . mcra gene fragment provides more information than the 16s rrna gene ; even if the saturation rates are similar between the mcra gene fragment and the complete 16s rrna gene , the number of differences per site in the mcra fragment is 2 - 3 times higher than that in the full - length 16s rrs . therefore , assignment of genera with mcra sequences offers more conclusive resolution than assignment with 16s rrna gene sequences . the mutation rates and selective pressures of the apra and mcra metabolic gene markers and of the structural 16s rrna gene are different ; however , phylogenetic studies done with partial sequences of apra and mcra belonging to the srp , sop , and ma lineages have established an agreement with the phylogenetic relationships based on 16s rrna gene sequences [ 13 , 18 ] . therefore , these functional gene markers can provide an estimate of the sr , so , and mt microbial diversity harbored in sediments of tirez lagoon . indeed , databases have been enriched in sequences of model strains for these two enzymes ; as a consequence , the apra and mcra gene markers also provide us information to identify srp , sop , and ma selectively in complex microbial communities , for example , . the second aim of this study was to investigate whether the composition and distribution of the encoded amino acids in apra and mcra genes are indicative of haloadaptation to the hypersaline sediment . diverse lines of evidence report that halophilic microorganisms can bias their amino acid composition to deal with the multimolar salinities of their environment [ 21 , 22 ] . halophiles , where turgidity is maintained by the intracellular accumulation of k that is usually equilibrated with the presence of extracellular cl . therefore , an increase of the acidic nature of cytoplasmic proteins , which is offset by an overall decrease in basic amino acids , is needed to maintain an appropriate folding and functionality under osmotic stress [ 22 , 24 ] . in cytoplasmic proteins , it has been also pointed out a slight decrease in hydrophobicity as another amino acid haloadaptation [ 25 , 26 ] . halophiles build up concentrations of osmolytes ( also named osmoprotectants or compatible solutes ) to increase the intracellular osmolarity ; thereby maintaining the protein native states in spite of a highest energetic cost to manufacture the organic molecules . accordingly , only proteins in salt - out organisms exposed directly to the hypersaline medium exhibit an excess of acidic amino acids . all eukaryotes , most halophilic bacteria , and the halophilic methanogenic archaea ( such as methanohalobium evestigatum ) have evolved the the widely disparate taxonomic position of salt - in prokaryotes ( halobacteriales in archaea , salinibacter ruber , and halanaerobiales in bacteria ) suggests a convergent evolution of this osmoadaptation strategy . several studies have also reported that a high genomic cg content ( often upwards of 60% ) and a gc bias at the codon usage level are common adaptations to hypersaline environments , presumably to avoid uv - induced thymidine dimer formation and accumulation of mutations [ 21 , 23 ] . for example , the high gc composition ( 65.9% ) of halobacterium sp . nrc1 could reduce the chance of such lesions and its third position gc bias correlates with an overrepresentation of acidic residues ( i.e. , asp and glu ) . the unique exception to this general trend has been pointed out so far in another extreme halophile haloquadratum walsbyi shows a remarkably low genomic gc content ( 47.9% ) and a weak gc - bias at the codon usage level . given that other specific features of nucleotide selection may also be involved in the gc content of organisms , the gc - bias measurements are complementary to the amino acid composition but not decisive in order to infer the salt - in strategy . therefore , the findings of this study try to contribute to the knowledge of diversity and haloadaptation of the srp and mt thriving at rich sulfate sediment . additionally , tirez system is analog to the ocean of europa , satellite of jupiter , due to its sulfate abundance and neutral ph , and sulfates have been detected on mars indeed . thus , this knowledge will provide insight regarding the possible biological limits for life in other analogous places . sediment samples were collected from tirez lagoon , which has an area of < 1 km and it was originated after endorheic inflow under semiarid conditions . the lagoon is located in the southern subplateau of the iberian region of la mancha ( 39 32 42 n y 03 21 o ) . the salt content fluctuates from 6% ( w / v ) during winter to 35% ( w / v ) during spring . in summer , the system becomes an evaporite . temperature oscillation is about 40c and 7c , the mean annual thermal oscillation is 55c , and the annual mean rainfall is averaged at 400 mm . water drains through material from the triassic period ; dolomites and ca - sulfate marls are from the tertiary period . samples were collected in february and july 2005 and correspond to the winter and summer seasons , respectively . the winter and summer samplings were done by triplicate in three points at the lagoon ; all of them were located in the salt pan or lagoon basin because it is the region covered by salts in summer . the sample cores were obtained from sites separated from each other by several meters . in order to analyze seasonal changes in the lagoon , the summer samples were obtained from the holes - signals leaved by the winter ( flooded ) sampling . sediment cores were obtained with a ring kit core - sampler for soft soil to a depth of 40 cm . the sampled cores were cooled at 20c with jelly bags and kept until further processing . . eh and ph of the cores were measured with a probe connected to a potentiometer orion model 290a + thermo orion ( thermo fisher scientific ) . also , dissolved oxygen and temperature were measured with a sylant simplair f-15 oxymeter ( syland scientific gmbh ) . in order to determine the interstitial sulfide concentration , the sample cores from surface to 20 cm in depth were sonicated for 5 min ( labsonic b. braun sonicator ) . after centrifugation ( 14,000 rpm 10 min sorvall rc-5 ) , supernatants were mixed with zn acetate ( 2% ) and sulfide concentration was determined using the methylene blue method . the core samples used to analyze ion content ( cl , so4 , and nh4 ) and carbon : nitrogen ( c : n ) ratios were sampled from the surface to 20 cm in depth ; samples were dehydrated at 110c for 12 hrs for ionic chromatography and elemental analysis . ionic chromatography analysis was completed with 100 mg of pulverized samples diluted in 25 ml of filtered milliq water , whilst the elemental analysis was performed with dried and pulverized sediment . the sediments were assayed by chromatographic methods with an ic dionex dx-600 chromatograph and by spectrophotometric methods with a leco chns-932 elemental analyzer at the servicio interdepartamental de investigacin ( uam ) . srp organisms from winter samples were grown in a cysteine - reduced ( 4.12 mm ) medium for sulfate reducers , modified from raskin et al . , and contained glutamic acid ( 5.2 mm ) , glycine ( 0.2 mm ) , methanol ( 14 mm ) , methylamine ( 27 mm ) , peptone ( 250 mg / l ) , and yeast extract ( 250 mg / l ) . the salt content in sulfate - reducing media was 3.5% the tirez saltern . the inoculation was done with sedimentary slurry from samples collected in february 2005 ( winter season ) . cell culture growth was monitored through the count of the cell density with 4,6-diamino-2-phenylindole ( dapi ) , molecular probes ( invitrogen ) in a zeiss axiovert 200 m fluorescent microscope . the three cores with a weight of ~210 g were mixed with three volumes of pbs 1x at 4c to reduce microheterogeneities and to wash salts . integrity of bacterial cells after the treatment was confirmed by 4,6-diamino-2-phenylindole ( dapi ) 1 total genomic dna was extracted from supernatants of washed and centrifuged sediments ( 500 rpm for 1 min hettich mikro 22 r centrifuge to precipitate rocks ) . in order to collect cells from nonaxenic cultures , 100 ml of the samples were filtered onto 0.22 m of polycarbonate filters ( millipore ) . sediment and soil samples are characterized by the presence of inhibitors such as humic acids and exopolimeric substances , thus we used a specialized dna extraction kit ( fast dna spin kit for soil ) ( qbiogene , irving , calif , usa ) which has proved to retrieve a reliable dna extraction to obtain a broad and intense band patterns , in comparison with variants via phenol dna extraction , and it has been used for analyses of microbial diversity by dgge in sediments , for example , . total genomic dna was purified according to genomic dna purification jetquick kit ( genomed ) instructions . pcr reactions were performed in a mixture of 50 l containing : 2 l of template dna , 1 mm dntp 's , 0.5 m of each primer , 3 mm mgcl2 , 1x enzyme buffer , and 0.03 u/l amplitaq dna polymerase ( roche , molecular systems ) . the apra gene fragment of ~0.4 kb was amplified with the apsfw ( tggcagatmatgatymacggg with a gc clamp ) and apsrv ( gggccgtaaccgtccttgaa ) primer pairs . the following conditions were implemented : a first denaturing step at 94c for 3 min , the completion of 35 cycles of 30 s at 94c , an annealing at 60c for 55 s and at 72c for 1 min , and a final extension of 72c for 7 min . in the mcra gene fragment amplification , at first a 0.76 kb fragment was amplified with the primers me1 ( gcmatgcarathggwatgtc ) and me2 ( tcatkgcrtagttdggrtagt ) . the me - pcr reaction was carried out with an initial denaturing step at 94c ( 5 min ) , followed by 25 cycles of 1 min at 94c , an annealing at 57c for 1 min and at 72c for 2 min , and a final extension of 72c during 10 min . me - pcr product was used as template to amplify an internal 0.47 kb fragment ( figure s4 see in supplementary material available at doi : 10.1155/2011/753758 ) . nested pcr was performed with the primers mlf ( ggtggtgtmggattcacacartaygcwacagc ) and mlr ( ttcattgcrtagttwggrtagtt ) with a gc clamp applying the following conditions : a denaturing step of 5 min at 94c , 5 cycles at 95c for 40 s , 55c for 1 min , 72c for 90 s ( a ramp of 0.1c / s was included between the annealing to the extension steps ) , followed by 30 cycles of 95c for 40 s , 55c for 1 min , 72c for 90 s , and an extension of 72c for 7 min . the gc clamp was equivalent to 40 bp of gc at the 5 end in order to prevent a complete melting of the dna fragments . correct length pcr - dgge products were visualized on 0.5 g / ml ethidium - bromide - stained gels at 2% agarose . in order to generate a dgge pattern , an average of 5070 g of dna from pcr - dgge products were resolved using a d - code universal mutation detection system ( biorad laboratories ) in polyacrylamide gels with a horizontal denaturant gradient . all dgge patterns were achieved under standardized denaturant and electrophoretic conditions : constant temperature of 60c polyacrylamide composition ( acrylamide - n , n-methylene bisacrylamide , 37 : 1 ) containing 0100% of denaturants ( 7 m urea and 40% formamide deionized with mixed - bed resin ) , a running time of 4.5 hrs and a constant voltage of 200 v . high resolving band patterns from environmental and culture samples were obtained as follows ( denaturant composition is given in percentage ) : for apra gene fragments , 5080% from nonaxenic cultures , and 4070% from environmental samples . in mcra gene fragments from environmental samples , the performed gradients were done by duplicate at 4070% and 4060% to increase the resolution of distance among bands . the gels were incubated in ethidium bromide for 20 min and rinsed in distilled water for 30 min . all single bands were excised from the gel with scalpels and eluted in 10 l of milliq water to avoid desiccation ; hereafter , they were stored overnight at 4c . dna was extracted from polyacrylamide by electrophoresis in 2% agarose gels ( 40 ma ) . the agarose bands were filtered through glass fiber columns at 14,000 rpm for 2 min ( hettich mikro 22 r centrifuge ) . 510 l of the precipitate obtained were used as dna template for the band reamplification of mcra and apra genes under the same pcr conditions however , a minor fraction of the bands were reamplified . the mcra and apra gene pcr products were sequenced using primer pairs apsfw / apsrv and mlf / mlr in an abi 377 sequencer ( applied biosystems ) . nucleotide sequences were cleaned and assembled using dna baser software ( heracle software , germany , http://www.dnabaser.com/ ) . according to the review of fromin et al . , dgge reproducibility mainly relies on the dna extraction and/or pcr amplification steps ; therefore , the fingerprint analysis ( dgge ) was processed once . the reproducibility of dgge patterns has been tested previously by experimenting differences along the procedure , from sampling to pcr amplification conditions ; despite these modifications , the comparison of dgge patterns was consistent showing changes in band intensities only . thereby , the pattern of apra dgge gel from environmental samples was used to define a dendrogram . then , the bands were qualitatively scored as present / absent and no semiquantitative analyses were performed for band intensity ; the band clustering was performed with the maximum likelihood ( ml ) restriction analysis ( restml ) included in the phylip v.3.67 package . genbank accession numbersthe nucleotide sequence data reported here are available under the genbank accession numbers : eu722715eu722732 , hm466937hm466940 , hm466943hm466946 ( apra phylotypes ) , and eu091355eu091364 , hm466948 ( mcra phylotypes ) . the nucleotide sequence data reported here are available under the genbank accession numbers : eu722715eu722732 , hm466937hm466940 , hm466943hm466946 ( apra phylotypes ) , and eu091355eu091364 , hm466948 ( mcra phylotypes ) . the translations of the apra and mcra sequences into amino acids were defined using the translate tool with a standard code ( http://expasy.org/tools/dna.html/ ) . the best frames for all the apra and mcra fragments were firstly selected by the unstopped amino acid ( aa ) sequences and , secondly , by matching their best hits with those compiled in the nonredundant database of the genbank , which were detected through the blastp program ( e - value 10 ) . the final inferred aa sequences were compared against the swiss prot and genbank databases in order to obtain their homologous counterparts using the wu - blast program with a significant blastp e - value 10 . from a first approach , we also included apra and mcra sequences from reported environmental samples as seed sequences . a complete list of the sequences included in this study to reconstruct a phylogeny for the apra and mcra enzymes can be seen in supplementary material ( tables s1 and s2 , resp . ) . different filters were used , from the thousands of collected sequences , in order to choose the final candidates involved in the reconstruction of a phylogenetic hypothesis . in this sense , we firstly applied a redundant analysis at 90% identity using the cd - hit program . from the obtained sequences , a second analysis at 100% identity was done with the cd - hit program , which excludes redundant phylotypes ( subspecies and variants ) of the same species , warranting the diversity of sequences only by including strictly different species from the same or different genera . the apra phylotypes aps cw 1 ( eu722715 ) and aps cw 16 ( eu722724 ) showed 100% of identity , as well as the phylotypes aps cw 3 ( eu722716 ) and aps cw 10 ( eu722720 ) . only one sequence was taken as a representative of the identity cluster to reconstruct the phylogeny . in order to support a robust identification in the final phylogeny , we included species from the same genera for those cases in which the homologous counterparts are closely related to the phylotypes obtained in this work . when it was necessary , individual phylogenetic trees for the tirez phylotypes were done previously in order to improve their identification and to select the counterpart sequences for the final phylogeny . the aa sequences obtained from the previous approaches were then aligned using the clustalx program with default parameters . in order to identify an evolutionary signal from the sequence fragments obtained in this work and their homologous counterparts , we applied a manual and also an automatic approach to edit the alignment . first , we manually edited the alignment through the use of the bioedit program v.7.0.9 in order to include only the functional domains of the subunits of mcr and apr enzymes . the functional description of these domains is fully detailed in section 4 and in the supplementary material . the n - terminal domain of the -subunit of the apra ( apra_alpha_n ) harbors the fad cofactor - binding domain ( aa positions : 2261 and 394487 ) and the capping domains ( aa positions : 262393 ) . these functional domains have been characterized from archaeoglobus fulgidus in the reduced state ( fadred - aps , pdb i d : 1jnr ) and in the oxidized state ( fadox - aps , pdb i d : 2fja ) as well as in desulfovibrio gigas ( pdb i d : 3gyx ) . thus , a total of 100 aa sequences were included in the apra alignment , where 23 phylotypes are derived from this work . the apra alignment includes two of the nine ( the absent sites are asn - n , tyr - y , glu - e , val - v , gly - g , leu - l , and arg - r ) functional active sites of the apra_alpha_n domain : arg - r and trp - w , previously reported . see the apra alignment and catalytic sites in supplementary material ( figure s2 ) . on the other hand , the c - terminal domain of the -subunit of the mcra enzyme ( mcr_alpha_c ) harbors an all - alpha multihelical bundle domain ( pfam domain : pf02249 ) . this functional domain has been characterized in methanosarcina barkeri ( pdb i d : 1e6y , c - terminal domain : 328460 ) , methanothermobacter thermoautotrophicus ( pdb i d : 1mro , c - terminal domain : 315440 ) , and methanopyrus kandleri(pdb i d : 1e6v , c - terminal domain : 319444 ) . a total of 80 aa sequences , 11 of them derived from this work , were included in the mcra alignment . the mcra alignment includes five of the seven functional active sites of the mcr_alpha_c domain ( absent sites : asn - n and val - v ; present sites : phe - f , tyr - y , phe - f , tyr - y , and gly - g ) . see the mcra alignment and catalytic sites in the supplementary material ( figure s3 ) . finally , we readjusted a final alignment defining the informative sites and conserving the functional active sites of the enzymes previously described , through the use of the software gblocks v.0.91 . therefore , the final alignments were performed on a region of 137 aas for apra and 139 aas for mrca , from which 122 and 132 positions were involved in the phylogenetic analysis , respectively . in order to reconstruct a phylogenetic tree , a character - based approach for the apr_alpha_n and mcr_alpha_c phylogenetic reconstructions was developed using the protpars program in order to construct a maximum parsimony ( mp ) tree of sequences . pyrobaculum aerophilum and archaeoglobus fulgidus were used as outgroups in the apr_alpha_n phylogeny ; whereas methanopyrus kandleri was used as outgroup in the mcr_alpha_c phylogeny . a distance approach for the apr_alpha_n and mcr_alpha_c phylogenetic reconstructions was also developed using the seqboot program to generate 1000 bootstrapped datasets from the sequences , whereby the pseudoreplicates were used in the protdist program in order to generate a distance matrix through the jones - taylor - thornton ( jtt ) model of evolution . the evolutionary distances are in the units of the number of amino acid substitutions per site . the rate variation among sites was modeled with a gamma distribution estimated previously shape parameter = 0.9 for apra and = 0.8 for mcra . then , the distance matrix was used in the neighbor program to construct a neighbor - joining ( nj ) tree . the bootstrap consensus tree inferred from 100 replicates is taken to represent the evolutionary history of the taxa analyzed . branches corresponding to partitions reproduced in less than 60% bootstrap replicates were collapsed with the consensus program ( default parameters ) . the percentage of replicate trees ( > 50% ) in which the associated taxa clustered together in the bootstrap test ( 100 replicates ) is shown next to the branches . no major branching differences were detected between the mp and nj topologies obtained for both enzymes . we calculated the amino acid composition of all apra and mcra sequences used previously to construct the phylogenies . using the perl scripting language , a program was written to read each amino acid sequence ( fasta format ) and calculate the frequency for each amino acid . we also calculated two halophilia indicators from the amino acid composition of every sequence : the pab factor estimates the surplus of polar and acidic amino acid compared to polar and basic ones ( i.e. , pab = [ asx + glx ] [ arg + lys ] ) , and the ab ratio of the acidic amino acids glu and asp to the basic amino acids lys , his , and arg ( i.e. , ab = [ asp + glu ] : [ his + arg + lys ] ) . we divided the amino acid profiles from each marker into two different data sets in order to calculate an average and standard deviation of the samples . the first data set is based on salinity adaptation by dividing sequences in tirez , halophilic , and nonhalophilic species . the second data set is based on the species forming the major taxonomic groups in which the tirez phylotypes are phylogenetically allocated . for apra desulfovibrionales , desulfobacterales , peptococcales , and chromatiales ; whilst for mcra , methanomicrobiales and methanosarcinales . therefore , a spreadsheet was created using microsoft excel software for data tabulation and graph construction . we performed the corresponding nucleotide alignments for all apra and mcra sequences used previously to construct the phylogenies . in order to reduce the bias of the gc measurements by the use of sequences with different length and highly divergent regions ( i.e. , long indels ) , we manually edited and readjusted the final alignment . accordingly , highly and long divergent regions ( insertions and deletions ) were eliminated from the alignment . the final alignments only include the strict codon positions encoding for the functional domains of apra ( 375 nucleotide positions , 125 codons ) and mcra ( 399 nucleotide positions , 133 codons ) described on the phylogenetic analysis section . using the perl scripting language , a program was written to read each nucleotide sequence ( fasta format ) and calculate the total nucleotide percentages as well as at the three individual codon positions for each sequence . we divided the gc profiles from each gene marker into the same data sets used to analyze amino acid composition ( i.e. , salinity adaptation and taxonomic clades ) in order to calculate an average and standard deviation of the samples . a correction for the amino acid usage was applied by the calculation of the relative synonymous codon usage ( rscu ) values from the nucleotide datasets based on salinity adaptation : tirez , halophilic , and nonhalophilic species . the rscu for a particular codon ( i ) is given by : rscui = xi/ xi / n , where xi is the number of times the codon has been used for a given amino acid and n is the number of synonymous codons for that amino acid . rscu values are the number of times a particular codon is observed , relative to the number of times that the codon would be observed in the absence of any codon usage bias . in the absence of any codon usage bias , the rscu value would be 1.00 . a codon that is used less frequently than expected will have a value of less than 1.00 and vice versa for a codon that is used more frequently than expected . finally , a spreadsheet was created using microsoft excel software for data tabulation and graph construction . sulfide showed higher concentrations at the zone of 010 cm depth ( figure 1(a ) ) . the occurrence and distribution of sulfide along the depth profile can reflect a biogenic origin by the presence of sulfate - reducing bacteria ( srb ) in the hypersaline sediment . the concentration of h2s coincided with the presence of a black deposit of iron sulfide mainly in winter ( figure 2 ) . sulfate levels increased with depth , its concentration ranging at 0.2 m , and the highest values were detected at 1015 cm in depth ( 300 mm ) , just below the highest concentration zone of sulfide ( figure 1(a ) ) . the complete sediment profile was anoxic and in accordance with a negative redox potential ( figure 1(b ) ) . the redox potential and oxygen levels slightly increased in the deepest zones ( 1520 cm in depth ) . the redox conditions of most part of the sediment core were in the range of 300 and 200 mv , low enough to allow sr and mt activities . the lowest eh values were reached at 010 cm in depth and coincided with the increase in sulfide concentration ( figure 1(b ) ) . likewise , the highest nh4 concentration ( 46 m ) was observed at 1015 cm in depth ( figure 1(c ) ) . the cl : so4 proportion fluctuated between 0.1 and 0.3 , these ratios are lower than the values reported in the saltern and they reflect the athalassic nature of the system . sulfate concentration in tirez lagoon was lower than in the also athalassic chaka lake sediment ( 10 mm ) . even though chloride was undetermined in chaka lake sediment , its cl : so4 proportion is two times higher than the highest value registered at tirez lagoon . figure 1(d ) shows the ph course on sediment depth ; it is possible to observe the characteristic neutral ph of the system as well as a slight acidification , probably a consequence of biological volatile fatty acids ( vfa ) formation and sulfate reduction processes . the c : n ratio determined in the samples showed values characteristic of low photoautotrophic activity at the surface starting at > 6 at 05 cm depth ( figure 1(d ) ) . therefore , preferential nitrogen mineralizers should be found at the surface preceding carbon mineralizers at deeper zones . figure 1(e ) describes that divalent cations dominate over monovalents . finally , the ratio ( na + k)/(mg+ ca ) in tirez is between 1.8 and 0.09 , whilst in salt lake is > 9.0 . we applied a denaturing gradient gel electrophoresis ( dgge ) fingerprinting analysis through the use of two functional genes : adenosine-5-phosphosulfate reductase ( apr ) and the methyl coenzyme - m reductase ( mcr ) , in order to identify ecotypes from the sediments samples and nonaxenic cultures of tirez lagoon . apra dgge profiles are presented in figure 3 and mcra dgge profile in figure 4 . thus , we obtained sequences of diverse phylotypes from dgge profiles representing the bulk content of three sampling points of the lagoon salt pan . the bands were prefixed as aps and mcr ( from the gene marker ) and subfixed as cw ( from nonaxenic cultures obtained in winter ) and ew and es ( environmental sediment sampled in winter and summer , resp . ) . the apra dgge pattern from sediment profile ( figure 3(b ) ) revealed the presence of a more complex banding pattern in comparison with the profile from nonaxenic cultures ( figure 3(a ) ) . at 1525 cm depth , low yield or no pcr product given that a considerable number of environmental bands from the apra dgge profile could not be sequenced or specifically identified , probably due to the presence of residual pcr inhibitors such as humic acids coextracted with genomic dna that were not purified by the jetquick kit and that comigrate with dna in the polyacrylamide gel changes in population distribution were estimated through the use of p - analysis with maximum likelihood ( ml ) in phylip software in order to identify a significant clustering . the clustering pattern is shown in figure 3(c ) , and it was more in accordance with a disturbance due to seasonality instead of sediment depth . additionally , p value showed no significant differences between nodes w and s being p 0.05 as significant to reject the hypothesis that two population sets were derived from the same communities . from previous studies carried out in thalassic communities , where salt gradient is between 8 and 20% ( within the range of tirez ) , it has been reported that the rate of methanogenesis is below 0.1% of the total sulfate - reduction productivity . therefore , a lower abundance of ma was expected in tirez sedimentary community it is inferred from the lower cl : so4 ratio . in addition , the population size threshold for dgge detection is 1% . thereby , we had to perform a nested pcr from the mcra gene in order to improve the detection of the ma community in the sediment samples from tirez . we firstly obtained a 0.76 kb mcra fragment through the me primer pair . because such a length is inadequate to obtain a discernible dgge pattern and due to low yield in me amplicons , a small 0.47 kb mcra fragment nested in the me region was amplified through the ml primer pair ( supplementary material , figure s4 ) . in agreement with juottonen and collaborators , no differences in the diversity of ma organisms were expected from the use of me and ml pcr products . different dgge gradients for the ml - pcr products were tested in order to obtain the best pattern resolution . we detected two distinctive but adjacent bands in all dgge winter profiles ( e.g. , mcr - ew1 and mcr - ew2 ) obtained through several gradients ( figures 4(a ) and 4(c ) , 4070% and 4060% gradients , resp . ) . a pattern of bands in pairs is a result of the low dgge resolution , where two dna fragments differ in one or few bases due to the use of ambiguous primers . though me - ml primers are ambiguous ( see section 2 ) , the phylotypes were placed in different orders in methanogenic archaea . thereby , nucleotide sequences have similar electrophoretic mobilities but they represent different sequences and , thus , a double band was ruled out . we also obtained a dgge pattern from sediment sampled in flooded and dry seasons at different depths ( figure 4(c ) ) . it is interesting to note that the mcr - ew1 band ( marked in figures 4(a ) and 4(c ) ) appeared uniquely associated with flooded season at 05 cm in depth ( figure 4(c ) ) . phylogenetic reconstructions were done for the inferred amino acid ( aa ) sequences of apra and mcra gene markers and their homologous counterparts . we decided to analyze aa instead of nucleotides because the latter reduces the inherent variation seen in protein sequences , except for the third codon base . we defined two regions of unambiguously aligned aa , the first one located in the n - terminal domain ( 137 aas ) for the subunit of apra ( apra_alpha_n ) , and the second one located in the c - terminal domain ( 139 aas ) of the subunit of mcra ( mrca_alpha_c ) , both of them containing some of the catalytic sites involved in their metabolic role ( supplementary material , figures s2 and s3 , resp . ) . it is important to note that not only the phylogenetic topologies obtained for the apra_alpha_n and mcra_alpha_c sequences are robust , as can be seen by the significant bootstrap values in the main clustering branches , but also the internal groups are supported by the expected clustering of the mcra and apra crystals previously characterized for ( a ) the mcra in methanosarcina barkeri belonging to methanosarcinales , methanothermobacter thermoautotrophicus from methanobacteriales and methanopyrus kandleri in methanopyrales ; ( b ) the apra from archaeoglobus fulgidus in euryarchaeota , and desulfovibrio gigas in deltaproteobacteria . the phylogenetic analysis of the 25 apra tirez sequences is presented in figure 5 . this analysis included representative species from diverse srp and sop taxonomic groups such as euryarchaeota , crenarchaeota , firmicutes , , , and -proteobacteria ( supplementary material , table s1 ) . some of the environmental apra phylotypes were not resolved at genera level , and the result has been discussed for the next taxonomic rank . cultured and environmental srp populations were identified as follows : cultured phylotypes ( desulfohalobiaceae , peptococcaceae , and desulfobacteraceae ) and environmental phylotyeps ( desulfobacteraceae , and peptococcaceae ) . one cluster formed by three phylotypes ( aps - cw2 , -cw4 , and -cw5 ) was closely related to the halotolerant and alkaliphilic desulfonatronovibrio hydrogenovorans . interestingly , the summer sediment did not reveal the presence of species in the haloadapted desulfohalobiaceae . twelve phylotypes obtained from sediment ( summer and winter ) and enrichments were related to the acetoclastic and nonhalophilic species desulfonema magnum . two phylotypes ( aps - cw6 -es29 ) were identified as peptococcaceae . whilst the phylotype aps - cw6 was conclusively affiliated to desulfotomaculum solfataricum belonging to firmicutes , the phylotype aps - es29 was not resolved at genera level ; however , aps - es29 was allocated basal to the representative firmicutes taxa used in this study . actually , the affiliation of phylotype aps - es29 and other sop tirez phylotypes could become particularly uncertain given the well - known horizontal aps reductase ( apr ) gene transfer ( hgt ) events between the srps from firmicutes and -proteobacteria as well as between the sops from and -proteobacteria , respectively ( see figure 5 ) . both main apr - hgt events are identified in this work and are in accordance with previous phylogenetic studies . in four of the environmental sop phylotypes ( aps - ew7 , -ew8 , and -ew13 , aps - es28 ) , the assignment of the apra gene fragment could not be conclusive at species level ; thus , a detailed function in tirez 's system remains uncertain . the closest clade for three phylotypes was a group of noncultured microorganisms ( endosymbionts ) in hydrogenophilaceae in -proteobacteria . the phylotype from summer sediment ( aps - es28 ) remained unidentified at species level , and tree topology helped to designate it as -proteobacteria . the environmental phylotype aps - ew3 was conclusively affiliated to endosymbionts and close to thiotrichaceae and chromatiaceae in -proteobacteria . other three phylotypes were derived from enrichment ( aps - cw11 , -cw12 , and -cw13 ) and resulted with a short distance with the cultured haloalkaliphilic purple bacteria thioalkalivibrio ( ectothiorhodospiraceae ) in -proteobacteria . the phylogenetic reconstruction of the eleven mcra sequences obtained from the anoxic tirez sediments is shown in figure 6 . this analysis included representative ma species within methanomicrobiales , methanosarcinales , methanococcales , and methanobacteriales ( supplementary material , table s2 ) . the phylogenetic tree allowed the identification of mcra phylotypes belonging to the methanosarcinaceae and methanomicrobiaceae . in the same way , phylotype mcr - ew2 was closely related to methanolobus zinderi . finally , the phylotype mcr - ew1 closely clustered to the hydrogenotrophic and nonosmoadapted species methanoplanus petrolearius . salt - in halotolerant organisms have been related to adaptations to high intracellular concentration in order to favor an osmotic balance within an hypersaline environment [ 23 , 25 ] . given that the catalysis of apra and mcra enzymes occur in the cytoplasm , we were interested in determine whether tirez apra and mcra sequences show a bias when compared to their halophilic and nonhalophilic homologous counterparts . thus , we calculated the aa composition from the alignment used to reconstruct the phylogeny in order to estimate the hydrophobic ( gly , leu , val , ile , phe , met , ala , trp , and pro ) , polar ( ser , thr , cys , tyr , gln , and asn ) , basic ( his , arg , and lys ) and acidic ( glu and asp ) contents of the apra and mcra enzyme fragments analyzed in this study . additionally , we used the nucleotide alignment that covers the aa positions selected to reconstruct the phylogeny for each gene marker in order to estimate the general codon bias gc content and the relative synonymous codon usage ( rscu ) ( see section 2 ) . for this purpose , we divided the sequence profiles from each gene markers into two data sets the first one is based on salinity adaptation ( tirez , halophilic and nonhalophilic species ) and the second one is based on the major taxonomic groups in which the tirez phylotypes are phylogenetically allocated ( apra : desulfovibrionales , desulfobacterales , peptococcales , and chromatiales ; mcra : methanomicrobiales and methanosarcinales ) ( supplementary material , tables s1 and s2 , resp . ) . the degree of excess acidic amino acids and dearth of basic amino acids reflects the prevalence of the salt - in strategy and the amount of adaptation necessary to cope with the environmental stress . this can be quantified from two estimations : by calculating the surplus of polar and acidic amino acid compared to polar and basic ones ( i.e. , pab = [ asx + glx ] [ arg + lys ] ) and by the ratio of the acidic amino acids glu and asp to the basic amino acids lys , his , and arg ( i.e. , ab = [ asp + glu ] : [ his + arg + lys ] ) . on average , the amino acid composition measurements ( table 1 ) indicated that apra tirez phylotypes ( pab = 2.87 , ab = 0.62 ) were from similar to slightly higher in comparison with halophilic ( pab = 2.70 , ab = 0.59 ) and nonhalophilic sequences ( pab = 2.35 , ab = 0.62 ) . however , the observed differences in pab and ab indicators between tirez phylotypes and halophilic species are out of proportion to argue a salt - in signal in tirez phylotypes given that apra differences are more than ten times less the difference between escherichia coli and halobacterium salinarum or halomonas elongata and halobacterium salinarum . the total gc content of apra tirez phylotypes , halophiles , and nonhalophiles organisms is 57.60% , 55.70% , and 55.00% , respectively . the gc content of tirez phylotypes is higher than the reported for escherichia coli ( 50.3% ) but lower than the extreme halotolerant species from the dead sea metagenome ( 6267% ) and halobacterium salinarum ( 65.7% ) ( table 1 ) . a codon usage in apra tirez phylotypes is consistent with that expected , when corrected for gc composition ( figure 7 ) . in comparison to halophiles and nonhalophiles , apra tirez phylotypes show a significant overrepresentation of amino residues with a preferential use for a g or c in the third or first position : val ( guc ) , ser ( ucc ) , gln ( cag ) , lys ( aag ) , asn ( aac ) , asp ( gac ) , and glu ( gaa ) . even though arg ( cgg , agg ) , ala ( gcg ) , and cys ( ugc ) are underrepresented amino acids in apra tirez phylotypes as well as leu ( cug ) and gly ( ggc , ggg ) do not show compositional differences when compared with halophiles and nonhalophiles sequences ( supplementary material , figure s1b ) , all of them show a significant codon usage with gc bias ( rscu > 1.5 ) ( figure 7 ) . accordingly , the first , second , and third codon positions of apra tirez phylotypes have gc percentages of 54.2% , 42.4% , and 76.0% , respectively , and they agree with the gc content values previously reported in some salt - in halophiles ( table 1 ) , with high gc content and a third position gc bias [ 21 , 29 ] . similar trends on aa composition and gc content can be seen for the apra clades ( table s3 and figure s1 ) . a slighter segregation of the mcra tirez phylotypes from the nonhalophilic species is shown in table 1 . accordingly , the ab indicator for mcra phylotypes was slightly lower ( 1.50 ) in comparison with the average of halophilic ( 1.67 ) and nonhalophilic species ( 1.75 ) , whilst an opposite trend is shown with the pab indicator : 16.62 for tirez , 15.99 for halophiles , and 16.44 for nonhalophiles . in contrast to the apra tirez phylotypes , the total gc content ( 47.20% ) and the third codon gc bias ( 46.10% ) are significantly lower than the estimated for halophiles and nonhalophilic species ( table 1 ) . the gc content of the first ( 53.1% ) and second ( 42.4% ) codon positions does not change the trend of mcra tirez phylotypes ( supplementary material , figure s3c ) . nevertheless , an overrepresentation of amino residues in mcra tirez phylotypes with a preferential codon use ( in comparison to nonhalophiles sequences only ) can be pointed out for ile ( auu ) , pro ( cca ) , ala ( gca ) , tyr ( uau ) , and asn ( aac , aau ) . even though lys ( aaa ) , asp ( gau ) , ser ( ucc , ucu ) , and thr ( aca ) are underrepresented amino acids in mcra tirez phylotypes in comparison to halophiles and nonhalophiles sequences ( supplementary material , figure s1b ) , all of them show a preferential codon usage . furthermore , it is important to note that the aa composition and gc content trends for mcra clade profiles showed a differentiated tendency in contrast to the estimated average from all mcra tirez phylotypes ( supplementary material , table s3 ) . the first , second , and third codon position of methanomicrobiales present a high gc content values of 52.6% ( tirez 54.1% ) , 40.0% ( tirez 39.1% ) , and 73.1% ( tirez 77.4% ) , respectively . similarly , the polar and acidic content in methanomicrobiales ( pab = 17.12 and ab = 1.70 ) is interestingly higher than the bulk cell protein content reported for e. coli ( 15.85 ) and close to the haloadaptation threshold of h. elongata ( 17.56 ) . srp and ma are the frequent ecotypes responsible of major biogeochemical processes in sedimentary systems . a functional gene pcr - dgge approach was applied to identify these anaerobic ecotypes . regarding the sediment profile and community structure along time and depth , the bands identified in the apra dgge pattern from environmental samples are in agreement with the presence of black sediments below the evaporite layer observed in summer and winter seasons ( figure 2 ) . this mineral precipitation and the sulfide detected in the sediment ( figure 1(a ) ) are probably attributable to a dissimilatory sulfate reduction where ma were also detected ( figure 3(c ) ) . the use of a nested pcr implies additional amplification cycles , and , thus , it has been used to increase the visualization sensitivity of species present in low numbers by dgge . interestingly , our findings via this approach denote a predominance of the srp - sop ecotypes over ma , given that we performed the nested - pcr approach to obtain a positive pcr product of mcra gene fragment , whilst it was not necessary to apply it for the apra gene fragment . finally , a predominance of srp - sop ecotypes in tirez lake is in accordance with the high values of sulfate registered on the sediment . after the clustering analysis of sedimentary populations represented in the apra dgge pattern , the changes are better explained by a seasonal disturbance in accordance with the ephemeral lagoon . it is suggestible that population resilience is mainly regulated by changes in salinity because the main nodes indicate a partition into dry and flooded patterns ( figure 3(c ) ) ; note that salinity fluctuates from 6% ( w / v ) during winter to 35% ( w / v ) during spring . however , the strong temperature oscillation can be also associated with salinity over community composition . additionally , the p values ( > 0.05 ) indicate that the partition winter / summer is not significant enough to describe well - differentiated communities since flooded node and dry node are more clustered than expected by chance . interestingly , most of the sop , srp , and ma phylotypes obtained in this work were related to environmental sequences described from alkaliphilic or thalassic hypersaline systems [ 6 , 20 ] . were detected phylotypes ( aps - cw4 , -cw-5 , and -cw2 ) from desulfonatronovibrio hydrogenovorans , a lithoheterotrophic , halotolerant ( grows in a salinity range of 112% nacl ) , and alkaliphilic sulfate respirer . surprisingly , d. hydrogenovorans does not grow at ph of 7 and the highest ph of tirez is below 8.0 . desulfohalobiaceae species are commonly adapted to high osmolarity due to the anabolic metabolism of compatible solute synthesis and dependent on the use of lactate and hydrogen as electron donors . desulfohalobium retbaense is considered the neutrophilic and thalassic counterpart of d. hydrogenovorans , but it was not detected in tirez . gram - positive desulfotomaculum solfataricum ( aps - cw6 ) was detected in enrichments . another phylotype , aps - es29 , is also a member of peptococcaceae , but it could not be assigned to a specific genus . these phylotypes did not cluster with desulfotomaculum halophilum sequences , which tolerates up to 12% nacl . sulfate - reducing bacteria in peptococcaceae perform oxidation from a broad spectrum of electron donors such as lactate . compatible solutes in peptococcaceae have not been characterized ; however , the theoretical energy yield , for example , in medium supplied with lactate is g = 160 kj / mol , would give enough energy for the osmoprotectant synthesis or transport as , for example , desulfovibrio vulgaris ; d. vulgaris is trophically analog to desulfotomaculum species . d. vulgaris synthesizes sugars such as trehalose or accumulates amino acids such as glycine betaine and proline as compatible solutes as response to under salt stress . stress response in d. vulgaris is based on genes with homologous in diverse and distant species such as bacillus subtilis ; thus , the finding of peptococcaceae in tirez , under analog bioenergetic constraints , could be explained in the terms of the salt - out strategy ( see section 4 ) . the presence of methanohalobium evestigatum and methanolobus zinderi in the sulfate - rich and anoxic sediment is easily sustained by functional arguments , even in summer samples , because their metabolism requires methylated substrates ; thus , it is noncompetitive with srp . m. evestigatum and m. zinderi are theoretically productive in bioenergetic terms , enough to exhibit compatible solute synthesis . methanolobus zinderi was isolated from an estuary and grows at the higher rate and tolerates upper levels of divalent cations ( mg ) in comparison with monovalent na . this characteristic is remarkable because m. zinderi could be adequate to tirez given that divalent cation ca dominate over monovalents in the sediment ( figure 1(e ) ) . on the other hand , the increase of ammonium ( nh4 46 m ) at 1015 cm depth and the decrease of eh across the sediment profile ( figure 1 ) suggest the development of strict anaerobic and methylotrophic ma metabolisms . none of the genera detected in both seasons clustered with acetoclastic ma . the absence of acetoclastic ma in hypersaline systems has been widely accepted as a consequence of the low gibbs free energy dissipated from acetate as substrate . however , acetoclastic ma activity was reported in napoli mud volcano brines with 4.0 m chloride , where the cl : so4 ratio is 200 times higher than the observed in tirez . in tirez , the absence of acetoclastic ma is probably explained by substrate outcompetition , because the sulfate - reducing conditions prevail due to the high abundance of sulfate in tirez and to the putative adaptation of acetoclastic srp such as desulfonema magnum to the extreme sediment . some of the phylotypes from environmental and enrichment culturing were affiliated to endosymbionts ; its potential ecological role in the sediment is supported by the view that the sulfur cycle has been described in marine oligochaetes , where endosymbionts identified as proteobacterial microorganisms participate as sulfur oxidizers . therefore , it is plausible that the free - living and nonisolated relative populations in tirez sediment have an analogous metabolic role . three phylotypes from winter sediment and enrichments were affiliated to the chemoautotrophic genus thioalkalivibrio and sulfur oxidizing endosymbionts in /-proteobacteria clade ( figure 5 ) . these anaerobic ecotypes are expected to be found in the extremely saline sediment as much as the h2s is present ( figure 1(a ) ) ; in turn , h2s would be oxidized anaerobically by these purple bacteria given that low eh and partial o2 pressure were observed in the sampling site ( figure 1 ) . the discrepancy in the finding of thioalkalivibrio is due to its narrow range of optimal ph ( 9.510.0 ) , the fact that species in thioalkalivibrio are true alkaliphilic and are well adapted to athalassic soda lakes , that is , dominated by monovalent cations , and considering that other sulfur oxidizing and halophilic sop species such as thiomicrospira halophila or hallochromatium spp . unfortunately , the sop tirez phylotype from summer sediment was not identified at species level . it has been argued that hypersaline environments are inappropriate for the biological development of anaerobic acetate oxidation as a consequence of the low negative balance of the standard g yielded by this dissimilatory metabolism and due to the high maintenance energy needed for the synthesis / accumulation of compatible solute under high osmotic conditions . however , at high sulfate concentrations , desulfonema magnum populations were unequivocally detected in the evaportitic sediment and winter sediment samples ( environmental and derived from enrichment culturing ) at 015 cm depth under an extreme salinity stress of 35% salts . this acetate - oxidizing desulfobacteraceae has not been described in hypersaline systems and was the most abundant phylotype identified in tirez lagoon . d. magnum has an optimal salinity about 2.5% nacl and has been described in marine microbial mats . previous studies have shown that desulfobacteraceae are present in thalassic hypersaline basins and athalassic soda lakes . this is a notable finding for the understanding of carbon cycle in extreme hypersaline ecotypes because under extreme conditions there is a decline in organic matter remineralization ; thereby , organisms encoding the corresponding apra gene probably face the salinity changes . halophilic species from desulfobacterales have not been isolated ; desulfobacter halotolerans is member of desulfobacterales but has an optimum growth with only of 1 - 2% nacl . nevertheless , very little is known about the mechanisms involved in energy conservation that allow acetoclastic srp organisms to survive in extreme saline conditions . salt - in osmoadaptation has been suggested for desulfobacteraceae ecotypes identified in soda lakes to compensate saline stress . possibly , desulfonema , being an acetoclastic sr , exerts additional energy conserving mechanisms ( as in the case of ma and acetogenic bacteria ) consisting in extra transference of electrons from membrane complexes dependent on h or na pumping . such process is likely to occur in the acetoclastic desulfobacteraceae desulfobacterium autrotrophicum whose conservation mechanism of chemiosmotic energy is analogous to that in homoacetogenic bacteria . a mcra phylotype from the hydrogenotrophic methanoplanus petrolearius was detected in the surface dgge profile from winter sediment at 05 cm depth ( figure 4 ) , when salt content in the saltern is averaged at 6% w / v . this organism has a maximum tolerance at 5% and an optimal growth at 13% nacl . it is feasible that the m. petrolearius salt tolerance determines its absence in summer samples and is correlated with the low energy yielded by the methanogenic pathway based on h2 and formate as electron donors . mt activity based on these substrates has a low theoretical energy yielded ( g ) . methanoplanus clones , which have been reported in thalassic hypersaline sediments but at 2.2 m cl and sulfate below the detection limit . in order to adjust to lower water activities of the environment and the resulting decrease in cytoplasmic water , microorganisms must accumulate intracellular ions or organic solutes to reestablish the turgor pressure and preserve enzyme activity [ 27 , 88 ] . halophiles are adapted to hypersaline environments by a mechanism that involves at least equimolar extracellular and intracellular salt concentrations by a selective influx of potassium ions into the cytoplasm . strategy favors solubility and is energetically efficient , but unfolds proteins at high concentration . as a consequence , this halotolerant strategy requires that the entire intracellular machinery , that is , proteins , nucleic acids , s and their specific interactions with one another , must be adapted to high salt intracellular levels . the adaptations generally include an increase of the acidic nature of intracellular proteins and/or an increment of genomic cg content and a gc - bias at the codon usage level . nevertheless , paul et al . demonstrated common genomic and proteomic trends in halophiles that transcend the boundary of phylogenetic relationship and the genomic gc content of the species . accordingly , it has been suggested that distantly lineages adopted salt - in strategy independently by convergent evolution given its radical nature . all previous studies have estimated average trends of amino acid composition and gc content from selected sequences or enzymes in marine aerobic populations [ 21 , 57 ] or from completely sequenced genomes obtained from diverse aerobic environments [ 22 , 25 ] . even though salt - in strategy was recently proposed to explain the finding of the resilience desulfobacteraceae at hypersaline and alkaline lakes , this salt - adaptation strategy has been neither reported in species of the srp - sop nor in ma ; in part , given therefore , we consider it useful to use apra and mcra markers to test salt - in signals . an intuitive justification would be to expect a naturally biased selection for apra and mcra enzymes given their frequent or higher expression levels in the cytoplasm ( in comparison to other encoded genes at the genome ) in order to cope with their ecological and metabolic role on anaerobic and hypersaline sediments . our results can not be conclusive regarding the halotolerant strategies carried out by tirez phylotypes , until a large sequence data set can be achieved for these organisms . nevertheless , the amino acid composition , gc content , and preferential codon usage trends exhibited by the apra marker from tirez phylotypes suggest a plausible salt - in signal when compared to halophiles and non - haphiles . the increase in negatively charged ( asp and glu ) and polar ( ser , asn , and gln ) residues in apra tirez phylotypes can be explained by a codon usage with gc bias at the third position . the overrepresentation of these amino acid residues and their preferential codon usage are consistent with reports on salt - in adaptation [ 22 , 25 ] . similarly , a higher frequency of val in apra tirez phylotypes compared to nonhalophiles and halophiles supports the observation of madern et al . and paul et al . , but disagree with earlier propositions on underrepresentation of all strong hydrophobic residues in halophiles . we also report a slight decrement of the basic residue arg in apra tirez phylotypes . the role of arg in haloadaptation is quite controversial ; its increment in halophilic species can be expected by mutational bias given that five of the six codons have a bias towards gc nucleotides ; however , arg has been also reported in a consistent decrement in specific haloadapted species [ 29 , 57 ] . even though the slight increment of lys observed in apra tirez phylotypes contradicts all previous propositions on underrepresentation of the most important basic residues in all salt - in halophiles [ 22 , 25 ] , it has been recently suggested that dipeptides like val - lys significantly contribute to the halostability in proteins . as described on results , the mcr - ew1 tirez phylotype allocated in methanomicrobiales shows an interestingly phylogenetic tendency to use amino acids , not initially biased by gc content or codon usage , that could be involved in a weak - moderate for example , slight increments of the polar residues asn , ser , and tyr , the negatively charged residues asp and glu , and the hydrophobic residues ala , ile , and pro in mcra methanomicrobiales phylotypes are in agreement with salt - in signals previously reported . charged amino acids prevent charged ions from attaching to proteins and thus they have a significant role in stabilizing proteins against salty conditions and keeping water molecules around these proteins [ 25 , 50 ] . similar to apra tirez phylotypes , we observed a decrement of arg and an increment of lys ( supplementary material , table s3 ) . the remaining mcra tirez phylotypes do not exhibit a clear tendency about expected aa composition , gc content , and codon usage bias to carry out salt - in haloadaptation . these phylotypes could compensate high salt extracellular concentrations through mechanisms independent of amino acid composition and gc content and that do not compromise the enzymatic activity . the salt - out strategy requires the accumulation of specific small - molecular - weight compounds ( i.e. , compatible solutes or osmolytes ) into the cytoplasm . salt - out signal can be expected on the mcra tirez phylotypes close clustered at methanohalobium evestigatum and methanohalobium sp . species belonging to the methanosarcinales . it is also well know that m. evestigatum uses methylated compounds such as methylamine and methanol to generate methane . these methylated substrates not only provide more energy to m. evestigatum than the use of others substrates for anabolic reactions , including the synthesis of compatible solutes , but also allow a tolerance up to 29.2% of nacl . in salt - out strategy , little or no adjustment is required to intracellular macromolecules ; in fact , the compatible solutes often act as more general stress protectants as well as just osmoprotectants . furthermore , halophiles do not live at constant salt concentrations ; but in many natural settings they are exposed to changing salinities due to evaporation or rain , and thus also the intracellular conditions change considerably . accordingly , enzyme activity on salt - in halophilic strategy will depend not only on the nature and concentration of the salt , but also on extensive genetic alterations as a prerequisite for adaptation to a saline intracellular environment [ 24 , 27 ] . tajima 's neutrality test for the apra and mcra enzyme fragments ( used in this study ) shows that both gene markers are evolving under positive selection ( dapra = 3.13 and dapra = 2.96 ) ( supplementary material , table s4 ) . this means that key functional enzymes of anaerobic microorganisms on tirez lagoon could undergone extensive genetic alterations that , if they help the organism to cope and adapt with a saline intracellular environment , could be clearly differentiated and fast fixed on the populations . two clear examples of this flexible genetic alterations and selective fixation can be seen on apra sequences of same species but that were obtained from different strains : thermodesulforhabdus norvegica dsm 9990 ( ef442952.1 ) ; af418159.1 , and archaeoglobus fulgidus dsm 4304 ( pdb : 1jnr - a ) ; pdb : 2fja - a , , which show interesting amino acid changes from basic ( lys and k ) to polar ( gln , q and asn , n ) residues ( see figure 5 and supplementary figure s2 ) . in spite of the considerable diversity in nucleotide content and amino acid composition of the apra and mcra enzyme fragments involved in all analyses , it can be seen a crucial conservation of catalytic sites ( arg - r - trp - w in apra and phe - f - tyr - y - phe - f - gly - g in mcra ) as well as of cofactor and nucleotide binding sites in both gene markers ( figures s1 and s2 for apra and mcra aa alignments , resp . ) . as previously reported for mcra , the same conservative trend holds true for most of the surrounding residues of the apra and mcra catalytic sites . probably , the amino acid conservation and/or the structural localization of these catalytic regions on apra and mcra gene markers underestimate the general trend composition of salt - in adaptation from moderately to high halotolerant organisms in tirez lagoon . in fact , it is not possible to figure out at the moment if the diversity , weakness , or absence of amino acid , gc content , and codon usage patterns reported for tirez phylotypes in this study are a consequence of a minor and biased coverage of their not completely sequenced genomes or if these inconclusive trends are true salt - in signals or a consequence of the use of complementary salt - adaptation strategies in bioenergetically constrained species , given that tirez phylotypes have a clear anaerobic mode of life on highly saline and sulfate sediments . accordingly , we do not discard the presence of mixed types of osmoadaptation in apra and mcra tirez phylotypes , where k accumulates to high levels ( salt - in ) along with neutral and negatively charged organic solutes ( salt - out ) , as previously reported for many slightly and moderately halophilic methanogens . for example , methanohalophilus portucalensis grows in 2.0 m nacl and its intracellular concentration of k is 0.76 m , indicating that concentration of intracellular k need not be the same as that of extracellular na . presumably , m. portucalensis uses three zwitterions and other osmolytes to balance osmotic pressure [ 92 , 96 ] . likewise , k plays an important role in the response of methanococcus thermolithotrophicus to hyperosmotic ( increased nacl ) or hypoosmotic ( decreased nacl ) shock . at the beginning of higher nacl extracellular concentration , m. thermolithotrophicus internalizes k until reach a new steady - state intracellular concentration the k--glutamate pair functions as a temporary osmolyte whilst the nonmetabolizable zwitterion ( ne - acetyl - b - lysine ) is synthesized and accumulated by m. thermolithotrophicus exclusively in response to high salt concentrations [ 96 , 97 ] . the characterization of srp , sop , and ma diversity in tirez lagoon contributes to the knowledge of anaerobic diversity of microorganisms in athalassohaline systems and has inferences on the survival and adaptation of life under steep salt gradients . a characterization of the anaerobic diversity in tirez lagoon is a first step to explain functional issues such as why not all anaerobic dissimilatory pathways occur optimally in extreme biotopes and whether an anaerobic way of life faces higher energetic constraints in hypersaline systems in terms of salt composition . any quantitative interpretation can be inferred because pcr - dgge fingerprint is an inconclusive source of information and fluorescence in situ hybridization ( fish ) , parallel experiments designed specifically to quantify -proteobacteria and methanogen populations along the sediment profile ( winter and sediment ) , failed to yield any positive result ( data not shown ) . the structure and activity of hydrogenotrophic methanogenesis and acetoclastic sulfidogenesis under thalassic hypersaline systems have been extensively studied [ 98 , 99 ] . but , it should be kept in mind that tirez sediment is a sulfate - rich system with a peculiar salt composition , considering that in the evaporitic period minerals such as gypsum ( caso42h2o ) , epsomite ( mgso4 7h2o ) , and hexahydrite ( mgso46h2o ) are deposited and dominate over halite ( nacl ) , and most importantly , the sulfate has a relevant role in anaerobic systems as electron acceptor . thus , present results might be of importance for the understanding of acetate mineralization as a key process for carbon cycling in extreme environments . in tirez sulfate - rich sediment , among all the detected phylotypes , desulfonema magnum and methanoplanus petrolearius are the ecotypes of major interest due to energetic constraints ; therefore , these ecotypes constitute a probable signal of haloadaptation in anaerobic populations . although it was possible to characterize several anaerobic prokaryotes involved in distinctive metabolic lineages across the tirez sediment , dgge and phylogenetic analyses revealed a poor srp , sop , and ma phylotype composition ; probably underestimated in comparison with other extreme systems . nevertheless , extant conditions in tirez , as well as in other hypersaline environments , enable the persistence of low energetic anaerobic metabolic capabilities such as the halanaerobiales fermenting bacteria ( manuscript in prep . ) , which use a well - adapted fermentation of organic compounds to produce co2/h2 and volatile fatty acids ( vfa ) such as acetate by the use of the salt - in strategy . typically , the carbon cycle in halophilic communities implicates low rates of carbon mineralization to co2 which explains the accumulation of acetate at salt saturation levels . in addition to h2 and acetate , methylated compounds as fermentation products of compatible solutes can be mineralized by ma [ 78 , 103 ] . the perspective for the nitrogen cycle is different in tirez , its completion is predictable given that it shares the characteristics of other hypersaline systems , where methylotrophic ma contribute to nitrogen mineralization . about the sulfur cycle , the sulfate - reducing microorganisms were identified in the sulfate - rich sediment and represent probable suppliers of sulfide for sulfur - oxidizing populations . this understanding is useful to infer possible biological processes in analogous systems such as europa because the ocean present in the satellite is rich in sulfates and divalent cations and probably it is also in anoxic state [ 31 , 104 ] . phylogenetic studies have been traditionally determined by physiological characterization of marine species , and the records of anaerobic phylotypes in hypersaline systems are dominated by thalassic species . tirez lagoon has sabkha properties thus , it is a brine of interest to analyze strong spectra in salinity . also , tirez lagoon is characterized by a low chloride / sulfate ratio ; this is remarkable considering that sulfate serves as terminal electron acceptor in the marine systems ; however , few biological descriptions have been made when this anion is abundant under hypersaline conditions . using the pcr - dgge fingerprint technique for the functional adenosine-5-phosphosulfate ( apra ) and the methyl coenzyme m reductase ( mcra ) gene markers , we have confirmed the occurrence of hydrogenotrophic methanogenic and acetoclastic sulfate - reducing organisms in tirez sediment . despite the steep osmotic change along the year in the lagoon , changes in composition of pcr - dgge dendrogram reflected weak differences on winter - summer community structure . the persistence of desulfobacteraceae phylotypes in summer sediment as well as the finding of methanomicrobiales at the hypersaline and sulfate - rich sediment is remarkable ( hydrogenotrophic ma are outcompeted by srp in high concentrations of sulfate ) . probably , these ecotypes are energetically constrained and , unfortunately , our findings on amino acid and nucleotide compositions can not be currently conclusive regarding the halotolerant strategies carried out by tirez phylotypes until a large sequence data set can be achieved for these uncultured , anaerobic and bioenergetically constrained organisms . nevertheless , it looks like apra gene marker could be a useful salt - in indicator for different environmental ( e.g. , marine versus sedimentary ) samples , not only because its amino acid overrepresentation and codon usage bias well correlate with those found in halophiles but also because apra gene marker could exhibit a preferential use of amino acid ( e.g. , val and lys ) on sediments in contrast to those found in marine and aerobic environments . similarly , mcra gene marker shows an unexpected amino acid and nucleotide composition with nonclear salt - in signals exhibited . however , we speculate that the diverse and not conclusive salt - in signals in these ecotypes ( perhaps due to the absence of complete sequenced mcra genes ) could reflect that whereas protective osmolytes salt - out can be produced by ma tirez populations in response to salt stress , probably also a weak salt - in strategy may contribute to adaptation of osmotic stress on sedimentary ma tirez populations . an extended understanding for acetoclastic sulfate reducing activity under high osmolarity conditions is needed in order to elucidate mechanisms that are involved in the biological carbon mineralization . on the long term , the findings of this work will provide valuable information to determine habitable conditions of europa , the most interesting moon of jupiter for the astrobiology field , as an anoxic and hypersaline environment . l. montoya , i. lozada - chvez , i. marn and r. amils conceived the study . l. montoya , i. marn , r. amils and n. rodriguez were involved in the fieldwork . l. montoya and i. lozada - chvez performed the analysis and interpretation of data , and wrote the paper . all authors read , improved , and approved the final paper .

our goal was to examine the composition of methanogenic archaea ( ma ) and sulfate - reducing ( srp ) and sulfur - oxidizing ( sop ) prokaryotes in the extreme athalassohaline and particularly sulfate - rich sediment of tirez lagoon ( spain ) . thus , adenosine-5-phosphosulfate ( aps ) reductase ( apra ) and methyl coenzyme m reductase ( mcra ) gene markers were amplified given that both enzymes are specific for srp , sop , and ma , respectively . anaerobic populations sampled at different depths in flooded and dry seasons from the anoxic sediment were compared qualitatively via denaturing gradient gel electrophoresis ( dgge ) fingerprint analysis . phylogenetic analyses allowed the detection of srp belonging to desulfobacteraceae , desulfohalobiaceae , and peptococcaceae in -proteobacteria and firmicutes and sop belonging to chromatiales / thiotrichales clade and ectothiorhodospiraceae in -proteobacteria as well as ma belonging to methylotrophic species in methanosarcinaceae and one hydrogenotrophic species in methanomicrobiaceae . we also estimated amino acid composition , gc content , and preferential codon usage for the apra and mcra sequences from halophiles , nonhalophiles , and tirez phylotypes . even though our results can not be currently conclusive regarding the halotolerant strategies carried out by tirez phylotypes , we discuss the possibility of a plausible salt - in signal in srp and sop as well as of a speculative complementary haloadaptation between salt - in and salt - out strategies in ma .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion Authors' Contribution

a similar pattern has been described for sulfate - reducing prokaryotes : acetoclastic sulfate reducers ( g = 47.6 kj mol substrate ) , most of them belonging to desulfobacteraceae , cease to tolerate high osmolarity conditions , for example , desulfobacter halotolerans grows up to 13% nacl ; on the other hand , desulfohalobiaceae members have higher salt tolerances ( up to 25% nacl ) and grow with h2/co2 ( g = 152.2 kj mol ) or lactate ( g = 160 kj / mol ) . one interesting ephemeral and sulfate - rich system is tirez lagoon , or sabkha , because it is subjected to flooding / desiccation regimes , located in la mancha , an arid region in spain . given this scenario , the primary objective of this study was to characterize the composition of the anaerobic populations in the ephemeral and sulfate rich tirez lagoon . the identification of environmental sulfate reducing prokaryotes ( srp ) and sulfur oxidizing prokaryotes ( sop ) can be performed by enrichment culturing and molecular ecology fingerprinting ; however , a characterization of methanogenic archaea ( ma ) through isolation techniques is problematic given their slow growth rates . the use of molecular ecology fingerprinting techniques such as denaturing gradient gel electrophoresis ( dgge ) from pcr - amplified genes is informative to assess the temporal and spatial qualitative diversity in natural samples , and it also requires fewer sequencing resources in comparison to clone libraries and/or metagenomic analysis . in the dissimilatory pathways of sulfate reduction and sulfur oxidation , dissimilatory sulfite reductase ( dsr ) and adenosine-5-phosphosulfate ( aps ) reductase ( apr ) are considered as key enzymes . for the sulfur - oxidizing pathway the alpha subunits of apr and dsr enzymes are found in all known srp and most of sop . regarding the methanogenesis pathway , the methyl coenzyme - m reductase ( mcr ) ( ec : 2.8.4.1 ) catalyses the reduction of a methyl group bound to coenzyme - m , with the concomitant release of methane . the mutation rates and selective pressures of the apra and mcra metabolic gene markers and of the structural 16s rrna gene are different ; however , phylogenetic studies done with partial sequences of apra and mcra belonging to the srp , sop , and ma lineages have established an agreement with the phylogenetic relationships based on 16s rrna gene sequences [ 13 , 18 ] . indeed , databases have been enriched in sequences of model strains for these two enzymes ; as a consequence , the apra and mcra gene markers also provide us information to identify srp , sop , and ma selectively in complex microbial communities , for example , . all eukaryotes , most halophilic bacteria , and the halophilic methanogenic archaea ( such as methanohalobium evestigatum ) have evolved the the widely disparate taxonomic position of salt - in prokaryotes ( halobacteriales in archaea , salinibacter ruber , and halanaerobiales in bacteria ) suggests a convergent evolution of this osmoadaptation strategy . given that other specific features of nucleotide selection may also be involved in the gc content of organisms , the gc - bias measurements are complementary to the amino acid composition but not decisive in order to infer the salt - in strategy . the translations of the apra and mcra sequences into amino acids were defined using the translate tool with a standard code ( http://expasy.org/tools/dna.html/ ) . the best frames for all the apra and mcra fragments were firstly selected by the unstopped amino acid ( aa ) sequences and , secondly , by matching their best hits with those compiled in the nonredundant database of the genbank , which were detected through the blastp program ( e - value 10 ) . from a first approach , we also included apra and mcra sequences from reported environmental samples as seed sequences . a complete list of the sequences included in this study to reconstruct a phylogeny for the apra and mcra enzymes can be seen in supplementary material ( tables s1 and s2 , resp . ) these functional domains have been characterized from archaeoglobus fulgidus in the reduced state ( fadred - aps , pdb i d : 1jnr ) and in the oxidized state ( fadox - aps , pdb i d : 2fja ) as well as in desulfovibrio gigas ( pdb i d : 3gyx ) . we calculated the amino acid composition of all apra and mcra sequences used previously to construct the phylogenies . we also calculated two halophilia indicators from the amino acid composition of every sequence : the pab factor estimates the surplus of polar and acidic amino acid compared to polar and basic ones ( i.e. a correction for the amino acid usage was applied by the calculation of the relative synonymous codon usage ( rscu ) values from the nucleotide datasets based on salinity adaptation : tirez , halophilic , and nonhalophilic species . we applied a denaturing gradient gel electrophoresis ( dgge ) fingerprinting analysis through the use of two functional genes : adenosine-5-phosphosulfate reductase ( apr ) and the methyl coenzyme - m reductase ( mcr ) , in order to identify ecotypes from the sediments samples and nonaxenic cultures of tirez lagoon . at 1525 cm depth , low yield or no pcr product given that a considerable number of environmental bands from the apra dgge profile could not be sequenced or specifically identified , probably due to the presence of residual pcr inhibitors such as humic acids coextracted with genomic dna that were not purified by the jetquick kit and that comigrate with dna in the polyacrylamide gel changes in population distribution were estimated through the use of p - analysis with maximum likelihood ( ml ) in phylip software in order to identify a significant clustering . thereby , we had to perform a nested pcr from the mcra gene in order to improve the detection of the ma community in the sediment samples from tirez . we also obtained a dgge pattern from sediment sampled in flooded and dry seasons at different depths ( figure 4(c ) ) . phylogenetic reconstructions were done for the inferred amino acid ( aa ) sequences of apra and mcra gene markers and their homologous counterparts . it is important to note that not only the phylogenetic topologies obtained for the apra_alpha_n and mcra_alpha_c sequences are robust , as can be seen by the significant bootstrap values in the main clustering branches , but also the internal groups are supported by the expected clustering of the mcra and apra crystals previously characterized for ( a ) the mcra in methanosarcina barkeri belonging to methanosarcinales , methanothermobacter thermoautotrophicus from methanobacteriales and methanopyrus kandleri in methanopyrales ; ( b ) the apra from archaeoglobus fulgidus in euryarchaeota , and desulfovibrio gigas in deltaproteobacteria . cultured and environmental srp populations were identified as follows : cultured phylotypes ( desulfohalobiaceae , peptococcaceae , and desulfobacteraceae ) and environmental phylotyeps ( desulfobacteraceae , and peptococcaceae ) . actually , the affiliation of phylotype aps - es29 and other sop tirez phylotypes could become particularly uncertain given the well - known horizontal aps reductase ( apr ) gene transfer ( hgt ) events between the srps from firmicutes and -proteobacteria as well as between the sops from and -proteobacteria , respectively ( see figure 5 ) . in four of the environmental sop phylotypes ( aps - ew7 , -ew8 , and -ew13 , aps - es28 ) , the assignment of the apra gene fragment could not be conclusive at species level ; thus , a detailed function in tirez 's system remains uncertain . given that the catalysis of apra and mcra enzymes occur in the cytoplasm , we were interested in determine whether tirez apra and mcra sequences show a bias when compared to their halophilic and nonhalophilic homologous counterparts . thus , we calculated the aa composition from the alignment used to reconstruct the phylogeny in order to estimate the hydrophobic ( gly , leu , val , ile , phe , met , ala , trp , and pro ) , polar ( ser , thr , cys , tyr , gln , and asn ) , basic ( his , arg , and lys ) and acidic ( glu and asp ) contents of the apra and mcra enzyme fragments analyzed in this study . for this purpose , we divided the sequence profiles from each gene markers into two data sets the first one is based on salinity adaptation ( tirez , halophilic and nonhalophilic species ) and the second one is based on the major taxonomic groups in which the tirez phylotypes are phylogenetically allocated ( apra : desulfovibrionales , desulfobacterales , peptococcales , and chromatiales ; mcra : methanomicrobiales and methanosarcinales ) ( supplementary material , tables s1 and s2 , resp . ) however , the observed differences in pab and ab indicators between tirez phylotypes and halophilic species are out of proportion to argue a salt - in signal in tirez phylotypes given that apra differences are more than ten times less the difference between escherichia coli and halobacterium salinarum or halomonas elongata and halobacterium salinarum . the total gc content of apra tirez phylotypes , halophiles , and nonhalophiles organisms is 57.60% , 55.70% , and 55.00% , respectively . the gc content of tirez phylotypes is higher than the reported for escherichia coli ( 50.3% ) but lower than the extreme halotolerant species from the dead sea metagenome ( 6267% ) and halobacterium salinarum ( 65.7% ) ( table 1 ) . even though arg ( cgg , agg ) , ala ( gcg ) , and cys ( ugc ) are underrepresented amino acids in apra tirez phylotypes as well as leu ( cug ) and gly ( ggc , ggg ) do not show compositional differences when compared with halophiles and nonhalophiles sequences ( supplementary material , figure s1b ) , all of them show a significant codon usage with gc bias ( rscu > 1.5 ) ( figure 7 ) . accordingly , the first , second , and third codon positions of apra tirez phylotypes have gc percentages of 54.2% , 42.4% , and 76.0% , respectively , and they agree with the gc content values previously reported in some salt - in halophiles ( table 1 ) , with high gc content and a third position gc bias [ 21 , 29 ] . in contrast to the apra tirez phylotypes , the total gc content ( 47.20% ) and the third codon gc bias ( 46.10% ) are significantly lower than the estimated for halophiles and nonhalophilic species ( table 1 ) . even though lys ( aaa ) , asp ( gau ) , ser ( ucc , ucu ) , and thr ( aca ) are underrepresented amino acids in mcra tirez phylotypes in comparison to halophiles and nonhalophiles sequences ( supplementary material , figure s1b ) , all of them show a preferential codon usage . stress response in d. vulgaris is based on genes with homologous in diverse and distant species such as bacillus subtilis ; thus , the finding of peptococcaceae in tirez , under analog bioenergetic constraints , could be explained in the terms of the salt - out strategy ( see section 4 ) . the presence of methanohalobium evestigatum and methanolobus zinderi in the sulfate - rich and anoxic sediment is easily sustained by functional arguments , even in summer samples , because their metabolism requires methylated substrates ; thus , it is noncompetitive with srp . even though salt - in strategy was recently proposed to explain the finding of the resilience desulfobacteraceae at hypersaline and alkaline lakes , this salt - adaptation strategy has been neither reported in species of the srp - sop nor in ma ; in part , given therefore , we consider it useful to use apra and mcra markers to test salt - in signals . our results can not be conclusive regarding the halotolerant strategies carried out by tirez phylotypes , until a large sequence data set can be achieved for these organisms . nevertheless , the amino acid composition , gc content , and preferential codon usage trends exhibited by the apra marker from tirez phylotypes suggest a plausible salt - in signal when compared to halophiles and non - haphiles . the increase in negatively charged ( asp and glu ) and polar ( ser , asn , and gln ) residues in apra tirez phylotypes can be explained by a codon usage with gc bias at the third position . the overrepresentation of these amino acid residues and their preferential codon usage are consistent with reports on salt - in adaptation [ 22 , 25 ] . as described on results , the mcr - ew1 tirez phylotype allocated in methanomicrobiales shows an interestingly phylogenetic tendency to use amino acids , not initially biased by gc content or codon usage , that could be involved in a weak - moderate for example , slight increments of the polar residues asn , ser , and tyr , the negatively charged residues asp and glu , and the hydrophobic residues ala , ile , and pro in mcra methanomicrobiales phylotypes are in agreement with salt - in signals previously reported . the remaining mcra tirez phylotypes do not exhibit a clear tendency about expected aa composition , gc content , and codon usage bias to carry out salt - in haloadaptation . in salt - out strategy , little or no adjustment is required to intracellular macromolecules ; in fact , the compatible solutes often act as more general stress protectants as well as just osmoprotectants . tajima 's neutrality test for the apra and mcra enzyme fragments ( used in this study ) shows that both gene markers are evolving under positive selection ( dapra = 3.13 and dapra = 2.96 ) ( supplementary material , table s4 ) . in spite of the considerable diversity in nucleotide content and amino acid composition of the apra and mcra enzyme fragments involved in all analyses , it can be seen a crucial conservation of catalytic sites ( arg - r - trp - w in apra and phe - f - tyr - y - phe - f - gly - g in mcra ) as well as of cofactor and nucleotide binding sites in both gene markers ( figures s1 and s2 for apra and mcra aa alignments , resp . ) probably , the amino acid conservation and/or the structural localization of these catalytic regions on apra and mcra gene markers underestimate the general trend composition of salt - in adaptation from moderately to high halotolerant organisms in tirez lagoon . in fact , it is not possible to figure out at the moment if the diversity , weakness , or absence of amino acid , gc content , and codon usage patterns reported for tirez phylotypes in this study are a consequence of a minor and biased coverage of their not completely sequenced genomes or if these inconclusive trends are true salt - in signals or a consequence of the use of complementary salt - adaptation strategies in bioenergetically constrained species , given that tirez phylotypes have a clear anaerobic mode of life on highly saline and sulfate sediments . accordingly , we do not discard the presence of mixed types of osmoadaptation in apra and mcra tirez phylotypes , where k accumulates to high levels ( salt - in ) along with neutral and negatively charged organic solutes ( salt - out ) , as previously reported for many slightly and moderately halophilic methanogens . the characterization of srp , sop , and ma diversity in tirez lagoon contributes to the knowledge of anaerobic diversity of microorganisms in athalassohaline systems and has inferences on the survival and adaptation of life under steep salt gradients . but , it should be kept in mind that tirez sediment is a sulfate - rich system with a peculiar salt composition , considering that in the evaporitic period minerals such as gypsum ( caso42h2o ) , epsomite ( mgso4 7h2o ) , and hexahydrite ( mgso46h2o ) are deposited and dominate over halite ( nacl ) , and most importantly , the sulfate has a relevant role in anaerobic systems as electron acceptor . in tirez sulfate - rich sediment , among all the detected phylotypes , desulfonema magnum and methanoplanus petrolearius are the ecotypes of major interest due to energetic constraints ; therefore , these ecotypes constitute a probable signal of haloadaptation in anaerobic populations . although it was possible to characterize several anaerobic prokaryotes involved in distinctive metabolic lineages across the tirez sediment , dgge and phylogenetic analyses revealed a poor srp , sop , and ma phylotype composition ; probably underestimated in comparison with other extreme systems . about the sulfur cycle , the sulfate - reducing microorganisms were identified in the sulfate - rich sediment and represent probable suppliers of sulfide for sulfur - oxidizing populations . using the pcr - dgge fingerprint technique for the functional adenosine-5-phosphosulfate ( apra ) and the methyl coenzyme m reductase ( mcra ) gene markers , we have confirmed the occurrence of hydrogenotrophic methanogenic and acetoclastic sulfate - reducing organisms in tirez sediment . the persistence of desulfobacteraceae phylotypes in summer sediment as well as the finding of methanomicrobiales at the hypersaline and sulfate - rich sediment is remarkable ( hydrogenotrophic ma are outcompeted by srp in high concentrations of sulfate ) . probably , these ecotypes are energetically constrained and , unfortunately , our findings on amino acid and nucleotide compositions can not be currently conclusive regarding the halotolerant strategies carried out by tirez phylotypes until a large sequence data set can be achieved for these uncultured , anaerobic and bioenergetically constrained organisms .

within the group of cranial vascular lesions , brain arteriovenous malformations ( bavms ) represent an abnormal communication between pial arteries supplying brain tissue and pial veins draining brain tissue . such abnormal communications can be as simple as to consist of a single fistulous connection , generally denoted a pial arteriovenous fistula , or consist of an extensive network of abnormal intervening vessels ; a so - called nidus . the latter is usually of a compact or glomerular nature but may also be of a more diffuse or proliferative nature [ 2 , 3 ] . the imaging characteristics of bavms will depend on various aspects of the lesion , such as the location , nature , and size of the nidus , whether or not recruited arterial feeders have increased in size to accommodate for the fistulous flow , whether or not the resulting fistulous flow has led to venous ectasias , whether the shunt interferes with blood supply to the brain by means of arterial steal or venous congestion , and whether prior bleeding has occurred . many of these characteristics can be noted on planar imaging , such as computed tomography ( ct ) or magnetic resonance imaging ( mri ) , especially in larger lesions . however , the gold standard to demonstrate an arteriovenous shunt , which is necessary to demonstrate small lesions and to distinguish mimicking lesions like proliferative angiopathy [ 3 , 4 ] , is catheter - based digital subtraction angiography ( dsa ) . dsa further helps to identify the involved feeding arteries and draining veins and to determine possible endovascular treatment strategies [ 2 , 5 ] . in addition , dsa can provide those angioarchitectural features that are important for treatment decision making . unfortunately , dsa is relatively expensive and time - consuming and carries a rather high incidence of silent embolic events and a small risk of transient or permanent neurologic deterioration [ 79 ] . although the complication rate is most likely especially low in the relatively young , and otherwise healthy , avm patient population , a noninvasive alternative angiographic method is of interest . in recent years , mri angiography ( mra ) and ct angiography ( cta ) have shown sufficient spatial resolution to answer many clinical neurovascular questions [ 1013 ] . however , these vessel - cast techniques lacked the temporal resolution necessary to demonstrate an arteriovenous shunt ( i.e. , early venous filling ) or to visualize feeding and draining patterns ( e.g. , high flow versus low flow shunts , presence or absence of venous rerouting ) . more recently , time - resolved techniques have emerged for both mra and cta [ 15 , 16 ] . aside from generating cross - sectional images , these datasets enable visualization of blood flow dynamics in cranial vessels with the first pass of an intravenous ( iv ) contrast bolus . hence , it is conceivable that they may replace dsa in cases in which time - resolved imaging is required . we aimed to evaluate the advantages and disadvantages of whole head time - resolved cta ( 4d - cta ) in the clinical setting of a consecutive series of patients with an untreated bavm , for whom dsa imaging was already available . approval for this study was obtained from our institutional research ethics board in november 2008 , and patients were admitted to the study between november 2008 and march 2010 . patients were included if dsa demonstrated an untreated bavm . exclusion criteria were patient age of below 18 years , current treatment for diabetes mellitus , known allergy for iodinated contrast agents , and renal failure ( indicated by a baseline serum creatinine above 133 mol / l ) . after obtaining informed consent , 4d - cta imaging was performed . all dsa and 4d - cta studies were anonymized and a panel of three readers ( authors pw , pt , and tk ) performed a consensus reading . for each patient , the panel was blinded for the dsa results at the time of reading the 4d - cta . subsequently , a standardized scoring sheet was filled out for each diagnostic study ( see below ) . diagnostic intra - arterial dsa was performed with standard biplane fluoroscopy equipment ( infinix , toshiba medical systems , japan ; or lcl - p , ge healthcare , uk ) . since patients were only included in the study after dsa had demonstrated a bavm , the angiographic protocol was not influenced by the study . it generally consisted of bilateral injections of the internal carotid arteries ( ica ) , external carotid arteries ( eca ) and at least one vertebral artery ( va ) . during each injection anterior posterior ( ap ) and lateral projections were obtained at 3 fps . all 4d - cta examinations were performed using an aquilion one multidetector ct scanner ( toshiba medical systems , japan ) , equipped with 320 0.5 mm detector rows covering 16 cm of volume per rotation . imaging was performed in a manner previously described [ 16 , 17 ] . in short , intravenous infusion of 60 ml of nonionic contrast medium , followed by 20 ml of saline , was followed by a dynamic acquisition sequence with a gantry rotation speed of 1 rotation per second . the dynamic acquisition sequence consisted of one mask volume ( 80 kvp , 300 mas ) and 22 dynamic volumes ( 80 kvp , 120 mas ) . after subtracting the mask volume from the dynamic volumes , a total of 7,040 ( 22 320 ) images were stored in dicom files . using these files , the standard scanner software generated time - resolved ( arterial to venous ) maximum intensity projections ( mips ) at different viewing angles . both the 4d - cta examinations and the dsa examinations were scored with the same scoring sheet . items to be scored included presence or absence of a shunt , grading according to the spetzler martin grade ( size , eloquence of the location , presence of deep venous drainage ) but also additional angioarchitectural features that are routinely reported upon in our institutions when reading out bavm studies on dsa . these included for the arterial aspect of the angiographies : feeding arteries , presence of dural supply , arterial enlargement , flow - related aneurysms and presence of a transfer of the watershed ( i.e. , indirect supply to an arterial territory via leptomeningeal collaterals ) . concerning the nidus , we evaluated the nidus type ( micro bavm or fistula versus macro bavm ) and the flow volume ; concerning the venous aspect of the angiographies stagnation of contrast material , venous outflow obstruction and the presence of venous pouches were sought after . approval for this study was obtained from our institutional research ethics board in november 2008 , and patients were admitted to the study between november 2008 and march 2010 . patients were included if dsa demonstrated an untreated bavm . exclusion criteria were patient age of below 18 years , current treatment for diabetes mellitus , known allergy for iodinated contrast agents , and renal failure ( indicated by a baseline serum creatinine above 133 mol / l ) . after obtaining informed consent , 4d - cta imaging was performed . all dsa and 4d - cta studies were anonymized and a panel of three readers ( authors pw , pt , and tk ) performed a consensus reading . for each patient , the panel was blinded for the dsa results at the time of reading the 4d - cta . subsequently , a standardized scoring sheet was filled out for each diagnostic study ( see below ) . diagnostic intra - arterial dsa was performed with standard biplane fluoroscopy equipment ( infinix , toshiba medical systems , japan ; or lcl - p , ge healthcare , uk ) . since patients were only included in the study after dsa had demonstrated a bavm , the angiographic protocol was not influenced by the study . it generally consisted of bilateral injections of the internal carotid arteries ( ica ) , external carotid arteries ( eca ) and at least one vertebral artery ( va ) . during each injection anterior posterior ( ap ) and lateral projections were obtained at 3 fps . all 4d - cta examinations were performed using an aquilion one multidetector ct scanner ( toshiba medical systems , japan ) , equipped with 320 0.5 mm detector rows covering 16 cm of volume per rotation . imaging was performed in a manner previously described [ 16 , 17 ] . in short , intravenous infusion of 60 ml of nonionic contrast medium , followed by 20 ml of saline , was followed by a dynamic acquisition sequence with a gantry rotation speed of 1 rotation per second . the dynamic acquisition sequence consisted of one mask volume ( 80 kvp , 300 mas ) and 22 dynamic volumes ( 80 kvp , 120 mas ) . after subtracting the mask volume from the dynamic volumes , a total of 7,040 ( 22 320 ) images were stored in dicom files . using these files , the standard scanner software generated time - resolved ( arterial to venous ) maximum intensity projections ( mips ) at different viewing angles . both the 4d - cta examinations and the dsa examinations were scored with the same scoring sheet . items to be scored included presence or absence of a shunt , grading according to the spetzler martin grade ( size , eloquence of the location , presence of deep venous drainage ) but also additional angioarchitectural features that are routinely reported upon in our institutions when reading out bavm studies on dsa . these included for the arterial aspect of the angiographies : feeding arteries , presence of dural supply , arterial enlargement , flow - related aneurysms and presence of a transfer of the watershed ( i.e. , indirect supply to an arterial territory via leptomeningeal collaterals ) . concerning the nidus , we evaluated the nidus type ( micro bavm or fistula versus macro bavm ) and the flow volume ; concerning the venous aspect of the angiographies stagnation of contrast material , venous outflow obstruction and the presence of venous pouches were sought after . between november 2008 and march 2010 , 17 consecutive patients with an untreated bavm were included , 6 male and 11 female . table 1 shows the distribution of presenting symptoms and table 2 shows the characteristics of the bavms within our patient group , according to the items in the standardized scoring sheet , with the results of 4d - cta and dsa analysis . of the four patients who presented with hemorrhage , only one was imaged in the acute phase . the other three were imaged 4 months , 11 months , and 5 years after hemorrhage . table 1distribution of presenting symptoms of the patients enrolled in this studypresentationnhemorrhage4epilepsy5neurologic deficit2headache1incidental3unknown2total17table 2items scored by the reading panel for each diagnostic study and results of both 4d - cta and dsaitemoptions4d - ctadsashunt detected ( early venous filling)yes / no1717bavm diagnosedyes / no1717spetzler martin grade size(1 ) <3 eloquence(0 ) non - eloquent location55(1 ) eloquent location1212 drainage(0 ) superficial drainage only109(1 ) deep drainage present78arterial feeding territoriesanterior circulation ( ac)1515posterior circulation ( pc)56(or both)(3)(4)arterial feedersac deep branches from the anterior cerebral artery11 cortical branches from the anterior cerebral artery54 deep branches from the middle cerebral artery54 cortical branches from the middle cerebral artery1011 branches from the internal carotid artery , anterior choroidal artery or ophthalmic system00pc branches from the vertebral artery or pica00 branches from the basilar artery or aica00 branches from the superior cerebellar artery11 deep branches from the posterior cerebral artery12 cortical branches from the posterior cerebral artery33(or any combination of these)dural artery supplyyes / no11watershed transferyes / no36arterial enlargementyes / no1012flow related aneurysm(s)yes / no00nidus typemicro ( < 1 cm ) or fistula / macro ( > 1 cm)5/121/16bavm flowlow flow / high flow6/116/11intranidal stasis of contrastyes / no00venous outflow obstructionyes / no00venous pouch(es)yes / no99pica posterior inferior cerebellar artery , aica anterior inferior cerebellar arteryin yes / no items , n represents the number of patients who scored yessince we were only interested in the diagnostic performance of each study , we excluded the spetzler martin category inoperable ( 6)4d - cta missed deep venous drainage in one patientin one case , the posterolateral choroidal artery feeding the bavm was mistakenly interpreted as a lenticulostriate perforator in the 4d - ctaindirect feeding through leptomeningeal collaterals ( watershed transfer ) was erroneously interpreted as direct feeding in one case in 4d - ctacortical branches of the anterior and middle cerebral artery feeding the bavm were interpreted by 4d - cta to be from the anterior cerebral artery only , in one casewatershed transfer with hypoxemia induced nonsprouting angiogenesis was missed by 4d - cta in three cases : these vessels were too small , interpreted as direct feeders ( see footnote e ) or interpreted as part of the nidus ( one case each)arterial enlargement of feeding vessel(s ) was too mild too be noticed by 4d - cta in two cases4d - cta underestimated nidus size in 4 cases , interpreting them as micro ( < 1 cm ) whereas dsa demonstrated them to be macro ( > 1 cm ) distribution of presenting symptoms of the patients enrolled in this study items scored by the reading panel for each diagnostic study and results of both 4d - cta and dsa pica posterior inferior cerebellar artery , aica anterior inferior cerebellar artery in yes / no items , n represents the number of patients who scored yes since we were only interested in the diagnostic performance of each study , we excluded the spetzler martin category inoperable ( 6 ) 4d - cta missed deep venous drainage in one patient in one case , the posterolateral choroidal artery feeding the bavm was mistakenly interpreted as a lenticulostriate perforator in the 4d - cta indirect feeding through leptomeningeal collaterals ( watershed transfer ) was erroneously interpreted as direct feeding in one case in 4d - cta cortical branches of the anterior and middle cerebral artery feeding the bavm were interpreted by 4d - cta to be from the anterior cerebral artery only , in one case watershed transfer with hypoxemia induced nonsprouting angiogenesis was missed by 4d - cta in three cases : these vessels were too small , interpreted as direct feeders ( see footnote e ) or interpreted as part of the nidus ( one case each ) arterial enlargement of feeding vessel(s ) was too mild too be noticed by 4d - cta in two cases 4d - cta underestimated nidus size in 4 cases , interpreting them as micro ( < 1 cm ) whereas dsa demonstrated them to be macro ( > 1 cm ) all 17 bavms were detected with 4d - cta . with regard to the spetzler martin grade , there was disagreement in one case , where deep drainage was not detected with 4d - cta . interestingly , four of the remaining seven cases with deep venous drainage showed this deep drainage to be indirect from rerouting of cortical veins draining the bavm , all of which were correctly identified with 4d - cta ( fig . 1 ) . 1example of a patient with a spetzler martin grade 3 bavm with indirect deep venous drainage , recognized in the lateral projection of the 4d - cta mip ( a ) and the dsa ( b ) . early drainage to the superior sagittal sinus ( large arrows ) precedes indirect drainage to the straight sinus ( small arrow ) through the sylvian vein ( large arrowhead ) and the basal vein of rosenthal ( small arrowhead ) . though this can be recognized in these images through differences in opacification , the analysis is simplified when studying the entire time - resolved series example of a patient with a spetzler martin grade 3 bavm with indirect deep venous drainage , recognized in the lateral projection of the 4d - cta mip ( a ) and the dsa ( b ) . early drainage to the superior sagittal sinus ( large arrows ) precedes indirect drainage to the straight sinus ( small arrow ) through the sylvian vein ( large arrowhead ) and the basal vein of rosenthal ( small arrowhead ) . though this can be recognized in these images through differences in opacification , the analysis is simplified when studying the entire time - resolved series though there was agreement in all cases in the size classification used in the spetzler martin grade , the subclassification of small bavms into micro bavms ( < 1 cm , including pial fistulas ) versus macro bavms ( > 1 cm ) was not agreed upon in all cases ( fig . 2 ) . five lesions were denoted micro based on the 4d - cta , while dsa identified only a single fistulous micro bavm whereas the other four lesions were noted to be small bavms with a nidus size larger than 1 cm . martin grade 1 bavm , superficially in the right temporal area . based on the 4d - cta the nidus was scored as micro ( < 1 cm ) whereas the dsa demonstrated a macro nidus ( > 1 cm ) . lateral mip of the arterial phase of the 4d - cta ( a ) demonstrates two obvious draining veins ( large arrowheads ) and one faint one ( small arrowhead ) . an axial slice at the level of the presumed nidus in the same phase ( b ) suggests the nidus to be larger than suggested by the mip ( arrows ) . lateral projection of the late arterial phase of the dsa ( c ) demonstrates the nidus more clearly ( arrows ) . the rather diffuse nature of the nidus , possibly due to disruption by the presenting hemorrhage 4 months earlier , was thought to be the cause of poor detection by 4d - cta example of a patient with a small spetzler based on the 4d - cta the nidus was scored as micro ( < 1 cm ) whereas the dsa demonstrated a macro nidus ( > 1 cm ) . lateral mip of the arterial phase of the 4d - cta ( a ) demonstrates two obvious draining veins ( large arrowheads ) and one faint one ( small arrowhead ) . an axial slice at the level of the presumed nidus in the same phase ( b ) suggests the nidus to be larger than suggested by the mip ( arrows ) . lateral projection of the late arterial phase of the dsa ( c ) demonstrates the nidus more clearly ( arrows ) . the rather diffuse nature of the nidus , possibly due to disruption by the presenting hemorrhage 4 months earlier , was thought to be the cause of poor detection by 4d - cta with regard to arterial feeding territories ( anterior and/or posterior circulation ) , there was disagreement in one patient . in this case , the posterolateral choroidal artery feeding the bavm was mistakenly interpreted as a lenticulostriate perforator in the 4d - cta ( fig . looking at the feeding branches within the anterior and posterior circulation , discrepancies were found in two additional patients . one of these demonstrated indirect feeding from the anterior cerebral artery through pial collaterals to the middle cerebral artery territory . the other showed a lesion fed by cortical branches of the anterior and middle cerebral artery , while the 4d - cta suggested feeding from cortical branches of the anterior cerebral artery only . 3example of a patient with a spetzler martin grade 3 bavm in the right parietal lobe . a 4d - cta mip in the arterial phase ( a ) suggests an enlarged lenticulostriate perforator ( arrow ) feeding the deeper part of the nidus . the right ica injection of the dsa in the same phase and projection ( b ) does not confirm this feeder . the right va injection of the dsa in the same phase and projection ( c ) shows the vessel to be the right posterolateral choroidal artery ( arrow ) . the false interpretation of the 4d - cta , even when oblique mips were used , is due to the non - selective opacification of all cranial arteries with this technique example of a patient with a spetzler martin grade 3 bavm in the right parietal lobe . a 4d - cta mip in the arterial phase ( a ) suggests an enlarged lenticulostriate perforator ( arrow ) feeding the deeper part of the nidus . the right ica injection of the dsa in the same phase and projection ( b ) does not confirm this feeder . the right va injection of the dsa in the same phase and projection ( c ) shows the vessel to be the right posterolateral choroidal artery ( arrow ) . the false interpretation of the 4d - cta , even when oblique mips were used , is due to the non - selective opacification of all cranial arteries with this technique secondary dural and transosseous feeding of the bavm was only seen in one patient and was correctly detected in the 4d - cta ( fig . 4 ) . arterial enlargements , indicating feeding vessels , were seen in 12 cases , but were too mild to be noticed in the 4d - cta in two of these . indirect feeding through enlarged leptomeningeal collaterals from one cortical territory to another , also referred to as watershed transfer related to hypoxemia induced nonsprouting angiogenesis , could be seen in six cases , but were only detected in the 4d - cta of three of these ( fig . these indirect feeders were either too small to be noticed , interpreted as direct feeders or interpreted as part of the nidus ( one case each ) . 4images of the only patient in our series with secondary dural and transosseous feeding of a bavm . the lateral mip of the 4d - cta in early arterial phase ( a ) clearly shows an enlarged occipital artery ( large arrowhead ) running up to the lesion , as well as smaller posterior meningeal branches ( small arrowhead ) from the ipsilateral va ( arrow ) . differentiation of a purely dural av shunt is possible , however , due to depiction of the enlarged posterior cerebral artery feeding an intraparenchymal nidus . the lateral projection of the eca injection ( b ) proves the eca contribution , by opacification of the bavm and early opacification of the draining vein ( arrow)fig . 5example of a patient with a spetzler martin grade 3 bavm ( same case as fig . 1 ) illustrating superiority of dsa over 4d - cta to demonstrate indirect feeding of a bavm , i.e. , watershed transfer . the ap mip of the 4d - cta ( a ) depicts a right frontal bavm ( arrow ) , fed by branches from the middle cerebral artery ( arrowhead ) . axial images from the 4d - cta in early venous phase ( b ) at the level of the nidus ( upper image ) and above the level of the nidus ( lower image ) allow identification of the compact nidus ( arrow ) and feeding vessels ( arrowheads ) , but identification of indirect feeders remains challenging . ap dsa images after injection of the right ica ( c ) and left ica ( d ) clearly aid this discrimination by demonstrating recruitment of leptomeningeal anastomoses ( arrow ) which have developed to enhance pial collateral supply from the anterior to the middle cerebral artery images of the only patient in our series with secondary dural and transosseous feeding of a bavm . the lateral mip of the 4d - cta in early arterial phase ( a ) clearly shows an enlarged occipital artery ( large arrowhead ) running up to the lesion , as well as smaller posterior meningeal branches ( small arrowhead ) from the ipsilateral va ( arrow ) . differentiation of a purely dural av shunt is possible , however , due to depiction of the enlarged posterior cerebral artery feeding an intraparenchymal nidus . the lateral projection of the eca injection ( b ) proves the eca contribution , by opacification of the bavm and early opacification of the draining vein ( arrow ) example of a patient with a spetzler martin grade 3 bavm ( same case as fig . 1 ) illustrating superiority of dsa over 4d - cta to demonstrate indirect feeding of a bavm , i.e. , watershed transfer . the ap mip of the 4d - cta ( a ) depicts a right frontal bavm ( arrow ) , fed by branches from the middle cerebral artery ( arrowhead ) . axial images from the 4d - cta in early venous phase ( b ) at the level of the nidus ( upper image ) and above the level of the nidus ( lower image ) allow identification of the compact nidus ( arrow ) and feeding vessels ( arrowheads ) , but identification of indirect feeders remains challenging . ap dsa images after injection of the right ica ( c ) and left ica ( d ) clearly aid this discrimination by demonstrating recruitment of leptomeningeal anastomoses ( arrow ) which have developed to enhance pial collateral supply from the anterior to the middle cerebral artery 4d - cta and dsa agreed on flow type in all cases , judging six bavms to be low flow and 11 to be neither 4d - cta nor dsa detected intranidal stasis of contrast material in any of the lesions . venous obstruction , i.e. , occlusion of a dural sinus or internal jugular vein , was not detected in any patient . the ap mip of the 4d - cta ( a ) and the dsa image in ap projection after left ica injection ( b ) , both in late arterial phase , demonstrate the single arterial feeding branches ( arrows ) , the level of the pial fistula ( asterisk ) and multiple draining veins ( small arrowheads ) draining into the superior sagittal sinus . the largest draining vein narrows significantly before reaching the sinus ( large arrowhead ) images of the only patient in our series with venous stenosis . the ap mip of the 4d - cta ( a ) and the dsa image in ap projection after left ica injection ( b ) , both in late arterial phase , demonstrate the single arterial feeding branches ( arrows ) , the level of the pial fistula ( asterisk ) and multiple draining veins ( small arrowheads ) draining into the superior sagittal sinus . we aimed to offer an evaluation of the performance of time - resolved cta in the diagnostic work - up of untreated bavms and recognize its potential capabilities and pitfalls in the evaluation of angioarchitectural details deemed to be important for the possible treatment strategies . to date , the major drawbacks of non - invasive time - resolved vascular imaging are the inferior spatial and temporal resolution compared to dsa . with time - resolved mra ( trmra ) , there is a trade - off between spatial resolution and temporal resolution . with 4d - cta , temporal and spatial resolution are independent parameters ; but there is a trade - off between temporal resolution , signal - to - noise ratio and radiation dose . for the 4d - cta protocol used in this study , radiation dose was 5.2 msv or less . with further refinements of imaging parameters or acceptance of dose increase even with current protocols , however , a combination of 1 s image intervals and 0.5 mm isotropic voxel size renders 4d - cta superior to current trmra . a comparison of radiation dose between 4d - cta and dsa is not easy , since the radiation dose received during dsa is highly variable . it depends on the fluoroscopy time per vessel studied , the number of vessels studied , fluoroscopic parameters , type of fluoroscopic equipment , and so forth . we calculated the radiation dose of four typical cerebral dsas , performed at our center . the calculations were based on monte carlo simulations by using the dose - area product values and beam geometry information . ninety - five percent of this dose was estimated to have been delivered to the cranium , with minor exposure of the thorax , abdomen , and pelvis . the present comparison with dsa demonstrated that early venous filling was recognized by 4d - cta in all cases , independent of nidus size . in addition , the correct distinction between the type of feeding artery ( dural versus pial arteries ) could be made . although the present series did not include other shunting lesions ( such as dural av fistulae ) or normal controls , the correct identification of these two features , suggests that 4d - cta can correctly identify the type of shunting lesion in most instances . however , the drawbacks of 4d - cta came into play when evaluating the angioarchitecture of each lesion in finer detail , as has also been shown in other types of arteriovenous shunting lesions . not only did such detailed evaluation suffer from impaired spatial resolution , temporal resolution and signal - to - noise ratio , but also from non - selectivity of the vessel opacification . this non - selectivity eliminates the ability to isolate certain vessels and leads to a crowded it is more difficult to determine the nature of certain vessels and their relationship to the lesion at hand . in the present series , these characteristics of 4d - cta led to misinterpretation of a feeding vessel ( one case ) , misinterpretation of indirect feeding through pial collaterals ( three cases ) , oversight of mild arterial enlargement ( two cases ) and oversight of deep venous drainage ( one case ) . furthermore , 4d - cta interpreted four bavms to be smaller than the estimation based on dsa ( < 1 cm rather than > 1 cm ) . however , large inter - observer discrepancies in nidus delineation are known to occur with dsa as well . we believe evaluation of the cross - sectional 4d - cta ( source ) images is essential when reading 4d - cta data , since these images may contain information which has been lost in subsequent 3d reconstructions . 4d - cta cross - sectional images may also be analyzed in a time - resolved fashion and may be helpful to recognize the nidus ( fig . 2 ) and distinguish it from surrounding feeders ( fig . 5 ) . moreover , they can be used to observe structural changes not directly visible in the mips or in dsa imaging , such as hydrocephalus or a hematoma , and may aid surgical planning . other additional image sets that can be reconstructed from the same dynamic imaging sequence are perfusion maps , which may be used to assess the microcirculation of tissues surrounding the bavm . this microcirculation may be impaired by vascular steal phenomena or venous hypertension , which may impact on planning and risk estimation of bavm treatment [ 21 , 22 ] . although the capabilities of 4d - cta in the assessment of bavms appear promising , we feel caution is appropriate when attempting to change present imaging paradigms . firstly , no conclusion can be drawn with regard to flow related arterial aneurysms or intranidal ( pseudo- ) aneurysms , which may represent a target for endovascular treatment after intracranial hemorrhage [ 2 , 5 ] , since neither were present in this series . secondly , in our previous study regarding dural arteriovenous fistulas , a single lesion was missed due to its slow flow and the crowded appearance of the 4d - cta . it is conceivable that such errors may also occur when analyzing bavms , especially when small lesions with a low shunt volume are present . this question can only be answered by an unselected patient cohort in a prospective study that includes patients in whom a dsa is deemed necessary to rule out a shunting lesion so as to include patients with different types of pathology . until such a study is published , we suggest to use 4d - cta only as either a screening tool in the work - up of suspected patients , followed by dsa whenever negative , or as a follow - up tool when a lesion has been demonstrated previously and is managed conservatively . in this series of bavm patients , 4d - cta was able to detect all lesions . although some angioarchitectural details were missed or misinterpreted when compared to dsa , 4d - cta evaluation would have been sufficiently accurate to diagnose the shunt and classify it . we believe treatment planning may benefit from the accompanying availability of cross - sectional imaging in different planes and perfusion maps , which can be calculated from the presented data set . although these results await validation in a prospective unselected patient cohort , 4d - cta seems to be a valuable new adjunct in the non - invasive diagnostic work - up and follow - up of bavm patients .

introductionwe aimed to evaluate the use of time - resolved whole - head ct angiography ( 4d - cta ) in patients with an untreated arteriovenous malformation of the brain ( bavm ) , as demonstrated by catheter angiography ( dsa).methodsseventeen patients with a dsa - proven bavm were enrolled . these were subjected to 4d - cta imaging using a 320 detector row ct scanner . using a standardized scoring sheet , all studies were analyzed by a panel of three readers . this panel was blind to the dsa results at the time of reading the 4d-cta.results4d-cta detected all bavms . with regard to the spetzler martin grade , 4d - cta disagreed with dsa in only one case , where deep venous drainage was missed . further discrepancies between 4d - cta and dsa analyses included underestimation of the nidus size in small lesions ( four cases ) , misinterpretation of a feeding vessel ( one case ) , misinterpretation of indirect feeding through pial collaterals ( three cases ) and oversight of mild arterial enlargement ( two cases ) . 4d - cta correctly distinguished low - flow from high - flow lesions and detected dural / transosseous feeding ( one case ) , venous narrowing ( one case ) and venous pouches ( nine cases).conclusionin this series , 4d - cta was able to detect all bavms . although some angioarchitectural details were missed or misinterpreted when compared to dsa , 4d - cta evaluation was sufficiently accurate to diagnose the shunt and classify it . moreover , 4d - cta adds cross - sectional imaging and perfusion maps , helpful in treatment planning . 4d - cta appears to be a valuable new adjunct in the non - invasive diagnostic work - up of bavms and their follow - up when managed conservatively .

Introduction Materials and methods Patient selection and data collection DSA examination 4D-CTA examination Angiographical evaluation Results Discussion Conclusion

the imaging characteristics of bavms will depend on various aspects of the lesion , such as the location , nature , and size of the nidus , whether or not recruited arterial feeders have increased in size to accommodate for the fistulous flow , whether or not the resulting fistulous flow has led to venous ectasias , whether the shunt interferes with blood supply to the brain by means of arterial steal or venous congestion , and whether prior bleeding has occurred . we aimed to evaluate the advantages and disadvantages of whole head time - resolved cta ( 4d - cta ) in the clinical setting of a consecutive series of patients with an untreated bavm , for whom dsa imaging was already available . all dsa and 4d - cta studies were anonymized and a panel of three readers ( authors pw , pt , and tk ) performed a consensus reading . for each patient , the panel was blinded for the dsa results at the time of reading the 4d - cta . items to be scored included presence or absence of a shunt , grading according to the spetzler martin grade ( size , eloquence of the location , presence of deep venous drainage ) but also additional angioarchitectural features that are routinely reported upon in our institutions when reading out bavm studies on dsa . concerning the nidus , we evaluated the nidus type ( micro bavm or fistula versus macro bavm ) and the flow volume ; concerning the venous aspect of the angiographies stagnation of contrast material , venous outflow obstruction and the presence of venous pouches were sought after . after obtaining informed consent , 4d - cta imaging was performed . all dsa and 4d - cta studies were anonymized and a panel of three readers ( authors pw , pt , and tk ) performed a consensus reading . for each patient , the panel was blinded for the dsa results at the time of reading the 4d - cta . all 4d - cta examinations were performed using an aquilion one multidetector ct scanner ( toshiba medical systems , japan ) , equipped with 320 0.5 mm detector rows covering 16 cm of volume per rotation . both the 4d - cta examinations and the dsa examinations were scored with the same scoring sheet . items to be scored included presence or absence of a shunt , grading according to the spetzler martin grade ( size , eloquence of the location , presence of deep venous drainage ) but also additional angioarchitectural features that are routinely reported upon in our institutions when reading out bavm studies on dsa . concerning the nidus , we evaluated the nidus type ( micro bavm or fistula versus macro bavm ) and the flow volume ; concerning the venous aspect of the angiographies stagnation of contrast material , venous outflow obstruction and the presence of venous pouches were sought after . between november 2008 and march 2010 , 17 consecutive patients with an untreated bavm were included , 6 male and 11 female . table 1 shows the distribution of presenting symptoms and table 2 shows the characteristics of the bavms within our patient group , according to the items in the standardized scoring sheet , with the results of 4d - cta and dsa analysis . table 1distribution of presenting symptoms of the patients enrolled in this studypresentationnhemorrhage4epilepsy5neurologic deficit2headache1incidental3unknown2total17table 2items scored by the reading panel for each diagnostic study and results of both 4d - cta and dsaitemoptions4d - ctadsashunt detected ( early venous filling)yes / no1717bavm diagnosedyes / no1717spetzler martin grade size(1 ) <3 eloquence(0 ) non - eloquent location55(1 ) eloquent location1212 drainage(0 ) superficial drainage only109(1 ) deep drainage present78arterial feeding territoriesanterior circulation ( ac)1515posterior circulation ( pc)56(or both)(3)(4)arterial feedersac deep branches from the anterior cerebral artery11 cortical branches from the anterior cerebral artery54 deep branches from the middle cerebral artery54 cortical branches from the middle cerebral artery1011 branches from the internal carotid artery , anterior choroidal artery or ophthalmic system00pc branches from the vertebral artery or pica00 branches from the basilar artery or aica00 branches from the superior cerebellar artery11 deep branches from the posterior cerebral artery12 cortical branches from the posterior cerebral artery33(or any combination of these)dural artery supplyyes / no11watershed transferyes / no36arterial enlargementyes / no1012flow related aneurysm(s)yes / no00nidus typemicro ( < 1 cm ) or fistula / macro ( > 1 cm)5/121/16bavm flowlow flow / high flow6/116/11intranidal stasis of contrastyes / no00venous outflow obstructionyes / no00venous pouch(es)yes / no99pica posterior inferior cerebellar artery , aica anterior inferior cerebellar arteryin yes / no items , n represents the number of patients who scored yessince we were only interested in the diagnostic performance of each study , we excluded the spetzler martin category inoperable ( 6)4d - cta missed deep venous drainage in one patientin one case , the posterolateral choroidal artery feeding the bavm was mistakenly interpreted as a lenticulostriate perforator in the 4d - ctaindirect feeding through leptomeningeal collaterals ( watershed transfer ) was erroneously interpreted as direct feeding in one case in 4d - ctacortical branches of the anterior and middle cerebral artery feeding the bavm were interpreted by 4d - cta to be from the anterior cerebral artery only , in one casewatershed transfer with hypoxemia induced nonsprouting angiogenesis was missed by 4d - cta in three cases : these vessels were too small , interpreted as direct feeders ( see footnote e ) or interpreted as part of the nidus ( one case each)arterial enlargement of feeding vessel(s ) was too mild too be noticed by 4d - cta in two cases4d - cta underestimated nidus size in 4 cases , interpreting them as micro ( < 1 cm ) whereas dsa demonstrated them to be macro ( > 1 cm ) distribution of presenting symptoms of the patients enrolled in this study items scored by the reading panel for each diagnostic study and results of both 4d - cta and dsa pica posterior inferior cerebellar artery , aica anterior inferior cerebellar artery in yes / no items , n represents the number of patients who scored yes since we were only interested in the diagnostic performance of each study , we excluded the spetzler martin category inoperable ( 6 ) 4d - cta missed deep venous drainage in one patient in one case , the posterolateral choroidal artery feeding the bavm was mistakenly interpreted as a lenticulostriate perforator in the 4d - cta indirect feeding through leptomeningeal collaterals ( watershed transfer ) was erroneously interpreted as direct feeding in one case in 4d - cta cortical branches of the anterior and middle cerebral artery feeding the bavm were interpreted by 4d - cta to be from the anterior cerebral artery only , in one case watershed transfer with hypoxemia induced nonsprouting angiogenesis was missed by 4d - cta in three cases : these vessels were too small , interpreted as direct feeders ( see footnote e ) or interpreted as part of the nidus ( one case each ) arterial enlargement of feeding vessel(s ) was too mild too be noticed by 4d - cta in two cases 4d - cta underestimated nidus size in 4 cases , interpreting them as micro ( < 1 cm ) whereas dsa demonstrated them to be macro ( > 1 cm ) all 17 bavms were detected with 4d - cta . with regard to the spetzler martin grade , there was disagreement in one case , where deep drainage was not detected with 4d - cta . interestingly , four of the remaining seven cases with deep venous drainage showed this deep drainage to be indirect from rerouting of cortical veins draining the bavm , all of which were correctly identified with 4d - cta ( fig . 1example of a patient with a spetzler martin grade 3 bavm with indirect deep venous drainage , recognized in the lateral projection of the 4d - cta mip ( a ) and the dsa ( b ) . though this can be recognized in these images through differences in opacification , the analysis is simplified when studying the entire time - resolved series example of a patient with a spetzler martin grade 3 bavm with indirect deep venous drainage , recognized in the lateral projection of the 4d - cta mip ( a ) and the dsa ( b ) . though this can be recognized in these images through differences in opacification , the analysis is simplified when studying the entire time - resolved series though there was agreement in all cases in the size classification used in the spetzler martin grade , the subclassification of small bavms into micro bavms ( < 1 cm , including pial fistulas ) versus macro bavms ( > 1 cm ) was not agreed upon in all cases ( fig . five lesions were denoted micro based on the 4d - cta , while dsa identified only a single fistulous micro bavm whereas the other four lesions were noted to be small bavms with a nidus size larger than 1 cm . lateral mip of the arterial phase of the 4d - cta ( a ) demonstrates two obvious draining veins ( large arrowheads ) and one faint one ( small arrowhead ) . an axial slice at the level of the presumed nidus in the same phase ( b ) suggests the nidus to be larger than suggested by the mip ( arrows ) . the rather diffuse nature of the nidus , possibly due to disruption by the presenting hemorrhage 4 months earlier , was thought to be the cause of poor detection by 4d - cta example of a patient with a small spetzler based on the 4d - cta the nidus was scored as micro ( < 1 cm ) whereas the dsa demonstrated a macro nidus ( > 1 cm ) . lateral mip of the arterial phase of the 4d - cta ( a ) demonstrates two obvious draining veins ( large arrowheads ) and one faint one ( small arrowhead ) . an axial slice at the level of the presumed nidus in the same phase ( b ) suggests the nidus to be larger than suggested by the mip ( arrows ) . the rather diffuse nature of the nidus , possibly due to disruption by the presenting hemorrhage 4 months earlier , was thought to be the cause of poor detection by 4d - cta with regard to arterial feeding territories ( anterior and/or posterior circulation ) , there was disagreement in one patient . one of these demonstrated indirect feeding from the anterior cerebral artery through pial collaterals to the middle cerebral artery territory . 3example of a patient with a spetzler martin grade 3 bavm in the right parietal lobe . a 4d - cta mip in the arterial phase ( a ) suggests an enlarged lenticulostriate perforator ( arrow ) feeding the deeper part of the nidus . the right va injection of the dsa in the same phase and projection ( c ) shows the vessel to be the right posterolateral choroidal artery ( arrow ) . the false interpretation of the 4d - cta , even when oblique mips were used , is due to the non - selective opacification of all cranial arteries with this technique example of a patient with a spetzler martin grade 3 bavm in the right parietal lobe . a 4d - cta mip in the arterial phase ( a ) suggests an enlarged lenticulostriate perforator ( arrow ) feeding the deeper part of the nidus . the right va injection of the dsa in the same phase and projection ( c ) shows the vessel to be the right posterolateral choroidal artery ( arrow ) . the false interpretation of the 4d - cta , even when oblique mips were used , is due to the non - selective opacification of all cranial arteries with this technique secondary dural and transosseous feeding of the bavm was only seen in one patient and was correctly detected in the 4d - cta ( fig . indirect feeding through enlarged leptomeningeal collaterals from one cortical territory to another , also referred to as watershed transfer related to hypoxemia induced nonsprouting angiogenesis , could be seen in six cases , but were only detected in the 4d - cta of three of these ( fig . these indirect feeders were either too small to be noticed , interpreted as direct feeders or interpreted as part of the nidus ( one case each ) . the lateral mip of the 4d - cta in early arterial phase ( a ) clearly shows an enlarged occipital artery ( large arrowhead ) running up to the lesion , as well as smaller posterior meningeal branches ( small arrowhead ) from the ipsilateral va ( arrow ) . axial images from the 4d - cta in early venous phase ( b ) at the level of the nidus ( upper image ) and above the level of the nidus ( lower image ) allow identification of the compact nidus ( arrow ) and feeding vessels ( arrowheads ) , but identification of indirect feeders remains challenging . ap dsa images after injection of the right ica ( c ) and left ica ( d ) clearly aid this discrimination by demonstrating recruitment of leptomeningeal anastomoses ( arrow ) which have developed to enhance pial collateral supply from the anterior to the middle cerebral artery images of the only patient in our series with secondary dural and transosseous feeding of a bavm . the lateral mip of the 4d - cta in early arterial phase ( a ) clearly shows an enlarged occipital artery ( large arrowhead ) running up to the lesion , as well as smaller posterior meningeal branches ( small arrowhead ) from the ipsilateral va ( arrow ) . the lateral projection of the eca injection ( b ) proves the eca contribution , by opacification of the bavm and early opacification of the draining vein ( arrow ) example of a patient with a spetzler martin grade 3 bavm ( same case as fig . 1 ) illustrating superiority of dsa over 4d - cta to demonstrate indirect feeding of a bavm , i.e. axial images from the 4d - cta in early venous phase ( b ) at the level of the nidus ( upper image ) and above the level of the nidus ( lower image ) allow identification of the compact nidus ( arrow ) and feeding vessels ( arrowheads ) , but identification of indirect feeders remains challenging . ap dsa images after injection of the right ica ( c ) and left ica ( d ) clearly aid this discrimination by demonstrating recruitment of leptomeningeal anastomoses ( arrow ) which have developed to enhance pial collateral supply from the anterior to the middle cerebral artery 4d - cta and dsa agreed on flow type in all cases , judging six bavms to be low flow and 11 to be neither 4d - cta nor dsa detected intranidal stasis of contrast material in any of the lesions . the ap mip of the 4d - cta ( a ) and the dsa image in ap projection after left ica injection ( b ) , both in late arterial phase , demonstrate the single arterial feeding branches ( arrows ) , the level of the pial fistula ( asterisk ) and multiple draining veins ( small arrowheads ) draining into the superior sagittal sinus . the ap mip of the 4d - cta ( a ) and the dsa image in ap projection after left ica injection ( b ) , both in late arterial phase , demonstrate the single arterial feeding branches ( arrows ) , the level of the pial fistula ( asterisk ) and multiple draining veins ( small arrowheads ) draining into the superior sagittal sinus . we aimed to offer an evaluation of the performance of time - resolved cta in the diagnostic work - up of untreated bavms and recognize its potential capabilities and pitfalls in the evaluation of angioarchitectural details deemed to be important for the possible treatment strategies . to date , the major drawbacks of non - invasive time - resolved vascular imaging are the inferior spatial and temporal resolution compared to dsa . a comparison of radiation dose between 4d - cta and dsa is not easy , since the radiation dose received during dsa is highly variable . the present comparison with dsa demonstrated that early venous filling was recognized by 4d - cta in all cases , independent of nidus size . in the present series , these characteristics of 4d - cta led to misinterpretation of a feeding vessel ( one case ) , misinterpretation of indirect feeding through pial collaterals ( three cases ) , oversight of mild arterial enlargement ( two cases ) and oversight of deep venous drainage ( one case ) . we believe evaluation of the cross - sectional 4d - cta ( source ) images is essential when reading 4d - cta data , since these images may contain information which has been lost in subsequent 3d reconstructions . 4d - cta cross - sectional images may also be analyzed in a time - resolved fashion and may be helpful to recognize the nidus ( fig . although the capabilities of 4d - cta in the assessment of bavms appear promising , we feel caution is appropriate when attempting to change present imaging paradigms . until such a study is published , we suggest to use 4d - cta only as either a screening tool in the work - up of suspected patients , followed by dsa whenever negative , or as a follow - up tool when a lesion has been demonstrated previously and is managed conservatively . in this series of bavm patients , 4d - cta was able to detect all lesions . although some angioarchitectural details were missed or misinterpreted when compared to dsa , 4d - cta evaluation would have been sufficiently accurate to diagnose the shunt and classify it . we believe treatment planning may benefit from the accompanying availability of cross - sectional imaging in different planes and perfusion maps , which can be calculated from the presented data set . although these results await validation in a prospective unselected patient cohort , 4d - cta seems to be a valuable new adjunct in the non - invasive diagnostic work - up and follow - up of bavm patients .

starting from the first description of alternative splicing and constitutive splicing processes in 1977 [ 13 ] , the importance of this process that guarantees the correct flow of information from transcription to translation in eukaryotic cells has continued to grow exponentially . in particular , one major branch of research in this area has the aim to investigate and characterize the cellular macromolecular machine ( i.e. , the spliceosome ) that is physically responsible for the cutting and joining of intronsexons by catalyzing two transesterification reactions [ 4 , 5 ] and the mechanisms that ensure its fidelity . as a result , research in spliceosome composition and functioning has been complemented by studies aiming to understand the sequences and molecules that determine under which conditions a particular exon or intron is selectively recognized and included in the mature transcript . therefore , after the basic elements that define introns and exons and are composed by donor and acceptor splice sites plus the branch point sequence , there was the discovery of enhancer and silencer elements that can affect either positively or negatively the way these basic elements are recognized by the spliceosome [ 79 ] . usually , enhancer and silencer elements are bound by members of the sr and hnrnp protein families , respectively . the recruitment of these proteins to specific sites is crucial for their activity as depending on their position with respect to the basic elements their roles can be antagonistic or , in some case , agonistic [ 10 , 11 ] . global analyses of the ways these factors cooperate and influence each other in shaping the splicing process has only recently begun to shed light on how they promote or hinder exon recognition in a co - ordinated manner [ 1214 ] . rather unexpectedly , these studies have shown that sr proteins interact not only with alternatively spliced exons but also with constitutively spliced exons , and that their major role consists in recruiting the splicing machinery to splice sites [ 15 , 16 ] . on the other hand , hnrnp proteins bind to nascent pre - mrna and influence splicing decision through a complex and finely tuned network of protein / protein interactions . the mechanisms of hnrnps actions on splicing is generally less defined than those of sr proteins . in addition to these elements , we now also know that splicing choices are affected by a myriad of other factors , ranging from chromatin modifications [ 18 , 19 ] , transcriptional factors , rna secondary structure , short noncoding rnas [ 22 , 23 ] , and various cellular stresses . in parallel to these mechanistic studies , another branch of splicing research has also addressed the functional importance of alternative splicing processes in biological pathways and in particular the way that alternative splicing isoforms of proteins can acquire different or even antagonistic biological properties [ 25 , 26 ] . because of this ability to expand the proteome of cells , alternative splicing has represented a very useful and powerful tool that allows cells to execute the various expression programs which underlie many fundamental needs of higher organisms : from general needs such as controlling normal development and tissue - specific expression of proteins , to highly specialized processes such as dna damage response or microrna biogenesis [ 2733 ] . moreover , the rearrangements that a pre - mrna undergoes during the splicing process are also advantageous in terms of providing a longer half - life and better translational capacity , something that has recently begun to be exploited by the biotechnology industry . considering all these necessities and advantages , it is therefore not surprising that the number of genes that are subject to alternative splicing in eukaryotic organisms has been steadily growing . indeed , it was recently estimated that more than 90% of mammalian protein coding genes can produce at least one splicing - derived isoform expressed at potentially significant biological levels . the complexity of this system , however , also puts spliceosome functioning at risk of being impaired by the occurrence of single - point mutations in splicing regulatory elements , deletions / insertions , genomic rearrangements , and alterations at the splicing factor expression level . any of these alterations can result in a variety of aberrant splicing outcomes that usually include aberrant exon skipping , cryptic splice site selection , intron retention , and pseudoexon activation [ 37 , 38 ] . as expected , many of these changes can lead directly to the occurrence of disease in humans , and it has now been estimated that a sizable proportion of all gene mutations leading to disease can be directly connected with the presence of a splicing defect [ 7 , 39 ] . importantly , the introduction of novel technologies that allow fast profiling of the transcriptome seems promising for simplifying investigation of which genomic variability and plasticity events allow cancer cells to tailor specific functional units from the available exons of a gene . for example , recent rna sequencing of the breast cancer transcriptome has revealed many new splicing alterations that were not previously described . in particular , for example , it was reported that 423 primary transcripts ( derived from 377 genes ) were differentially spliced in triple - negative breast cancer samples ( generating 496 novel isoforms ) . by analyzing non - triple - negative breast cancer samples , the same study found that 270 and 460 primary transcripts ( derived from 242 and 387 differentially spliced genes , resp . ) it has become clear that in several human pathologies , including cancer , the alternative splicing profile is aberrantly modified in a specific manner and in ways that can favor the growth and survival of cancer cells [ 4144 ] . the generation of these aberrant splicing profiles can occur in many ways , such as through the re - expression of developmentally regulated isoforms that had previously been shut off following early developmental stages [ 45 , 46 ] , by affecting the splicing profiles of genes that are implicated in tumor progression [ 47 , 48 ] , or through other mechanisms that generally have anti - apoptotic / metastatic consequences and that can affect response to therapies [ 49 , 50 ] . in addition , several studies have defined that cancer - associated splicing alterations arise not only from mutations in the cancer - related genes but derive also from variations in the expression and/or activity of splicing regulatory factors . for example , it has been well established that the levels of sr- and hnrnp - proteins undergo changes associated with transformation and progression of cancers [ 44 , 52 ] . strikingly , it has been recently reported that some members of the sr protein family of splicing factors and other components of the spliceosomal cellular machinery can actually act as oncoproteins and play a direct role in promoting tumor origin and progression [ 5355 ] and that external stimuli such as hypoxia can cause the aberrant redistribution of important splicing factors such as tra2 and promote the expression of tumor - promoting splicing isoforms . the case of bcl - x ( bcl2l1 ) gene , belonging to the bclii family and implicated in the control of mitochondrial breakdown during apoptosis , is emblematic of this concept . for example , two splicing bcl - x isoforms can arise from the use of two alternative 5 splice sites within exon 2 and lead to the synthesis of a short apoptosis - promoting protein ( bcl - xs ) and to a long antiapoptotic form ( bcl - xl ) . different splicing factors , including sam68 , hnrnpa1 , sf2/asf , hnrnp f / h , hnrnp k , sap155 , and srp30c have been found to be involved in the selection of the two competing alternative 5 splice sites that give rise to these two isoforms [ 5861 ] . in addition , recent studies have shown that the elongation and splicing - related factor tcerg1 can bind to the bcl- x pre - mrna and promote the proapoptotic bcl - xs 5 splice site in a promoter - dependent manner . finally , the production of the proapoptotic bcl - xs splice variant seems to be improved by the core ( y14 and eif4a3 ) and auxiliary ( rnps1 , acinus , and sap18 ) components of the exon junction complex ( ejc ) , suggesting that ejc - associated components can regulate apoptosis at the alternative splicing level and represent a further level of vulnerability of cancer . therefore , one of the most interesting research areas in this field consists in the identification of cancer - specific splice variants or the aberrant expression of splicing - affecting proteins that could lead to their generation . examples of both these events , in fact , have already been shown to occur in some individual types of cancer , such as breast and ovarian cancer [ 64 , 65 ] . another interesting research area in aberrant splicing events connected with tumors is the occurrence of particular types of splicing defects that involve the inclusion of new sequences ( known as pseudoexons ) in the mature mrna of cancer - related genes . rather more rarely , the opposite has also been shown to occur : the aberrant recognition of intronic sequences ( pseudointrons ) within normal exons . in this review , we have also decided to provide particular attention to these events as they are probably more common than previously considered and have not yet been the subject of particular attention . one of the reasons why these two events are particularly interesting is that these types of defects are ideally suited for novel therapeutic effector molecules that are based in rna biology . in the case of pseudoexons and pseudointrons , in fact , the major advantage of targeting this type of inclusion events is that the antisense oligonucleotides would be targeted against normal intronic sequences and thus would not remain bound to the mature mrna ( possibly to interfere with later stages of rna processing such as export / translation ) . in the pre - mrna splicing field , the term pseudoexon has been introduced to describe exonic - like sequences that are present within intronic regions but are ignored by the spliceosomal machinery . a closer look at these sequences has often provided a reason for their inability to be recognized as normal exons : the presence of intrinsic defects in their apparently viable donor and acceptor sites or of silencer elements [ 6769 ] and the formation of inhibiting rna secondary structures [ 7072 ] . from a functional point of view , in most cases of pseudoexon insertion , the presence of an extraneous exon within the mature mrna causes either the disruption of the translational reading frame or the insertion of novel amino acid sequences following translation . as a result , the normal biological properties of the resulting protein are very likely disrupted , and this can be associated with the development of disease . unfortunately , it is still quite hard to identify reliable pseudoexon insertion events in human genes implicated in cancers by just performing a general interrogation of databases ( matching 22719 ensembl protein coding genes versus 31057 entries of cangem gene list ) . in fact , at present , it is only possible to retrieve strong candidates for alternative splicing events in protein coding genes ( table 1 ) . as expected , a similar situation was seen when pseudogenes were investigated ( defined as genomic dna sequences similar to normal genes but nonfunctional , although some can still be transcribed ) . in this case , inspection of 14775 pseudogenes returned a list of alternative splicing hits from which it is not easy to distinguish real events in expressed pseudogenes ( table 1 ) . more recently , some bioinformatic studies have tried to extrapolate the presence of pseudoexons in cancer tissues by developing new analysis methods of genechip gene expression array data [ 74 , 75 ] . in this way , by comparing normal cerebellum and medulloblastoma tumors , it was possible to predict 811 significantly different expressed pseudoexons ( derived from 577 genes ) . in addition , when nonmetastatic and metastatic medulloblastomas were compared , 13 pseudoexonic sequences were significantly expressed in a differential manner ( derived from 8319 strong candidates ) . however , it should be noted that no experimental validation was carried out to support these predictions . therefore , presently , manual annotations remain the most reliable system for identifying real pseudoexons . fortunately , the scientific community has recently identified a certain number of cases where pseudoexon inclusion has been validated in detail . for this reason , table 2 reports all the cryptic exons described in the literature that are localized within genes whose expression was altered in cancer . first of all , regarding the mechanisms underlying pseudoexon activation , it is interesting to note that these phenomena are caused by inherited mutations resulting in the insertion of intronic sequences in the mature mrna . in this respect , it is interesting to note that among the genes presenting pseudoexon awakening directly associated with cancer origin ( table 2 ) , the creation of new splicing donor ( 12 events ) and acceptor ( 7 events ) sites represents the more frequent occurrence ( 48% and 28% of the listed events , resp . ) . some of the most paradigmatic examples of cancer genes such as brca1 , brca2 , nf1 , and atm are included in these two sets . on the other hand , among pseudoexons described in cancer - related genes ( table 3 ) , the creation of 5 splice sites and of 3 splice sites comprise 55% and 15% of the listed records , respectively . moreover , pseudoexon insertion can also be triggered by the deletion of nearby donor or acceptors splice sites ( 4 events in both lists ) , highlighting the importance of the genetic milieu in splicing decisions . a deeper look into the mechanisms underlying pseudoexon activation has revealed the involvement of hnrnps in regulation of these events . in particular , the partial inclusion of a pseudoexon of nf1 gene has been found to arise from a novel intronic mutation c.31279a > g intron 30 , creating a new acceptor splice site and activation of a cryptic 5 splice site . it has been found that both ptb and nptb play an active role in the pseudoexon splicing by repressing its inclusion . this finding is particularly interesting since it further supports the hypothesis that ptb / nptb might have a general role in repressing weak exons and in particular most of pseudoexons . another example of mutation causing pseudoexon activation consists in the creation of novel branch site ( table 2 , one event ) . this latter case is associated with a peculiar mutation ( ivs5 ds + 232 ga ) that leads to pseudoexon inclusion in nf2 gene by creating a consensus branch point . in particular , it has been found that the g > a transition , occurring at position 18 from the acceptor site of nf2 ivs5 , works in combination with existing consensus splice acceptor and donor sites and causes the formation of an extra exon 5a in the nf2 gene that introduces a premature stop codon . finally , in spite of being lowly represented , gross genomic rearrangements can bring together or expose splice site sequences that would normally be very distant from each other or absent form the original sequence ( one event ) . pseudoexon activation can also be caused by mutations that cause the creation / deletion of splicing regulatory elements ( sres , 6 events ) . among these events , one of the most representative examples of the complex regulatory networks that can underlie pseudoexon insertion is represented by the identification of an unusual splicing defect directly related with neoplasia ( figure 1 ) . in the case of this particular event , a patient affected by ataxia - telangiectasia was found to carry a 4 nt - deletion ( gtaa ) within intron 20 of atm gene . this deletion , termed intron - splicing processing element ( ispe ) , caused the inclusion of a 65 nucleotide the mechanism through which this occurs has been well characterized in recent studies . in normal conditions , the repression of the 3ss in the wild - type pseudoexon sequence ( atm wt ) was shown to be a direct consequence of u1snrnp binding in correspondence to the ispe sequence . the importance of this u1snrnp binding was twofold : first of all , to sterically hinder 3ss recognition by u2snrnp and secondly , to also inhibit binding of the srsf1 splice factor to the stem - loop sequence formed by this pseudoexon ( figure 1(b ) ) [ 71 , 72 ] . as a result , this internal u1snrnp binding event to the ispe sequence causes an unproductive u2snrnp association with the 3ss that results in the complete inhibition of pseudoexon inclusion in normal conditions ( figure 1(b ) ) . following the deletion of the guaa motif observed in the patient , u1snrnp binding to the ispe is relieved , and srsf1 is also free to bind to the internal enhancer site that becomes available ( figure 1(c ) ) . taken together , these two events result in efficient recruitment of u2snrnp to the branchsite and a better recruitment of u1snrnp to the rather inefficient 5gc splice site that lead to efficient inclusion of the pseudoexon in the mature atm mrna . this example clearly shows the complexity of pseudoexon activation events that relies on a variety of factors well beyond the simple initial mutation ( i.e. , 5ss or 3ss creation ) , and that can include the eventual presence of enhancer and silencer elements within the cryptic exon or intro , rna secondary structures , and probably several other mechanisms that have not yet been described in detail . pseudointrons ( psis ) represent an intriguing set of intron - like sequences localized within exons that can undergo alternative splicing and that therefore can be included in or excluded from the orf within the final mrna species . although the examples of pseudointrons reported in the review are not associated with activating mutations , we have included them because of the peculiarity of events insofar that they do not represent simple intron retention events , but rather alternatively spliced intraexonic sequences whose behaviour can resemble that of introns under particular circumstances ( e.g. , occurrence of previous splicing events in the processed transcript ) . it is this regulated use of the intraexonic splice sites that fits well with the idea that these intraexonic splice site represent false introns , even in the absence of genetic alterations . in general , however , pseudointrons are less well described compared withpseudoexons probably because of the difficulty in detecting them using available technology ( a situation that may well soon change for the better because of the introduction of more sensitive and comprehensive technical approaches such as rna sequencing ) . at the moment , only three examples of psi have been characterized , and for at least two of these pseudointrons there are data supporting their relevance in the pathogenesis of cancer . fibronectin ( fn ) is an extracellular matrix protein whose functions range from cell adhesion and migration to wound healing and oncogenic transformation . the functional complexity of fn is related to the structural diversity arising from cell type - specific alternative splicing . the fibronectin pre - mrna undergoes alternative splicing primarily at three sites : two extra domain exons encoding extra structural repeats and a region of nonhomologous sequence called the type - iii connecting segment ( iiics ) . the iiics domain is divided into three subdomains of 75 bp , 192 bp , and 93 bp , respectively , that can be alternatively spliced , thus generating up to fivevariant mrnas in humans [ 147 , 148 ] . the subdomains of 75 bp and 93 bp encode for two cell - specific binding sites , cs1 ( residues 125 of the iiics ) , and cs5 ( residues 90109 of the iiics ) , respectively , that interact with the integrin 41 with different affinity [ 149 , 150 ] . indeed , the cs1 site has approximately 20-fold higher affinity for integrins than the cs5 site . because of its intraexonic localization and splicing behavior , the 93 bp segment can be considered a pseudointron ( figure 2(a ) ) . different tissue - specific and disease - associated changes in the variants of the iiics region have been reported [ 152154 ] , and the cs5 pseudointron is upregulated in foetal tissue and in adult liver . for the purpose of this review , it is important to note that alternative splicing of the iiics - cs5 subdomain can influence the onset and progression of cancers by affecting fibronectin activities related to tissue organization and cell - ecm interactions . in keeping with this hypothesis , in vitro studies have shown that cs5 can modulate the spreading of melanoma cells . acetylcholinesterase is a serine specific hydrolase that hydrolyzes acetylcholine , and its main function is related to the clearance of the neurotransmitter from the synaptic cleft . nonetheless , the observation that the expression of ache is not limited to cholinergic tissues has suggested the presence of additional functions . from the splicing process point of view , mammal ache has been shown to undergo alternative splicing resulting in expression of 3 isoforms , called r ( readthrough ) , h ( hydrophobic ) , and t / s ( tailed or synaptic ) , that differ in their subcellular localization , tissue distribution , and developmental pattern of expression . whereas , ache - h or ache - t is the most abundant and common isoforms , the ache - r expression is low and restricted in specific cell lines , cell differentiation stage , or muscle development [ 159 , 160 ] . interestingly , the ache - r mrna arises by retention of pseudointron4 , that leads to generation of the e1-e2-e3-e4-i4-e5 mrna transcript ( figure 2(b ) ) . since pseudointron 4 includes a stop codon when it is included in mature rna , the resulting protein will be prematurely terminated by a c - terminus translated from the 5 region of i4 . this variant is expected to remain monomeric . in general , the ache - r transcript can be coexpressed with the ache - t or ache - h transcripts , and variations in their relative levels seem to be important for modulation of ache functions , as well as for the pathogenesis of neurological and autoimmune disorders [ 161 , 162 ] . intriguingly , recent studies have found that ache - r mrna accumulates in primary human astrocytomas , and that its presence is correlated with their grade of aggressiveness . in human u87 mg glioblastoma cells , it was found that ache - r protein variant can support proliferation of glioblastoma tumors by forming a complex with the scaffold protein rack1 and protein kinase ce . in addition , increased levels of an n - terminally extended n - ache - r isoform were detected in human testicular tumors indicating that the generation of the ache - r variant can be associated with the utilization of an alternative promoter in the transformed cells . finally , related functional studies have suggested that both ache - r variants ( ache - r and n - ache - r ) might be crucial for increasing the cellular atp levels and might support selective metabolic advantages as well as genotoxic resistance by altering p73 gene expression . although it is not yet clear whether the ache - r ( as well as the other ache splicing variants ) can be related directly to tumorigenesis , the observed downregulation of all these splicing isoforms in colorectal carcinoma suggests a crucial role for tumor development . a third example of pseudointron possibly implicated in cancer pathogenesis is found within the thrombopoietin ( thpo ) gene . thpo is the most important cytokine for regulation of platelet production , and its expression has also been reported in skeletal muscle , ovary , testis , and central nervous system ( cns ) [ 167169 ] . the thpo gene shows a complex pattern of alternative splicing ; among the six splice variants that can be generated , three of them ( thpo-3 , thpo-5 , and thpo-6 ) show the peculiar alternative splicing of 116 nucleotides within exon 6 ( that can therefore be classified as a pseudointron ) ( figure 2(c ) ) . contrary to the fibronectin iiics - c5 and ache - r pseudointrons , the involvement in cancerogenesis of the variants deriving from the alternative splicing of the 116 nt pseudointron is less characterized . nonetheless , functional studies have demonstrated that the protein translated from mouse thpo-3 is not secreted efficiently [ 172174 ] . these results have suggested that thpo-3 might be implicated in the fine regulation of thpo full - length levels and in the modulation of several biological functions beyond thrombopoiesis . interestingly , parallel studies have also shown an altered expression pattern of the thpo-3 , thpo-5 , and thpo-6 splicing variants in human carcinomas . as a result , these observations have led researchers to hypothesize that alterations in alternative splicing of the 116 nt - thpo pseudointron might be employed as biomarker of tumor formation or progression . in conclusion , although currently there are few examples of pseudointron removal , at least three of these events have been associated with different levels of cancerogenesis , ranging from transformation to cancer progression . therefore , it is advisable that future high throughput screening analysis should be able to better associate the alternative splicing process of these pseudointrons with tumoral events . in particular , with regard to their use as possible biomarkers of transformation or of cancer staging / grading . regarding therapies , it is likely that all this information on the importance of alternative splicing for tumor development and progression may soon become extremely important for the development of novel therapeutic effectors in the fight against cancer [ 176 , 177 ] . indeed , one of the reasons why these defects are particularly interesting is the opportunity they provide for rna therapies and the chance to actually make the jump from the bench to the bedside . currently , the field of rna therapy is rapidly growing especially in the inhibition of undesirable splice site choices by the use of antisense oligos [ 178180 ] . this technique , pioneered by dominski and kole , is based on the use of suitably modified antisense oligonucleotides that target specific sequences within introns and exons and block the recognition by their cellular binding factors . therefore , they are ideally suited to block unwanted splice sites that may become activated following their creation or activation . it is interesting that the role of using antisense strategies to regulate exon inclusion also occurs in physiological conditions . an example of this is snorna hbii-52 in the regulation of exon vb inclusion in the serotonin receptor 2c . to this moment , a few pilot studies aimed at inhibiting the inclusion of a pseudoexon in patient cell lines have already been reported . in particular , antisense oligonucleotides have been used to target newly created 5 or 3 splice sites deep within intronic regions of the nf1 gene to restore the normal splicing profile [ 91 , 92 ] . similar approaches have also been successfully used to target deep intronic mutations causing pseudoexon activation within brca2 intron 12 ( c.6937 + 594t > g ) , atm intron 11 ( c.1236405c > t ) , and atm ivs19 ( c.2639384a > g ) . recent advances in antisense - mediated exon skipping for dmd suggest that not only the splice sites but also exonic splicing enhancer sites or branch points might represent potential targets for inducing pseudoexon skipping [ 183 , 184 ] , and that the action of asos might be potentiated by coadministration of drugs , as shown for duchenne muscular dystrophy . an intriguing variation on the theme of the antisense oligonucleotides , successfully tested for inhibiting the 5 splice site of bcl - xl , might consist in the use of tailed oligonucleotides containing a portion annealing to sequences immediately upstream of the target donor splice site joined to a nonhybridizing 5 tail that includes binding sites for the hnrnp a1/a2 proteins . in spite of being prominent , antisense technologies are not the only possibility available for targeting these aberrant splicing events . an alternative to antisense usage is the development of sirna molecules capable of targeting the newly created system . in this manner , only the mrnas containing the pseudoexon might be selectively degraded leaving any residual normal mrna to be properly processed . the disadvantage of simply degrading the pseudoexon - bearing transcript ( rather than acting on the splicing event itself ) is that everything depends on the level of pseudoexon inclusion ( less than 100% ) and on the status of the other allele ( i.e. , whether it is normally expressed or not ) . finally , small molecule compounds might be also considered as possible therapeutic options in order to prevent the recognition of pseudoexons or modulate the recognition of pseudointrons . for example , it has been shown that sodium butyrate , an histone deacetylase inhibitor known to upregulate the expression of htra2-beta1 and sc35 , promotes skipping of the pseudoexon activated by the cftr 3849 + 10kbc > t mutation and can restore functional cftr channels . in addition , the splicing of different nf1 skipped exons as a result of mutations in cis - acting sequences has been restored with administration of the small molecule kinetin . more recently , a growing body of research indicates that tale nucleases ( talens ) have been used with great success in a number of organisms to generate site - specific dna variations . as a result , this approach is another good candidate for an innovative therapeutic anticancer strategy for correction of deep intronic mutations that create novel splice sites . of course , crossing the difficult gap between the bench to the bedside will not be immediate even in the presence of highly efficient therapeutic molecules , whether antisense oligos , sirnas , or others . indeed , before any of these can be used in humans to treat cancer , several additional factors need to be considered . first of all , there is the development of an efficient and safe carrier system to deliver these compounds into the human body . secondly , these systems will need to be optimized in order to achieve the best balance between successful delivery , intrinsic toxicity ( if any ) , and avoidance of undesired immune responses ( in the case of antisense oligos ) . finally , even when all these achievements are met , there will still be the need to optimize recurrent - administration protocols to determine the uptake levels , clearance , and accumulation in various tissues ( this is an often overlooked , since none of these methods will cause permanent correction of mrna splicing defects ) . nonetheless , the first results of this exciting news strategy are available , and there is the distinct possibility that rna - based treatment of cancer may soon enter the application stage .

in all eukaryotic organisms , pre - mrna splicing and alternative splicing processes play an essential role in regulating the flow of information required to drive complex developmental and metabolic pathways . as a result , eukaryotic cells have developed a very efficient macromolecular machinery , called the spliceosome , to correctly recognize the pre - mrna sequences that need to be inserted in a mature mrna ( exons ) from those that should be removed ( introns ) . in healthy individuals , alternative and constitutive splicing processes function with a high degree of precision and fidelity in order to ensure the correct working of this machinery . in recent years , however , medical research has shown that alterations at the splicing level play an increasingly important role in many human hereditary diseases , neurodegenerative processes , and especially in cancer origin and progression . in this minireview , we will focus on several genes whose association with cancer has been well established in previous studies , such as atm , brca1/a2 , and nf1 . in particular , our objective will be to provide an overview of the known mechanisms underlying activation / repression of pseudoexons and pseudointrons ; the possible utilization of these events as biomarkers of tumor staging / grading ; and finally , the treatment options for reversing pathologic splicing events .

1. Introduction 2. Aberrant Splicing Events in Cancer Genes 3. Pseudoexon Activation in Cancer 4. Pseudointron Activation in Cancer 5. Therapeutic Outlooks

starting from the first description of alternative splicing and constitutive splicing processes in 1977 [ 13 ] , the importance of this process that guarantees the correct flow of information from transcription to translation in eukaryotic cells has continued to grow exponentially . in particular , one major branch of research in this area has the aim to investigate and characterize the cellular macromolecular machine ( i.e. , the spliceosome ) that is physically responsible for the cutting and joining of intronsexons by catalyzing two transesterification reactions [ 4 , 5 ] and the mechanisms that ensure its fidelity . as a result , research in spliceosome composition and functioning has been complemented by studies aiming to understand the sequences and molecules that determine under which conditions a particular exon or intron is selectively recognized and included in the mature transcript . global analyses of the ways these factors cooperate and influence each other in shaping the splicing process has only recently begun to shed light on how they promote or hinder exon recognition in a co - ordinated manner [ 1214 ] . rather unexpectedly , these studies have shown that sr proteins interact not only with alternatively spliced exons but also with constitutively spliced exons , and that their major role consists in recruiting the splicing machinery to splice sites [ 15 , 16 ] . on the other hand , hnrnp proteins bind to nascent pre - mrna and influence splicing decision through a complex and finely tuned network of protein / protein interactions . in addition to these elements , we now also know that splicing choices are affected by a myriad of other factors , ranging from chromatin modifications [ 18 , 19 ] , transcriptional factors , rna secondary structure , short noncoding rnas [ 22 , 23 ] , and various cellular stresses . in parallel to these mechanistic studies , another branch of splicing research has also addressed the functional importance of alternative splicing processes in biological pathways and in particular the way that alternative splicing isoforms of proteins can acquire different or even antagonistic biological properties [ 25 , 26 ] . because of this ability to expand the proteome of cells , alternative splicing has represented a very useful and powerful tool that allows cells to execute the various expression programs which underlie many fundamental needs of higher organisms : from general needs such as controlling normal development and tissue - specific expression of proteins , to highly specialized processes such as dna damage response or microrna biogenesis [ 2733 ] . moreover , the rearrangements that a pre - mrna undergoes during the splicing process are also advantageous in terms of providing a longer half - life and better translational capacity , something that has recently begun to be exploited by the biotechnology industry . considering all these necessities and advantages , it is therefore not surprising that the number of genes that are subject to alternative splicing in eukaryotic organisms has been steadily growing . the complexity of this system , however , also puts spliceosome functioning at risk of being impaired by the occurrence of single - point mutations in splicing regulatory elements , deletions / insertions , genomic rearrangements , and alterations at the splicing factor expression level . any of these alterations can result in a variety of aberrant splicing outcomes that usually include aberrant exon skipping , cryptic splice site selection , intron retention , and pseudoexon activation [ 37 , 38 ] . as expected , many of these changes can lead directly to the occurrence of disease in humans , and it has now been estimated that a sizable proportion of all gene mutations leading to disease can be directly connected with the presence of a splicing defect [ 7 , 39 ] . importantly , the introduction of novel technologies that allow fast profiling of the transcriptome seems promising for simplifying investigation of which genomic variability and plasticity events allow cancer cells to tailor specific functional units from the available exons of a gene . in particular , for example , it was reported that 423 primary transcripts ( derived from 377 genes ) were differentially spliced in triple - negative breast cancer samples ( generating 496 novel isoforms ) . it has become clear that in several human pathologies , including cancer , the alternative splicing profile is aberrantly modified in a specific manner and in ways that can favor the growth and survival of cancer cells [ 4144 ] . the generation of these aberrant splicing profiles can occur in many ways , such as through the re - expression of developmentally regulated isoforms that had previously been shut off following early developmental stages [ 45 , 46 ] , by affecting the splicing profiles of genes that are implicated in tumor progression [ 47 , 48 ] , or through other mechanisms that generally have anti - apoptotic / metastatic consequences and that can affect response to therapies [ 49 , 50 ] . for example , it has been well established that the levels of sr- and hnrnp - proteins undergo changes associated with transformation and progression of cancers [ 44 , 52 ] . strikingly , it has been recently reported that some members of the sr protein family of splicing factors and other components of the spliceosomal cellular machinery can actually act as oncoproteins and play a direct role in promoting tumor origin and progression [ 5355 ] and that external stimuli such as hypoxia can cause the aberrant redistribution of important splicing factors such as tra2 and promote the expression of tumor - promoting splicing isoforms . different splicing factors , including sam68 , hnrnpa1 , sf2/asf , hnrnp f / h , hnrnp k , sap155 , and srp30c have been found to be involved in the selection of the two competing alternative 5 splice sites that give rise to these two isoforms [ 5861 ] . in addition , recent studies have shown that the elongation and splicing - related factor tcerg1 can bind to the bcl- x pre - mrna and promote the proapoptotic bcl - xs 5 splice site in a promoter - dependent manner . finally , the production of the proapoptotic bcl - xs splice variant seems to be improved by the core ( y14 and eif4a3 ) and auxiliary ( rnps1 , acinus , and sap18 ) components of the exon junction complex ( ejc ) , suggesting that ejc - associated components can regulate apoptosis at the alternative splicing level and represent a further level of vulnerability of cancer . therefore , one of the most interesting research areas in this field consists in the identification of cancer - specific splice variants or the aberrant expression of splicing - affecting proteins that could lead to their generation . examples of both these events , in fact , have already been shown to occur in some individual types of cancer , such as breast and ovarian cancer [ 64 , 65 ] . another interesting research area in aberrant splicing events connected with tumors is the occurrence of particular types of splicing defects that involve the inclusion of new sequences ( known as pseudoexons ) in the mature mrna of cancer - related genes . in this review , we have also decided to provide particular attention to these events as they are probably more common than previously considered and have not yet been the subject of particular attention . in the case of pseudoexons and pseudointrons , in fact , the major advantage of targeting this type of inclusion events is that the antisense oligonucleotides would be targeted against normal intronic sequences and thus would not remain bound to the mature mrna ( possibly to interfere with later stages of rna processing such as export / translation ) . in the pre - mrna splicing field , the term pseudoexon has been introduced to describe exonic - like sequences that are present within intronic regions but are ignored by the spliceosomal machinery . from a functional point of view , in most cases of pseudoexon insertion , the presence of an extraneous exon within the mature mrna causes either the disruption of the translational reading frame or the insertion of novel amino acid sequences following translation . as a result , the normal biological properties of the resulting protein are very likely disrupted , and this can be associated with the development of disease . in fact , at present , it is only possible to retrieve strong candidates for alternative splicing events in protein coding genes ( table 1 ) . in this case , inspection of 14775 pseudogenes returned a list of alternative splicing hits from which it is not easy to distinguish real events in expressed pseudogenes ( table 1 ) . more recently , some bioinformatic studies have tried to extrapolate the presence of pseudoexons in cancer tissues by developing new analysis methods of genechip gene expression array data [ 74 , 75 ] . fortunately , the scientific community has recently identified a certain number of cases where pseudoexon inclusion has been validated in detail . for this reason , table 2 reports all the cryptic exons described in the literature that are localized within genes whose expression was altered in cancer . first of all , regarding the mechanisms underlying pseudoexon activation , it is interesting to note that these phenomena are caused by inherited mutations resulting in the insertion of intronic sequences in the mature mrna . in this respect , it is interesting to note that among the genes presenting pseudoexon awakening directly associated with cancer origin ( table 2 ) , the creation of new splicing donor ( 12 events ) and acceptor ( 7 events ) sites represents the more frequent occurrence ( 48% and 28% of the listed events , resp . ) some of the most paradigmatic examples of cancer genes such as brca1 , brca2 , nf1 , and atm are included in these two sets . on the other hand , among pseudoexons described in cancer - related genes ( table 3 ) , the creation of 5 splice sites and of 3 splice sites comprise 55% and 15% of the listed records , respectively . a deeper look into the mechanisms underlying pseudoexon activation has revealed the involvement of hnrnps in regulation of these events . in particular , the partial inclusion of a pseudoexon of nf1 gene has been found to arise from a novel intronic mutation c.31279a > g intron 30 , creating a new acceptor splice site and activation of a cryptic 5 splice site . it has been found that both ptb and nptb play an active role in the pseudoexon splicing by repressing its inclusion . this finding is particularly interesting since it further supports the hypothesis that ptb / nptb might have a general role in repressing weak exons and in particular most of pseudoexons . in particular , it has been found that the g > a transition , occurring at position 18 from the acceptor site of nf2 ivs5 , works in combination with existing consensus splice acceptor and donor sites and causes the formation of an extra exon 5a in the nf2 gene that introduces a premature stop codon . finally , in spite of being lowly represented , gross genomic rearrangements can bring together or expose splice site sequences that would normally be very distant from each other or absent form the original sequence ( one event ) . among these events , one of the most representative examples of the complex regulatory networks that can underlie pseudoexon insertion is represented by the identification of an unusual splicing defect directly related with neoplasia ( figure 1 ) . this deletion , termed intron - splicing processing element ( ispe ) , caused the inclusion of a 65 nucleotide the mechanism through which this occurs has been well characterized in recent studies . in normal conditions , the repression of the 3ss in the wild - type pseudoexon sequence ( atm wt ) was shown to be a direct consequence of u1snrnp binding in correspondence to the ispe sequence . the importance of this u1snrnp binding was twofold : first of all , to sterically hinder 3ss recognition by u2snrnp and secondly , to also inhibit binding of the srsf1 splice factor to the stem - loop sequence formed by this pseudoexon ( figure 1(b ) ) [ 71 , 72 ] . as a result , this internal u1snrnp binding event to the ispe sequence causes an unproductive u2snrnp association with the 3ss that results in the complete inhibition of pseudoexon inclusion in normal conditions ( figure 1(b ) ) . following the deletion of the guaa motif observed in the patient , u1snrnp binding to the ispe is relieved , and srsf1 is also free to bind to the internal enhancer site that becomes available ( figure 1(c ) ) . pseudointrons ( psis ) represent an intriguing set of intron - like sequences localized within exons that can undergo alternative splicing and that therefore can be included in or excluded from the orf within the final mrna species . although the examples of pseudointrons reported in the review are not associated with activating mutations , we have included them because of the peculiarity of events insofar that they do not represent simple intron retention events , but rather alternatively spliced intraexonic sequences whose behaviour can resemble that of introns under particular circumstances ( e.g. in general , however , pseudointrons are less well described compared withpseudoexons probably because of the difficulty in detecting them using available technology ( a situation that may well soon change for the better because of the introduction of more sensitive and comprehensive technical approaches such as rna sequencing ) . at the moment , only three examples of psi have been characterized , and for at least two of these pseudointrons there are data supporting their relevance in the pathogenesis of cancer . the fibronectin pre - mrna undergoes alternative splicing primarily at three sites : two extra domain exons encoding extra structural repeats and a region of nonhomologous sequence called the type - iii connecting segment ( iiics ) . different tissue - specific and disease - associated changes in the variants of the iiics region have been reported [ 152154 ] , and the cs5 pseudointron is upregulated in foetal tissue and in adult liver . for the purpose of this review , it is important to note that alternative splicing of the iiics - cs5 subdomain can influence the onset and progression of cancers by affecting fibronectin activities related to tissue organization and cell - ecm interactions . acetylcholinesterase is a serine specific hydrolase that hydrolyzes acetylcholine , and its main function is related to the clearance of the neurotransmitter from the synaptic cleft . from the splicing process point of view , mammal ache has been shown to undergo alternative splicing resulting in expression of 3 isoforms , called r ( readthrough ) , h ( hydrophobic ) , and t / s ( tailed or synaptic ) , that differ in their subcellular localization , tissue distribution , and developmental pattern of expression . interestingly , the ache - r mrna arises by retention of pseudointron4 , that leads to generation of the e1-e2-e3-e4-i4-e5 mrna transcript ( figure 2(b ) ) . since pseudointron 4 includes a stop codon when it is included in mature rna , the resulting protein will be prematurely terminated by a c - terminus translated from the 5 region of i4 . in general , the ache - r transcript can be coexpressed with the ache - t or ache - h transcripts , and variations in their relative levels seem to be important for modulation of ache functions , as well as for the pathogenesis of neurological and autoimmune disorders [ 161 , 162 ] . in addition , increased levels of an n - terminally extended n - ache - r isoform were detected in human testicular tumors indicating that the generation of the ache - r variant can be associated with the utilization of an alternative promoter in the transformed cells . the thpo gene shows a complex pattern of alternative splicing ; among the six splice variants that can be generated , three of them ( thpo-3 , thpo-5 , and thpo-6 ) show the peculiar alternative splicing of 116 nucleotides within exon 6 ( that can therefore be classified as a pseudointron ) ( figure 2(c ) ) . contrary to the fibronectin iiics - c5 and ache - r pseudointrons , the involvement in cancerogenesis of the variants deriving from the alternative splicing of the 116 nt pseudointron is less characterized . as a result , these observations have led researchers to hypothesize that alterations in alternative splicing of the 116 nt - thpo pseudointron might be employed as biomarker of tumor formation or progression . in conclusion , although currently there are few examples of pseudointron removal , at least three of these events have been associated with different levels of cancerogenesis , ranging from transformation to cancer progression . therefore , it is advisable that future high throughput screening analysis should be able to better associate the alternative splicing process of these pseudointrons with tumoral events . in particular , with regard to their use as possible biomarkers of transformation or of cancer staging / grading . regarding therapies , it is likely that all this information on the importance of alternative splicing for tumor development and progression may soon become extremely important for the development of novel therapeutic effectors in the fight against cancer [ 176 , 177 ] . currently , the field of rna therapy is rapidly growing especially in the inhibition of undesirable splice site choices by the use of antisense oligos [ 178180 ] . in particular , antisense oligonucleotides have been used to target newly created 5 or 3 splice sites deep within intronic regions of the nf1 gene to restore the normal splicing profile [ 91 , 92 ] . in this manner , only the mrnas containing the pseudoexon might be selectively degraded leaving any residual normal mrna to be properly processed . the disadvantage of simply degrading the pseudoexon - bearing transcript ( rather than acting on the splicing event itself ) is that everything depends on the level of pseudoexon inclusion ( less than 100% ) and on the status of the other allele ( i.e. finally , small molecule compounds might be also considered as possible therapeutic options in order to prevent the recognition of pseudoexons or modulate the recognition of pseudointrons . for example , it has been shown that sodium butyrate , an histone deacetylase inhibitor known to upregulate the expression of htra2-beta1 and sc35 , promotes skipping of the pseudoexon activated by the cftr 3849 + 10kbc > t mutation and can restore functional cftr channels . in addition , the splicing of different nf1 skipped exons as a result of mutations in cis - acting sequences has been restored with administration of the small molecule kinetin . as a result , this approach is another good candidate for an innovative therapeutic anticancer strategy for correction of deep intronic mutations that create novel splice sites . indeed , before any of these can be used in humans to treat cancer , several additional factors need to be considered . secondly , these systems will need to be optimized in order to achieve the best balance between successful delivery , intrinsic toxicity ( if any ) , and avoidance of undesired immune responses ( in the case of antisense oligos ) . finally , even when all these achievements are met , there will still be the need to optimize recurrent - administration protocols to determine the uptake levels , clearance , and accumulation in various tissues ( this is an often overlooked , since none of these methods will cause permanent correction of mrna splicing defects ) . nonetheless , the first results of this exciting news strategy are available , and there is the distinct possibility that rna - based treatment of cancer may soon enter the application stage .

pelvic floor dysfunction is an often used term that covers many different conditions including pelvic organ prolapse ( pop ) , urinary incontinence ( ui ) , and anorectal dysfunction . pop has a significant negative impact on women s quality of life , affects physical well - being , psychological and sexual functioning , and causes occupational and social restraints . up to 20 % of the general female population will have symptoms of pop and/or ui significant enough to require surgery [ 25 ] . both conditions often occur concurrently : up to 40 % of pop patients have preoperative concomitant ui [ 1 , 6 ] . of the women undergoing pop surgery , the highest recurrence rates are reported in the anterior vaginal compartment [ 2 , 7 ] . important risk factors for the development of pop are a family history of pop and vaginal delivery , and over the past decade levator ani muscle injury emerged as a major contributing factor in pop pathophysiology . this type of injury is only observed in women who have given birth vaginally or have at least entered the second stage of labor . risk factors for levator ani muscle injury are forceps delivery , length of second stage of labor , and large fetal head circumference [ 912 ] . besides being associated with an increased risk of pop , levator ani muscle injury has been reported to lead to an increased risk of recurrence after pop surgery [ 13 , 14 ] . the prevalence of levator ani muscle injury is reported to be up to 36 % in vaginally parous women and presents as a detachment , i.e. , avulsion , of the pubovisceral component of the levator ani muscle from the pubis [ 1517 ] . these pubovisceral avulsions can be observed as a complete loss of connection to the pubis or as a partial detachment with apparent loss of muscle bulk , both either unilateral or bilateral . pubovisceral avulsions can be visualized using magnetic resonance ( mr ) imaging or three - dimensional ( 3-d ) perineal ultrasonography . mr imaging is a noninvasive diagnostic tool that allows for detailed evaluation of all soft tissue structures of the pelvic support system . perineal ultrasonography is increasingly used in urogynecology and provides four - dimensional ( 4-d , i.e. , 3-d + temporal information ) assessment of the pelvic floor during routine clinical practice . both mr imaging and perineal ultrasonography require post - processing of the obtained image data , for which significant training is needed . which of these diagnostic methods is best for diagnosing pubovisceral avulsions is still under investigation . various studies have been conducted with regard to pubovisceral avulsions and the associated risks and specific symptoms . the aims of this systematic literature review were to assess whether the detection of pubovisceral avulsions by mr imaging or perineal ultrasonography was clinically relevant in women with pelvic floor dysfunction and to evaluate if there was a relation with anatomy and symptoms . a systematic literature search was performed by a clinical researcher ( kl ) and a senior librarian . the electronic databases pubmed , embase , and cinahl were searched from inception up to 27 september 2011 . the search and selection of the literature were restricted to publications written in western languages . to capture all relevant articles on the clinical relevance of pubovisceral avulsions , as diagnosed by mr imaging or perineal ultrasonography , we chose the following strategy : search term combinations were adapted for each database and consisted of medical subject headings ( mesh ) , thesaurus terms and cinahl headings , text words and word variations for the terms pelvic floor , mr imaging , ultrasonography , and physical examination . the entire strings of search terms are depicted in appendices 1 , 2 , and 3 . due to the large variability of terms that indicate pubovisceral avulsions , e.g. , detachment , disconnection , tearing off , and severing , we did not attempt to include the corresponding terms in the search strategy but used this as an inclusion criterion . hereby , the initial search was as sensitive as possible . articles identified by the literature search were included in our systematic review in case they reported on pubovisceral avulsions diagnosed by at least one of the two diagnostic methods : mr imaging and perineal ultrasonography . articles were included if they concerned clinical studies that provided data on pop status , pelvic floor symptoms , or recurrence of pop after surgery . pelvic floor symptoms had to be documented using standardized questions or validated ( quality of life ) questionnaires . recurrence of pop after surgery and pop status had to be documented with a standardized method , such as the pelvic organ prolapse quantification ( pop - q ) or baden - walker system , or stated as number of reoperations . all studies were evaluated by title and abstract according to the inclusion and exclusion criteria by kl . if necessary , full text articles were evaluated . after this preselection , a final decision on inclusion or exclusion was made in consensus with an experienced pelvic floor specialist ( kk ) . reference lists of relevant retrieved studies were cross - checked to identify additional studies that had been overlooked in the database search . the full text articles were evaluated to collect data on study design , aim of the study , sample size , study population , control group , parity , age , diagnostic method(s ) , pop staging , previous prolapse surgeries , number of reoperations , and the method(s ) of pelvic floor symptom assessment . to report pubovisceral avulsions , scoring systems have previously been developed by other research groups . in mr imaging , defect severity is scored in both muscle sides separately , ranging from 0 ( no defect ) to 3 ( complete muscle loss ) . a summed score for the two sides ( 06 ) can then be assigned and grouped as no defect ( 0 ) , minor defect ( 13 ) , or major defect ( 46 , or a unilateral score of 3 ) . for perineal ultrasonography , the integrity of the pubovisceral muscle is evaluated in the axial plane using multislice imaging , i.e. , tomographic ultrasound imaging ( tui ) . a set of eight tomographic slices are evaluated at intervals of 2.5 mm , in which both muscle sides are scored separately , resulting in a defect score ranging from 0 ( no defect ) to 16 ( complete bilateral avulsion ) . a complete avulsion is diagnosed if the reference slice , i.e. , the slice that represents the plane of minimum hiatal dimensions , as well as the two slices immediately cranial to this plane show an avulsion . partial avulsion is diagnosed when any of the slices are abnormal , without the patient being classified as having a complete avulsion [ 21 , 22 ] . the embase and cinahl searches found an additional 1,171 and 119 articles , respectively . in total , 3,134 articles were checked for eligibility . eight studies reported on detection of pubovisceral avulsion using mr imaging [ 13 , 15 , 2328 ] . twelve studies used perineal ultrasonography as the diagnostic method [ 14 , 17 , 22 , 2937 ] . apart from these 20 studies that used one diagnostic method for the evaluation of pubovisceral avulsions , we identified 4 studies that compared the use of different diagnostic methods . of these , two studies compared perineal ultrasonography with palpation of the pubovisceral muscle defect [ 38 , 39 ] . in both studies , the detection of pubovisceral avulsions was the outcome measure of interest . one study compared mr imaging with palpation and one study compared ultrasonography with mr imaging . in these studies , the objective was to determine the agreement in the detection of abnormalities in the pubovisceral muscle . of the latter four studies that evaluated two diagnostic methods , only one compared their results with our outcomes of interest and was therefore the only article included in this review that used two different diagnostic methods . study designs , group characteristics , diagnostic methods , and outcome measures are summarized in table 1 [ 1315 , 17 , 2238 ] . apart from two studies published in 2003 [ 15 , 23 ] , all other articles have been published after 2005.fig . n number of articlestable 1characteristics of clinical studies reporting on diagnosing pubovisceral avulsionsfirst author and year of publicationn in studystudy populationage , yearsparity , numberstudy designdiagnostic methodoutcome measureabdool 2009 361urogynecological complaints55 [ 1989]88 % vaginally parousrcuspop anatomy and symptomsadekanmi 2003 70cases : symptomatic pop stage ii622 [ 15]ccmrpop anatomy and symptomscontrols : nulliparaenulliparouschantarasorn 2011 393urogynecological complaints54 [ 1889]2 [ 09]rcussymptoms of anorectal dysfunctiondelancey2003 240cases : primiparae with sui ( n = 80)30.0 ( 5.7)primiparousccmrurinary symptomscontrol groups : continent primiparae ( n = 80)29.8 ( 4.4)primiparousnulliparae ( n = 80)29.2 ( 5.5)nulliparous2007 286cases : pop stage ii ( n = 151)56.4 ( 12.9)3.0 ( 1.8)ccmrpop anatomy and symptomscontrols : pop < stage ii ( n = 135)56.6 ( 13.0)2.3 ( 1.5)dietz2006 333urogynecological complaints52.8 ( 13.3)3 [ 08]pcusurinary symptoms , pop anatomy and symptoms2007 262urogynecological complaints54 [ 2682]2.7 [ 16]rcusurinary symptoms , pop anatomy and symptoms2008 781urogynecological complaints53 [ 1589]2 [ 012]rcuspop anatomy and symptoms2008 107urogynecological complaints55.5 [ 1785]2 [ 08]pcus , palpationpop anatomy and symptoms2009 420urogynecological complaints55 [ 1787]3 [ 010]rcusurinary symptoms , pop anatomy and symptoms2010 753urogynecological complaints55 [ 1889]2 [ 010]rcuspop anatomy and symptoms2010 83status after anterior colporrhaphy61 [ 3186]3 [ 19]pcusrecurrence of popheilbrun 2010 206primiparae27.8 ( 6.1)primiparousccmrurinary symptoms , symptoms of anorectal dysfunction , pop anatomy and symptomslewicky - gaupp 2010 26cases : older women with fi ( n = 8)71.6 [ 6385]2.6 ( 1.2)ccmrsymptoms of anorectal dysfunctioncontrol groups : older continent ( n = 9)71.6 [ 6088]2.8 ( 1.7)younger continent ( n = 9)28.7 [ 2041]0.2 ( 0.4)model 2010 737urogynecological complaints54.9 [ 1789]2 [ 09]rcusrecurrence of popmorgan2007 286cases : pop stage ii ( n = 151)56.4 ( 12.9)3.0 ( 1.8)ccmrsymptoms of anorectal dysfunctioncontrols : pop stage i ( n = 135)56.6 ( 13.0 ) 2.3 ( 1.5)2010 151pop stage ii56.4 ( 12.9)3.0 ( 1.8)ccmrurinary symptoms2011 83primary surgery for pop stage ii58.5 ( 11.2)82.6 % of cases 2 childrenccmrpop anatomy and symptoms , recurrence of poprodrigo 2011 967urogynecological complaints56 [ 1790]90 % vaginally parousrcussymptoms of anorectal dysfunctionweemhoff 2011 152status after anterior colporrhaphy61.4 ( 10.1)2.3 ( 1.1)pcusrecurrence of popwong 2011 220status after anterior vaginal mesh64.4 [ 3289]3 [ 010]rcusrecurrence of popn number of patients , sd standard deviation , rc retrospective cohort , cc case - control , pc prospective cohort , us ultrasound , mr magnetic resonance imaging , pop pelvic organ prolapse , sui stress urinary incontinence , fi fecal incontinencemean ( sd ) or median [ range]conference abstract , ( so far ) no full text article available selection process of studies included in the systematic review . n number of patients , sd standard deviation , rc retrospective cohort , cc case - control , pc prospective cohort , us ultrasound , mr magnetic resonance imaging , pop pelvic organ prolapse , sui stress urinary incontinence , fi fecal incontinence mean ( sd ) or median [ range ] conference abstract , ( so far ) no full text article available tables 2 , 3 , 4 and 5 provide an overview of the studies assessing pubovisceral avulsions in relation to urinary symptoms , symptoms of anorectal dysfunction , pop anatomy and symptoms , and recurrence of pop after surgery , respectively.table 2urinary symptoms in women assessed for pubovisceral avulsionsfirst authorn with levator avulsionpercentage of women of study population with levator avulsionconclusiondelancey cases:2329 % primiparous women with sui were twice as likely to have a muscle abnormality than primiparae without sui . no avulsions were identified in nulliparous womencontrol groups : non - sui primiparae911 % nulliparae00 % dietz complete4614 % no association was found between complete avulsion and urodynamic findings or symptoms of bladder dysfunction except for frequency ( p = 0.02)dietz complete5019 % sixty - two percent of women with complete avulsions complained of sui . defect score was significantly higher in women with symptoms of urinary frequency ( p = 0.05)dietz complete10425 % women with complete avulsions were less likely to suffer from sui ( p < 0.001 ) and usi ( p = 0.065 ) , but more likely to show signs of voiding dysfunction ( p = 0.005)heilbrun major2010 % there was no relation between major avulsion and ( s)ui based on mesa compared to women with minor or no avulsionmorgan major8355 % sui symptoms were least present in women with major avulsions and most frequently reported by women with minor avulsionsminor2416 % n number of patients , ( s)ui ( stress ) urinary incontinence , p valuep value for difference between avulsion and outcome measure assessed , usi urodynamic stress incontinence , mesa medical , epidemiological , and social aspects of aging urinary symptoms in women assessed for pubovisceral avulsions n number of patients , ( s)ui ( stress ) urinary incontinence , p valuep value for difference between avulsion and outcome measure assessed , usi urodynamic stress incontinence , mesa medical , epidemiological , and social aspects of aging studies on the relation between urinary symptoms and pubovisceral avulsions yielded contradictory results ( table 2 ) . . found that the prevalence of pubovisceral avulsions was more than twice as high in primiparous women with stress urinary incontinence ( sui ) than in primiparous women without sui . reported that women with urogynecological complaints and pubovisceral avulsions more often suffered from urinary frequency and voiding dysfunction . they also found that women over the age of 50 were less likely to suffer from sui . studied women with pop stage ii and found that women with major defects had the lowest risk of sui symptoms , while this risk was the highest in women with minor defects . heilbrun et al . reported no association between complaints of ( s)ui and levator ani defect status . two of the available studies on anorectal dysfunction found that women with major or complete pubovisceral avulsions were more likely to report symptoms of anorectal dysfunction than women with minor or no avulsions ( table 3 ) . this association was found in women who had suffered an anal sphincter tear during delivery and older women . for women with pop stage ii and for women with urogynecological complaints , i.e. , women with symptoms of lower urinary tract and pelvic floor dysfunction , no significant relation between symptoms of anorectal dysfunction and pubovisceral avulsions could be found [ 27 , 30 ] . rodrigo et al . reported that the prevalence of rectal intussusception ( ri ) was higher among women with pubovisceral avulsions.table 3symptoms of anorectal dysfunction and the relation with pubovisceral avulsionsfirst authorn with levator avulsionpercentage of women of study population with levator avulsionconclusionchantarasorn complete bilateral3810 % both unilateral and bilateral complete avulsions were not associated with fi , fecal urgency , or flatus incontinencecomplete unilateral3910 % heilbrun major2010 % women with major avulsions had a higher prevalence of fi based on fisi questionnaire ( p = 0.006 ) compared to women with no or minor avulsionslewicky - gaupp oi675 % older women with fi were more likely to have avulsions than women without fioc222 % yc111 % overall935 % morgan cases : there was no relation between levator ani defect status and symptom severity of anal incontinence and difficult defecation among women with popmajor8355 % minor2416 % controls : major2116 % minor3022 % rodrigo complete20922 % levator ani avulsion was more common among women with ri ( p = 0.003 ) . on multivariable analysis , there was no independent effect of avulsionn number of patients , oi older incontinent , oc older continent , yc young continent , fi fecal incontinence , fisi fecal incontinence severity index , p valuep value for difference between avulsion and outcome measure assessed , pop pelvic organ prolapse , ri rectal intussusception symptoms of anorectal dysfunction and the relation with pubovisceral avulsions n number of patients , oi older incontinent , oc older continent , yc young continent , fi fecal incontinence , fisi fecal incontinence severity index , p valuep value for difference between avulsion and outcome measure assessed , pop pelvic organ prolapse , ri rectal intussusception eleven studies reported on pop anatomy and symptoms in relation to pubovisceral avulsions . all studies used the pop - q system to stage pop . except for morgan et al . , who did not find a difference in anterior , apical , and posterior pop - q data between women with and without major levator defects , all other ten studies did find a significant relation with women with pubovisceral avulsions reporting pop more often ( table 4 ) . this relation was strongest with regard to the anterior [ 14 , 17 , 22 , 24 , 29 , 31 , 38 ] and central vaginal compartment [ 14 , 17 , 29 , 31 ] , but was less apparent for the posterior vaginal compartment [ 24 , 29].table 4pop anatomy and symptoms in women assessed for pubovisceral avulsionsfirst authorn with levator avulsionpercentage of women of study population with levator avulsionconclusionabdool bilateral319 % cystocele and rectocele ( on pop - q ) were associated with both unilateral and bilateral avulsion . uterine prolapse was only related to bilateral avulsionunilateral4512 % adekanmi cases : women with symptomatic pop stage ii showed in 56 % of the cases a partial or complete avulsion . no avulsions were identified in nulliparous womenpartial1826 % complete2130 % controls00 % delancey cases : major avulsions were statistically significant related to pop status ( p < 0.001 ) and associated with an adjusted or of 7.3 ( 95 % ci 3.913.6)major8355 % minor2416 % controls : major2116 % minor3022 % dietz complete18123 % women with complete avulsions were twice as likely to have significant pop , especially cystocele and uterine prolapsedietz complete2120 % women with a palpated avulsion showed more cystocele descent both on ultrasound and on pop - qdietz complete4614 % women with complete avulsions had higher grades of pop of the anterior and central compartment . there was no association between complete avulsion and pop symptomsdietz complete5019 % defect score was associated with cystocele and uterine prolapse and pop symptomsdietz complete10425 % women with a complete avulsion were more likely to have pop of the anterior compartment ( p < 0.001)dietz complete22630 % a complete avulsion was strongly associated with symptoms of pop , significant pop on clinical assessment , and bladder descent on perineal ultrasonography ( all p < 0.001)heilbrun major2010 % pop - q points ba and bp were more often at or below the hymen in women with major avulsions compared to women with no or minor avulsionsmorgan major4655 % there was no difference in preoperative anterior , apical , and posterior pop - q data between women with and without a major avulsionpop pelvic organ prolapse , n number of patients , pop - q pelvic organ prolapse quantification , p valuep value for difference between avulsion and outcome measure assessed , or odds ratio , 95 % ci 95 % confidence interval , ba and bp most descended edge of anterior and posterior vaginal wall , respectively , relative to the hymen pop anatomy and symptoms in women assessed for pubovisceral avulsions pop pelvic organ prolapse , n number of patients , pop - q pelvic organ prolapse quantification , p valuep value for difference between avulsion and outcome measure assessed , or odds ratio , 95 % ci 95 % confidence interval , ba and bp most descended edge of anterior and posterior vaginal wall , respectively , relative to the hymen table 5 shows that recurrence of pop after surgery was related to pubovisceral avulsions in all available studies on the subject [ 28 , 32 , 34 , 35 , 37 ] . this association was merely seen in women with major pubovisceral avulsions as diagnosed by mr imaging or in women with a complete avulsion according to perineal ultrasonography . wong et al . have reported on the recurrence of pop in women with pubovisceral avulsions following anterior vaginal mesh surgery.table 5recurrence of pop after surgery in relation to pubovisceral avulsionsfirst authorn with levator avulsionpercentage of women of study population with levator avulsionfollow - up durationconclusiondietz complete2935 % 4.5 years [ 36.4]complete avulsion was associated with an rr of 3 to 4 for cystocele recurrencemodel complete15621 % nrcomplete avulsion was associated with an increased prevalence of significant pop and symptoms of pop after previous pop or anti- incontinence surgerymorgan major4655 % 42.3 days ( 12.0)women with major avulsions were less likely to have anterior compartment support at least 2 cm above the hymen after surgery compared to women with no or minor avulsionsweemhoff partial5939 % 31 months [ 1450]fifty - two percent of women with anatomical recurrence of cystocele had a complete avulsion compared to 31 % of women without anatomical recurrence . there was no difference in anatomical recurrence in relation to partial avulsioncomplete6341 % wong complete8338 % 2.1 years [ 6 weeks5.6 years]complete avulsion was associated with an or of 2.27 ( 95 % ci 1.234.21 ) for significant cystocele recurrence on ultrasound . this effect was significant for women after a specific type of mesh operationpop pelvic organ prolapse , n number of patients , sd standard deviation , nr not reported , rr relative risk , or odds ratio , 95 % ci 95 % confidence intervalmedian [ range ] or mean ( sd)conference abstract , ( so far ) no full text article available recurrence of pop after surgery in relation to pubovisceral avulsions pop pelvic organ prolapse , n number of patients , sd standard deviation , nr not reported , rr relative risk , or odds ratio , 95 % ci 95 % confidence interval median [ range ] or mean ( sd ) conference abstract , ( so far ) no full text article available in this systematic literature review , we assessed the clinical relevance of diagnosing pubovisceral avulsions in women with pelvic floor dysfunction . diagnostic methods of interest were mr imaging and perineal ultrasonography . the presence of pubovisceral avulsions was shown to be relevant with respect to pop symptoms and pop stage , especially in the anterior and central compartment . recurrence of pop after surgery was also related to avulsions according to the available studies . there was no clear relation between pubovisceral avulsions and sui , but in this respect , there might be a difference between minor and major defects . studies with regard to the relation between symptoms of anorectal dysfunction and pubovisceral avulsions yielded contradictory results . even though it has long been recognized that the levator ani muscle plays a critical role in pelvic organ support , in what way pregnancy and/or childbirth injure the pelvic floor has not been proven conclusively . pelvic floor injury might be caused by compression , stretching or tearing of nerves , muscles , and/or connective tissue [ 42 , 43 ] . evaluation of the importance of muscle integrity has gone through an exponential growth over the past decade with the assessment of pubovisceral avulsions initially being performed using mr imaging . the research group of professor delancey was the first to standardize the evaluation of mr images . however , nowadays a growing amount of studies use perineal ultrasonography to assess pubovisceral muscle integrity as this diagnostic method has the advantage over mr imaging of easier implementation in routine clinical care together with the benefits of significantly lower cost and superior availability . the standardization of perineal ultrasonography when evaluating pubovisceral avulsions was performed under the supervision of professor dietz . approximately half of the studies in this review focused on using mr imaging to detect pubovisceral avulsions while a slightly higher number of studies used perineal ultrasonography . there was a notable variance in the naming for the subdivision of the levator ani muscle of interest for this review . in 2004 , kearney et al . performed a literature search on the various descriptions and terminology for this muscle . they found that even though there was a great diversity regarding the terms chosen in the available literature , the number of origin and insertion pairs was relatively consistent among authors . overall , the levator ani muscle comprises three subdivisions , namely , the iliococcygeal , puborectal , and pubovisceral muscles . the pubovisceral muscle includes the puboanal , puboperineal , and pubovaginal muscles and , together with the puborectal muscle , originates from the pubis . another frequently used term for the pubovisceral muscle is pubococcygeus muscle ; however , this implies a connection between the pubis and coccyx while in fact the muscle originates from the pubis and inserts into the wall of the vagina and anorectum . it is thus our belief that pubovisceral muscle is the correct term to be used . we had some difficulty to decide on the inclusion of one study by adekanmi et al . . the muscle injuries that were evaluated differed from injuries as described in all other studies with a distinction being made by adekanmi et al . between central and lateral ( endopelvic ) subsequently published literature and correspondence with the authors , we came to the conclusion that the researchers had not studied pubovisceral avulsions as included in this review . the present review showed that the relation between pop and pubovisceral avulsions is eminent as all but one paper found a significant difference in pop incidence ( table 4 ) . this relation was strongest in the compartments with highest recurrence rates after pop surgery , namely , the anterior and central vaginal compartment . delancey et al . were the only researchers to perform a case - control study with group matching for pop status in which , after multivariable regression , avulsion was still identified as an independent risk factor for pop . we were not able to further assess the correlation between avulsions and pop stage , pop surgeries , and pop recurrences from the accumulated data in this review . however , as up to 30 % of pop surgeries are currently performed due to relapse of pop , pubovisceral avulsions seem clinically relevant as an independent risk factor for pop . it has been suggested that women with pubovisceral avulsions might benefit from primary vaginal mesh surgery [ 47 , 48 ] . however , wong et al . found that mesh implementation in these women did not fully compensate for the effect of pubovisceral avulsions on recurrence rates . evaluating pubovisceral muscle integrity in appropriate clinical pop outcome studies will be needed to further lead the way , especially with regard to different strategies in surgical repair of various pop stages . which diagnostic method , mr imaging or perineal ultrasonography , while the association between urinary symptoms and childbirth is beyond dispute , this is presumably not directly related to pubovisceral avulsions , since available studies have shown contradictory results . women with pubovisceral avulsions were less likely to suffer from sui ( subjective and/or objective as confirmed by urodynamics ) . when pubovisceral avulsions were divided into major and minor avulsions , it was found that women with major defects are less likely and women with minor defects are more likely to have lower urinary tract symptoms . the lack of a clear association between avulsions and sui might be due to the fact that these avulsions do not seem to affect urethral mobility as much as they affect bladder support [ 51 , 52 ] . moreover , morgan et al . suggested that damage to the pudendal nerve could explain the difference in frequency of urinary symptoms between major and minor defects . they proposed that with a minor injury the preservation of one side of the muscle or parts of both sides can result in an asymmetry that alters reflexive responses of the urethra , bladder , and pelvic floor leading to symptom exacerbation , while a complete injury may be symmetrical leading to the absence of a motor and sensory reflex and therefore potentially having a less dramatic effect on symptoms . electromyographic research to support this hypothesis is currently lacking . the fact that pubovisceral avulsions are caused by vaginal delivery is beyond question and the same accounts for the occurrence of sphincter tears due to vaginal delivery . as the latter association is found to be related to symptoms of anorectal dysfunction , e.g. , fecal incontinence ( fi ) , together with the similar etiology of pubovisceral avulsions and sphincter tears , we expected a relation between symptoms of anorectal dysfunction and pubovisceral avulsions . this expectation is strengthened by the finding of heilbrun et al . that women with major levator defects have a higher prevalence of anal sphincter tears . moreover , lewicky - gaupp et al . found that older women with fi were more likely to have levator defects than women without fi ( both younger and older ) and that this association remained significant after correcting for external anal sphincter tears . rodrigo et al . reported a higher prevalence of ri among a group of women with pubovisceral avulsions . after multivariable analysis , this relation came on the account of hiatal area size on valsalva and was not the effect of avulsions per se . due to the variation in diagnostic methods used , study populations included , and outcome measures assessed , it was not possible to pool the available data into reliable relative risk factors for pubovisceral avulsions . to perform a patient - specific risk analysis and to ultimately individualize therapy for pop , it is our recommendation to evaluate the integrity of the pubovisceral muscle in those women who are most affected by the consequences of pubovisceral avulsions , i.e. , women visiting tertiary urogynecological clinics . therefore , both standardized questionnaires regarding symptoms of pelvic floor dysfunction and clinical examination , e.g. , pop - q , should be registered in this population . another limitation was that all studies but one on perineal ultrasonography are coauthored by professor dietz ( sydney , australia ) . with regard to the technique of mr imaging , professor delancey ( ann arbor , mi , usa ) coauthored six of eight of the included studies . both researchers are renowned experts within their field , but external validation therefore seems relevant and is awaiting . little data are currently available on agreement between mr imaging and perineal ultrasonography in the evaluation of pubovisceral avulsions . regarding this , zhuang et al . they reported a substantial agreement for both agreement between methods ( cohen s kappa = 0.79 ) and for agreement regarding the extent of the avulsion ( cohen s kappa = 0.65 ) . to further evaluate the agreement between mr imaging and perineal ultrasonography with regard to pubovisceral avulsions and to obtain the level of agreement between observers , the translabial 3d - ultrasonography for diagnosing levator defects ( trudil ) study using palpation , a pubovisceral avulsion is diagnosed if there is a detachment of the pubovisceral muscle from its insertion on the pubis . palpation can be easily incorporated in the standard gynecological examination , but it has been reported to have a considerable learning curve with only moderate agreement between different observers [ 38 , 40 ] . no studies could be identified that used palpation of the pubovisceral muscle defect as a diagnostic method solely . three studies have , however , been published on the agreement between palpation and ultrasonography or mr imaging [ 3840 ] . in 2006 , dietz et al . evaluated palpation versus perineal ultrasonography in a cohort of 54 patients and found poor agreement ( cohen s kappa = 0.098 ) between both methods with only two avulsions diagnosed by both methods . the agreement between two observers performing palpation of the muscle defect and an independent blinded reviewer of ultrasonographic data showed moderate and fair agreement , respectively . agreement between palpation and mr imaging was reported to be moderate ( cohen s kappa = 0.444 ) . in the latter study , we believe that this might be explained by the fact that palpation appears to rely more on the comparison of findings with a supposedly intact contralateral side hereby making bilateral defects much more difficult to detect digitally than on imaging . overall , it seems that even though palpation of the muscle defect appears to be the easiest diagnostic method to implement in routine clinical care , the value of this method is limited . implementation of either perineal ultrasonography or mr imaging in the diagnostic workup of women with complaints of pelvic floor dysfunction appears more feasible and of added value . in conclusion , a clear relation exists between visualized pubovisceral avulsions and pop stage and symptoms of pop . recurrence rates after pop surgery the association between pubovisceral avulsions and urinary symptoms , and symptoms of anorectal dysfunction , was less apparent . ( ( ( ( ( ( ( ( ( ( magnetic resonance imaging ) ) or ( " mr imaging " ) ) or ( " mr studies " ) ) or ( " mr study " ) ) or ( mri ) ) ) or ( ( ( ( ( ( ( ( ultrasonography ) ) or ( ultrasonograph * ) ) or ( ultrasonic imaging ) ) or ( echograph * ) ) or ( sonograph * ) ) or ( ultrasound ) ) or ( echo ) ) ) or ( ( ( ( ( physical examination ) ) or ( palpation ) ) or ( digital detection ) ) or ( medical examination ) ) ) ) and ( ( ( ( ( ( ( pelvic floor ) ) or ( levator ani ) ) or ( pubovisc * ) ) or ( pubococ * ) ) or ( puborect * ) ) or ( puboperine * ) ) ( ( exp nuclear magnetic resonance imaging/ or ( magnetic and resonance and imaging ) or ( mr stud * ) or mri ) or ( exp echography/ or ultrasonic imaging or ultrasound or ultrasonograph * or echograph * or sonograph * or echo ) or ( exp physical examination/ or exp medical examination/ or digital detection or palpation or physical examination or medical examination ) ) and ( exp levator ani muscle/ or levator ani or pelvic floor or pelvis floor or pubovisc * or puborect * or pubococ * or puboperine * ) ( ( mh " magnetic resonance imaging " or ( magnetic and resonance and imaging ) or mri or mr imaging or mr studies or mr study or mr stud * ) or ( mh " ultrasonography + " or ultrasonography or ultrasound or echo or ultrasonic imaging or ultrasonograph * or sonograph * or echograph * ) or ( mh " physical examination + " or physical examination or medical examination or palpation or digital detection ) ) and ( mh " pelvic floor muscles " or levator ani or pelvic floor or pelvis floor or pubovisc * or pubococ * or puborect * )

the aims of this systematic literature review were to assess whether the detection of pubovisceral avulsions using magnetic resonance ( mr ) imaging or perineal ultrasonography was clinically relevant in women with pelvic floor dysfunction and to evaluate the relation with anatomy , symptoms , and recurrence after surgery . we performed a systematic literature review using three bibliographical databases ( pubmed , embase , and cinahl ) as data sources . clinical studies were included in which pubovisceral avulsions were studied in relation to pelvic organ prolapse ( pop ) stage , pelvic floor symptoms , and/or recurrence of pop after surgery . ultimately , 21 studies met the inclusion criteria . pop stage and recurrence of pop after surgery were strongly associated with pubovisceral avulsions . contradictory results were found regarding the relation between pubovisceral avulsions and urinary symptoms and symptoms of anorectal dysfunction . pubovisceral avulsions , as diagnosed by mr imaging or perineal ultrasonography , are associated with higher stages of pop and recurrence of pop after surgery .

Introduction Materials and methods Results Discussion Conclusion Appendix 1: full PubMed literature search terms Appendix 2: full Embase literature search terms Appendix 3: full CINAHL literature search terms

pelvic floor dysfunction is an often used term that covers many different conditions including pelvic organ prolapse ( pop ) , urinary incontinence ( ui ) , and anorectal dysfunction . pubovisceral avulsions can be visualized using magnetic resonance ( mr ) imaging or three - dimensional ( 3-d ) perineal ultrasonography . the aims of this systematic literature review were to assess whether the detection of pubovisceral avulsions by mr imaging or perineal ultrasonography was clinically relevant in women with pelvic floor dysfunction and to evaluate if there was a relation with anatomy and symptoms . the electronic databases pubmed , embase , and cinahl were searched from inception up to 27 september 2011 . to capture all relevant articles on the clinical relevance of pubovisceral avulsions , as diagnosed by mr imaging or perineal ultrasonography , we chose the following strategy : search term combinations were adapted for each database and consisted of medical subject headings ( mesh ) , thesaurus terms and cinahl headings , text words and word variations for the terms pelvic floor , mr imaging , ultrasonography , and physical examination . articles identified by the literature search were included in our systematic review in case they reported on pubovisceral avulsions diagnosed by at least one of the two diagnostic methods : mr imaging and perineal ultrasonography . articles were included if they concerned clinical studies that provided data on pop status , pelvic floor symptoms , or recurrence of pop after surgery . recurrence of pop after surgery and pop status had to be documented with a standardized method , such as the pelvic organ prolapse quantification ( pop - q ) or baden - walker system , or stated as number of reoperations . n number of articlestable 1characteristics of clinical studies reporting on diagnosing pubovisceral avulsionsfirst author and year of publicationn in studystudy populationage , yearsparity , numberstudy designdiagnostic methodoutcome measureabdool 2009 361urogynecological complaints55 [ 1989]88 % vaginally parousrcuspop anatomy and symptomsadekanmi 2003 70cases : symptomatic pop stage ii622 [ 15]ccmrpop anatomy and symptomscontrols : nulliparaenulliparouschantarasorn 2011 393urogynecological complaints54 [ 1889]2 [ 09]rcussymptoms of anorectal dysfunctiondelancey2003 240cases : primiparae with sui ( n = 80)30.0 ( 5.7)primiparousccmrurinary symptomscontrol groups : continent primiparae ( n = 80)29.8 ( 4.4)primiparousnulliparae ( n = 80)29.2 ( 5.5)nulliparous2007 286cases : pop stage ii ( n = 151)56.4 ( 12.9)3.0 ( 1.8)ccmrpop anatomy and symptomscontrols : pop < stage ii ( n = 135)56.6 ( 13.0)2.3 ( 1.5)dietz2006 333urogynecological complaints52.8 ( 13.3)3 [ 08]pcusurinary symptoms , pop anatomy and symptoms2007 262urogynecological complaints54 [ 2682]2.7 [ 16]rcusurinary symptoms , pop anatomy and symptoms2008 781urogynecological complaints53 [ 1589]2 [ 012]rcuspop anatomy and symptoms2008 107urogynecological complaints55.5 [ 1785]2 [ 08]pcus , palpationpop anatomy and symptoms2009 420urogynecological complaints55 [ 1787]3 [ 010]rcusurinary symptoms , pop anatomy and symptoms2010 753urogynecological complaints55 [ 1889]2 [ 010]rcuspop anatomy and symptoms2010 83status after anterior colporrhaphy61 [ 3186]3 [ 19]pcusrecurrence of popheilbrun 2010 206primiparae27.8 ( 6.1)primiparousccmrurinary symptoms , symptoms of anorectal dysfunction , pop anatomy and symptomslewicky - gaupp 2010 26cases : older women with fi ( n = 8)71.6 [ 6385]2.6 ( 1.2)ccmrsymptoms of anorectal dysfunctioncontrol groups : older continent ( n = 9)71.6 [ 6088]2.8 ( 1.7)younger continent ( n = 9)28.7 [ 2041]0.2 ( 0.4)model 2010 737urogynecological complaints54.9 [ 1789]2 [ 09]rcusrecurrence of popmorgan2007 286cases : pop stage ii ( n = 151)56.4 ( 12.9)3.0 ( 1.8)ccmrsymptoms of anorectal dysfunctioncontrols : pop stage i ( n = 135)56.6 ( 13.0 ) 2.3 ( 1.5)2010 151pop stage ii56.4 ( 12.9)3.0 ( 1.8)ccmrurinary symptoms2011 83primary surgery for pop stage ii58.5 ( 11.2)82.6 % of cases 2 childrenccmrpop anatomy and symptoms , recurrence of poprodrigo 2011 967urogynecological complaints56 [ 1790]90 % vaginally parousrcussymptoms of anorectal dysfunctionweemhoff 2011 152status after anterior colporrhaphy61.4 ( 10.1)2.3 ( 1.1)pcusrecurrence of popwong 2011 220status after anterior vaginal mesh64.4 [ 3289]3 [ 010]rcusrecurrence of popn number of patients , sd standard deviation , rc retrospective cohort , cc case - control , pc prospective cohort , us ultrasound , mr magnetic resonance imaging , pop pelvic organ prolapse , sui stress urinary incontinence , fi fecal incontinencemean ( sd ) or median [ range]conference abstract , ( so far ) no full text article available selection process of studies included in the systematic review . n number of patients , sd standard deviation , rc retrospective cohort , cc case - control , pc prospective cohort , us ultrasound , mr magnetic resonance imaging , pop pelvic organ prolapse , sui stress urinary incontinence , fi fecal incontinence mean ( sd ) or median [ range ] conference abstract , ( so far ) no full text article available tables 2 , 3 , 4 and 5 provide an overview of the studies assessing pubovisceral avulsions in relation to urinary symptoms , symptoms of anorectal dysfunction , pop anatomy and symptoms , and recurrence of pop after surgery , respectively.table 2urinary symptoms in women assessed for pubovisceral avulsionsfirst authorn with levator avulsionpercentage of women of study population with levator avulsionconclusiondelancey cases:2329 % primiparous women with sui were twice as likely to have a muscle abnormality than primiparae without sui . defect score was significantly higher in women with symptoms of urinary frequency ( p = 0.05)dietz complete10425 % women with complete avulsions were less likely to suffer from sui ( p < 0.001 ) and usi ( p = 0.065 ) , but more likely to show signs of voiding dysfunction ( p = 0.005)heilbrun major2010 % there was no relation between major avulsion and ( s)ui based on mesa compared to women with minor or no avulsionmorgan major8355 % sui symptoms were least present in women with major avulsions and most frequently reported by women with minor avulsionsminor2416 % n number of patients , ( s)ui ( stress ) urinary incontinence , p valuep value for difference between avulsion and outcome measure assessed , usi urodynamic stress incontinence , mesa medical , epidemiological , and social aspects of aging urinary symptoms in women assessed for pubovisceral avulsions n number of patients , ( s)ui ( stress ) urinary incontinence , p valuep value for difference between avulsion and outcome measure assessed , usi urodynamic stress incontinence , mesa medical , epidemiological , and social aspects of aging studies on the relation between urinary symptoms and pubovisceral avulsions yielded contradictory results ( table 2 ) . two of the available studies on anorectal dysfunction found that women with major or complete pubovisceral avulsions were more likely to report symptoms of anorectal dysfunction than women with minor or no avulsions ( table 3 ) . , women with symptoms of lower urinary tract and pelvic floor dysfunction , no significant relation between symptoms of anorectal dysfunction and pubovisceral avulsions could be found [ 27 , 30 ] . reported that the prevalence of rectal intussusception ( ri ) was higher among women with pubovisceral avulsions.table 3symptoms of anorectal dysfunction and the relation with pubovisceral avulsionsfirst authorn with levator avulsionpercentage of women of study population with levator avulsionconclusionchantarasorn complete bilateral3810 % both unilateral and bilateral complete avulsions were not associated with fi , fecal urgency , or flatus incontinencecomplete unilateral3910 % heilbrun major2010 % women with major avulsions had a higher prevalence of fi based on fisi questionnaire ( p = 0.006 ) compared to women with no or minor avulsionslewicky - gaupp oi675 % older women with fi were more likely to have avulsions than women without fioc222 % yc111 % overall935 % morgan cases : there was no relation between levator ani defect status and symptom severity of anal incontinence and difficult defecation among women with popmajor8355 % minor2416 % controls : major2116 % minor3022 % rodrigo complete20922 % levator ani avulsion was more common among women with ri ( p = 0.003 ) . on multivariable analysis , there was no independent effect of avulsionn number of patients , oi older incontinent , oc older continent , yc young continent , fi fecal incontinence , fisi fecal incontinence severity index , p valuep value for difference between avulsion and outcome measure assessed , pop pelvic organ prolapse , ri rectal intussusception symptoms of anorectal dysfunction and the relation with pubovisceral avulsions n number of patients , oi older incontinent , oc older continent , yc young continent , fi fecal incontinence , fisi fecal incontinence severity index , p valuep value for difference between avulsion and outcome measure assessed , pop pelvic organ prolapse , ri rectal intussusception eleven studies reported on pop anatomy and symptoms in relation to pubovisceral avulsions . , who did not find a difference in anterior , apical , and posterior pop - q data between women with and without major levator defects , all other ten studies did find a significant relation with women with pubovisceral avulsions reporting pop more often ( table 4 ) . there was no association between complete avulsion and pop symptomsdietz complete5019 % defect score was associated with cystocele and uterine prolapse and pop symptomsdietz complete10425 % women with a complete avulsion were more likely to have pop of the anterior compartment ( p < 0.001)dietz complete22630 % a complete avulsion was strongly associated with symptoms of pop , significant pop on clinical assessment , and bladder descent on perineal ultrasonography ( all p < 0.001)heilbrun major2010 % pop - q points ba and bp were more often at or below the hymen in women with major avulsions compared to women with no or minor avulsionsmorgan major4655 % there was no difference in preoperative anterior , apical , and posterior pop - q data between women with and without a major avulsionpop pelvic organ prolapse , n number of patients , pop - q pelvic organ prolapse quantification , p valuep value for difference between avulsion and outcome measure assessed , or odds ratio , 95 % ci 95 % confidence interval , ba and bp most descended edge of anterior and posterior vaginal wall , respectively , relative to the hymen pop anatomy and symptoms in women assessed for pubovisceral avulsions pop pelvic organ prolapse , n number of patients , pop - q pelvic organ prolapse quantification , p valuep value for difference between avulsion and outcome measure assessed , or odds ratio , 95 % ci 95 % confidence interval , ba and bp most descended edge of anterior and posterior vaginal wall , respectively , relative to the hymen table 5 shows that recurrence of pop after surgery was related to pubovisceral avulsions in all available studies on the subject [ 28 , 32 , 34 , 35 , 37 ] . this association was merely seen in women with major pubovisceral avulsions as diagnosed by mr imaging or in women with a complete avulsion according to perineal ultrasonography . have reported on the recurrence of pop in women with pubovisceral avulsions following anterior vaginal mesh surgery.table 5recurrence of pop after surgery in relation to pubovisceral avulsionsfirst authorn with levator avulsionpercentage of women of study population with levator avulsionfollow - up durationconclusiondietz complete2935 % 4.5 years [ 36.4]complete avulsion was associated with an rr of 3 to 4 for cystocele recurrencemodel complete15621 % nrcomplete avulsion was associated with an increased prevalence of significant pop and symptoms of pop after previous pop or anti- incontinence surgerymorgan major4655 % 42.3 days ( 12.0)women with major avulsions were less likely to have anterior compartment support at least 2 cm above the hymen after surgery compared to women with no or minor avulsionsweemhoff partial5939 % 31 months [ 1450]fifty - two percent of women with anatomical recurrence of cystocele had a complete avulsion compared to 31 % of women without anatomical recurrence . this effect was significant for women after a specific type of mesh operationpop pelvic organ prolapse , n number of patients , sd standard deviation , nr not reported , rr relative risk , or odds ratio , 95 % ci 95 % confidence intervalmedian [ range ] or mean ( sd)conference abstract , ( so far ) no full text article available recurrence of pop after surgery in relation to pubovisceral avulsions pop pelvic organ prolapse , n number of patients , sd standard deviation , nr not reported , rr relative risk , or odds ratio , 95 % ci 95 % confidence interval median [ range ] or mean ( sd ) conference abstract , ( so far ) no full text article available in this systematic literature review , we assessed the clinical relevance of diagnosing pubovisceral avulsions in women with pelvic floor dysfunction . studies with regard to the relation between symptoms of anorectal dysfunction and pubovisceral avulsions yielded contradictory results . which diagnostic method , mr imaging or perineal ultrasonography , while the association between urinary symptoms and childbirth is beyond dispute , this is presumably not directly related to pubovisceral avulsions , since available studies have shown contradictory results . , fecal incontinence ( fi ) , together with the similar etiology of pubovisceral avulsions and sphincter tears , we expected a relation between symptoms of anorectal dysfunction and pubovisceral avulsions . to perform a patient - specific risk analysis and to ultimately individualize therapy for pop , it is our recommendation to evaluate the integrity of the pubovisceral muscle in those women who are most affected by the consequences of pubovisceral avulsions , i.e. to further evaluate the agreement between mr imaging and perineal ultrasonography with regard to pubovisceral avulsions and to obtain the level of agreement between observers , the translabial 3d - ultrasonography for diagnosing levator defects ( trudil ) study using palpation , a pubovisceral avulsion is diagnosed if there is a detachment of the pubovisceral muscle from its insertion on the pubis . implementation of either perineal ultrasonography or mr imaging in the diagnostic workup of women with complaints of pelvic floor dysfunction appears more feasible and of added value . in conclusion , a clear relation exists between visualized pubovisceral avulsions and pop stage and symptoms of pop . recurrence rates after pop surgery the association between pubovisceral avulsions and urinary symptoms , and symptoms of anorectal dysfunction , was less apparent . ( ( ( ( ( ( ( ( ( ( magnetic resonance imaging ) ) or ( " mr imaging " ) ) or ( " mr studies " ) ) or ( " mr study " ) ) or ( mri ) ) ) or ( ( ( ( ( ( ( ( ultrasonography ) ) or ( ultrasonograph * ) ) or ( ultrasonic imaging ) ) or ( echograph * ) ) or ( sonograph * ) ) or ( ultrasound ) ) or ( echo ) ) ) or ( ( ( ( ( physical examination ) ) or ( palpation ) ) or ( digital detection ) ) or ( medical examination ) ) ) ) and ( ( ( ( ( ( ( pelvic floor ) ) or ( levator ani ) ) or ( pubovisc * ) ) or ( pubococ * ) ) or ( puborect * ) ) or ( puboperine * ) ) ( ( exp nuclear magnetic resonance imaging/ or ( magnetic and resonance and imaging ) or ( mr stud * ) or mri ) or ( exp echography/ or ultrasonic imaging or ultrasound or ultrasonograph * or echograph * or sonograph * or echo ) or ( exp physical examination/ or exp medical examination/ or digital detection or palpation or physical examination or medical examination ) ) and ( exp levator ani muscle/ or levator ani or pelvic floor or pelvis floor or pubovisc * or puborect * or pubococ * or puboperine * ) ( ( mh " magnetic resonance imaging " or ( magnetic and resonance and imaging ) or mri or mr imaging or mr studies or mr study or mr stud * ) or ( mh " ultrasonography + " or ultrasonography or ultrasound or echo or ultrasonic imaging or ultrasonograph * or sonograph * or echograph * ) or ( mh " physical examination + " or physical examination or medical examination or palpation or digital detection ) ) and ( mh " pelvic floor muscles " or levator ani or pelvic floor or pelvis floor or pubovisc * or pubococ * or puborect * )

this study was part of a large multinational trial comparing artesunate versus quinine in the treatment of severe falciparum malaria in african children ( aquamat , registration number isrctn50258054 ) , the results of which have been published elsewhere . this substudy was conducted at hospital central da beira ( hcb ) , beira , mozambique , between october 2005 and july 2010 . hcb is an 800-bed tertiary referral hospital in the port city of beira , which is built along the swampy grounds at the mouth of the pungwe river where malaria transmission is perennial and malaria is mesoendemic . the hiv burden in the city of beira is reflected in the hiv prevalence in pregnant women , reported as approximately 30% in 2004 . ethics approval , including confidential hiv and cd4 testing , was granted by the comit nacional de biotica para a sade in mozambique and the oxford tropical research ethics committee . children ( < 15 years ) and adults ( 15 years ) presenting with suspected severe malaria according to modified world health organization ( who ) clinical criteria were screened using a plasmodium lactate dehydrogenase ( pldh)based and plasmodium falciparum histidine - rich protein-2 ( pfhrp2)based rapid diagnostic test ( rdt ) and a peripheral blood slide [ 22 , 23 ] . patients with a positive pldh - based rdt were included in the trial , provided that full written informed consent was given by the patient or carer . severity criteria included decreased consciousness ( coma or severe prostration ) , convulsions , respiratory distress or acidotic breathing , shock , severe symptomatic anemia ( < 5 g / dl ) , hypoglycemia ( <3 mmol / l ) , hemoglobinuria , severe jaundice , or a convincing history of anuria or oliguria in adult patients . a venous blood sample was taken for peripheral blood parasite counts , hematocrit ( hct ) , quantitative assessment of plasma pfhrp2 ( a marker of total body parasite burden ) [ 24 , 25 ] , biochemistry and acid base parameters ( ec8 cartridge for i - stat handheld analyzer ) , as well as hiv testing and cd4 lymphocyte count . hiv antibody testing was performed according to the sequential test algorithm by the mozambican hiv testing guidelines , comprising of a screening test ( determine hiv-1/2 , abbott laboratories , abbott park , il ) followed by a confirmation test ( uni - gold hiv , trinity biotech plc , bray , ireland ) in case of a positive screening result . clinical staff and patients did not have access to the hiv-1 test results and laboratory staff did not have access to patient data . surviving patients or their guardians received voluntary counseling and testing after recovery , and if indicated , were offered further treatment and follow - up according to national guidelines . quality assessment of slide reading was performed at the mahidol - oxford tropical medicine research unit in bangkok on 1348/2017 ( 67% ) of screening slides and 100% of slides of enrolled patients , and was used as reference . quantitative parasite counts were calculated from thin film ( count per l = count per 1000 red blood cells 125.6 hct ) assuming an mcv of 80 fl or thick film ( count per l = count per 200 white blood cells [ wbc ] x 40 ) assuming a wbc of 8000/l [ 26 , 27 ] . data were analyzed with stata , version 12 ( statacorp , college station , tx ) . categorical variables were compared between hiv - negative and hiv - positive cases with or fisher 's exact test . normally distributed or log - normalized variables were compared using a student t test , the remainder using mann weight - for - age z scores as a measure of malnutrition were calculated using stata applications based on the who child growth standards [ 28 , 29 ] . strictly defined severe malaria was based on modified who criteria [ 19 , 22 ] . in hiv - positive patients , cd4 lymphocyte count or percentage - based immunological staging ( not significant , mild , advanced , severe ) was performed according to the who classification . to determine the prognostic significance of hiv coinfection or the who hiv immunological stage classification , a logistic regression model was constructed with death as the dependant variable and hiv infection in addition to established predictors of death as the independent variables , including coma , prostration , convulsions , hypoglycemia , respiratory distress , shock , hemoglobin ( g / dl ) , base excess ( be ; mmol / l ) , log blood urea nitrogen ( mg / dl ) , log parasitemia ( parasites/l ) , plasma pfhrp2 [ as log and ( log ) , ng / ml ] [ 19 , 24 , 25 , 3133 ] and weight - for - age z scores . since the case fatality rates between children and adults differed significantly ( p < .0001 ) and the number of adult participants was limited , only children were included in the logistic regression model , which was adjusted for age and antimalarial treatment ( artesunate or quinine ) [ 19 , 35 ] . using a stepwise approach , only covariates that were significant at p < .05 were retained in the final model . data were analyzed with stata , version 12 ( statacorp , college station , tx ) . categorical variables were compared between hiv - negative and hiv - positive cases with or fisher 's exact test . normally distributed or log - normalized variables were compared using a student t test , the remainder using mann weight - for - age z scores as a measure of malnutrition were calculated using stata applications based on the who child growth standards [ 28 , 29 ] . strictly defined severe malaria was based on modified who criteria [ 19 , 22 ] . in hiv - positive patients , cd4 lymphocyte count or percentage - based immunological staging ( not significant , mild , advanced , severe ) was performed according to the who classification . to determine the prognostic significance of hiv coinfection or the who hiv immunological stage classification , a logistic regression model was constructed with death as the dependant variable and hiv infection in addition to established predictors of death as the independent variables , including coma , prostration , convulsions , hypoglycemia , respiratory distress , shock , hemoglobin ( g / dl ) , base excess ( be ; mmol / l ) , log blood urea nitrogen ( mg / dl ) , log parasitemia ( parasites/l ) , plasma pfhrp2 [ as log and ( log ) , ng / ml ] [ 19 , 24 , 25 , 3133 ] and weight - for - age z scores . since the case fatality rates between children and adults differed significantly ( p < .0001 ) and the number of adult participants was limited , only children were included in the logistic regression model , which was adjusted for age and antimalarial treatment ( artesunate or quinine ) [ 19 , 35 ] . using a stepwise approach , only covariates that were significant at p < .05 were retained in the final model . between october 2005 and july 2010 , 896 adults were screened for severe malaria , out of whom 87 ( 9.7% ) had a positive pldh - based rdt , an inclusion criterion for the treatment trial ( figure 1 ) . out of these , 68 adults were enrolled in the treatment trial . in 1272 children with suspected severe malaria , 731 or 57.5% ( 724 positive pldh and pfhrp2 tests , 7 with positive pfhrp2-based test only ) had a positive rdt of which 655 were enrolled . in screened adult patients with quality assessment of the peripheral blood slide ( n = 513 ) , the sensitivity of the pfhrp2-based rdt compared to expert microscopy was 44.3% and the specificity was 95.2% . for the pldh - based rdt , sensitivity was 38.8% and specificity 98.5% . in children ( n = 835 ) , the sensitivities of these rdts were 92.6% and 85.3% , and the specificities were 64.5% and 83.3% , respectively . the combination of a negative rdt and a positive malaria slide was associated with low parasitemia . the parasite density ( geometric mean ; 95% confidence interval [ ci ] ) with a negative pfhrp2-based test and positive slide with reported parasite density was 232 ( 141383 ) parasites/l in adults ( n = 89 ) and 180 ( 90359 ) parasites/l in children ( n = 40 ; p = .56 ) . figure 1.study profile.abbreviations : hiv , human immunodeficiency virus ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 ; pldh , plasmodium lactate dehydrogenase ; rdt , rapid diagnostic test . patients with discordant hiv antibody test results ( n = 4 ) were excluded from the analysis . four children aged < 18 months were classified as hiv positive on the basis of their serostatus in absence of viral load testing and were included . seroprevalence was 74/655 ( 11% ) in children and 49/68 ( 72% ) in adults . hiv - positive status in coinfected patients was reported on admission in 9/74 ( 12% ) of children and 5/49 ( 10% ) of adults , of which 5 and 4 were on antiretroviral treatment , respectively . by history of the carer , 10/74 ( 14% ) of hiv - coinfected children had a prior chronic illness ( 6 tuberculosis , 2 tuberculosis with chronic otitis media , 2 chronic otitis media ) , versus 20/579 ( 3% ) of hiv - uninfected children ( 8 tuberculosis , 1 sequelae of previous cerebral malaria , 4 congenital heart disease , 1 chronic otitis media , 6 bronchial asthma , 2 no information ; p < .001 ) . in hiv - coinfected adults , 11/49 ( 22% ) had a history of chronic disease ( 5 tuberculosis , 1 hemiparesis after stroke , 1 epilepsy , 1 hepatitis , 3 herpes zoster ) versus 1/16 ( 6% ) ( hypertension , 3 no information ; p = .108 ) . table 1.baseline characteristics of children and adults with severe malaria according to hiv statuscharacteristicchildren < 15 yearsadults 15 yearshiv negative ( n = 581)hiv positive ( n = 74)p valuehiv negative ( n = 19)hiv positive ( n = 49)p valuefemale279 ( 48%)33 ( 45%).587 ( 37%)22 ( 45%).55age , years ( median , iqr)3.6 ( 2.55.0)5.0 ( 3.08.0).000118 ( 1623)27 2538.0002presenting symptoms coma441 ( 76%)54 ( 73%).5813 ( 68%)35 ( 71%).81 convulsions506 ( 87%)56 ( 76%).00810 ( 53%)18 ( 37%).23 prostration132 ( 23%)19 ( 26%).575 ( 26%)12 ( 24%)1.0 shock21 ( 4%)3 ( 4%).740 ( 0%)3 ( 6%).55 severe respiratory distress37 ( 6%)14 ( 19%)<.0011 ( 5%)6 ( 12%).66severe acidosis ( be 8 mmol / l)108 ( 22%)26 ( 38%).0041 ( 8%)12 ( 27%).26hypoglycemia32 ( 6%)8 ( 11%).071 ( 5%)2 ( 4%)1.0severe anemia with respiratory distress67 ( 12%)15 ( 21%).0280 ( 0%)3 ( 6%).55black water fever24 ( 4%)5 ( 7%).364 ( 21%)10 ( 20%)1.0severe jaundice15 ( 3%)5 ( 7%).062 ( 11%)4 ( 8%)1.0anuria / oliguria 0 ( 0%)2 ( 4%)1.0hyperparasitemia ( > 10%)106 ( 21%)21 ( 32%).0433 ( 21%)12 ( 29%).74strictly defined severe malaria533 ( 94%)66 ( 89%).0816 ( 94%)41 ( 89%)1.0abbreviations : be , base excess ; hiv , human immunodeficiency virus ; iqr , interquartile range . for children : compensated and decompensated shock combined . in adults : only decompensated shock . in adults only . based on who criteria . baseline characteristics of children and adults with severe malaria according to hiv status abbreviations : be , base excess ; hiv , human immunodeficiency virus ; iqr , interquartile range . for children : compensated and decompensated shock combined . expert slide readings with quantitative results were available from 575/655 ( 88% ) of enrolled children with known hiv status . geometric mean ( 95% ci ) parasitemia was 47 140 ( 37 98858 498 ) in hiv - negative ( n = 510 ) and 67 977 ( 37 143124 408 ) in hiv - positive children ( geometric mean ( 95% ci ) plasma pfhrp2 concentration assessed in 653/655 ( > 99% ) children was 831 ( 707975 ) ng / ml in hiv - negative ( n = 510 ) and 1395 ( 9112136 ) ng / ml in coinfected children ( n = 65 ; p = .0321 ; figure 2 ) . figure 2.comparison of the circulating peripheral blood parasitemia ( left y - axis ) and plasma pfhrp2 concentration as a measure of the total body parasite burden ( right y - axis ) , between human immunodeficiency virus ( hiv)negative ( , n = 510 ) and hiv - positive ( , n = 65 ) children with quantified plasma pfhrp2 and peripheral blood parasitemia . abbreviations : ci , confidence interval ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 . comparison of the circulating peripheral blood parasitemia ( left y - axis ) and plasma pfhrp2 concentration as a measure of the total body parasite burden ( right y - axis ) , between human immunodeficiency virus ( hiv)negative ( , n = 510 ) and hiv - positive ( , n = 65 ) children with quantified plasma pfhrp2 and peripheral blood parasitemia . abbreviations : ci , confidence interval ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 . hiv - coinfected children were older and differed in their clinical presentation from uninfected children with an increased frequency of severe acidosis and severe anemia ( both clinically and laboratory assessed ) and respiratory distress ( tables 1 and 2 ) . blood urea nitrogen ( bun ) concentrations were also higher in hiv - infected children . on physical examination , hiv - positive children had significantly lower weight - for - age z scores and more frequent oral candidiasis and lymphadenopathy . in the adult patients , table 2.clinical examination and laboratory assessments in patients with severe malaria according to hiv statusassessmentchildren < 15 yearsadults 15 yearshiv negative ( n = 581)hiv positive ( n = 74)p valuehiv negative ( n = 19)hiv positive ( n = 49)p valueclinical examinationweight - for - age z score1.0 ( 1.3)1.6 ( 1.1).0001 temperature ( c)38.2 ( 0.9)38.3 ( 1.0).7638.1 ( 1.2)38.2 ( 1.3).76blood pressure ( mmhg ) systolic103 ( 16)104 ( 16).48112 ( 16)113 ( 22).82 diastolic64 ( 13)65 ( 16).6569 ( 14)69 ( 13).89respiratory rate ( breaths / min)39 ( 10)39 ( 9).9826 ( 5)29 ( 8).14coma depth ( n , median , range ) bcs70 , 2 ( 05)7 , 2 ( 23).29 gcs511 , 8 ( 315)67 , 9 ( 315).28718,10 ( 314)48 , 8 ( 315).54oral candidiasis1 ( < 1%)9 ( 12%)<.00104 ( 9%).57lymphadenopathy16 ( 3%)20 ( 27%)<.0011 ( 6%)5 ( 10%).54laboratory assessments bun ( mg / dl)12 ( 1112)16 ( 1319)<.000123 ( 1535)32 ( 2540).18 hemoglobin ( g / dl)8.2 ( 2.7)7.5 ( 2.7).03311.3 ( 2.6)10.2 ( 3.2).16 ph7.40 ( 0.11)7.38 ( 0.14).267.39 ( 0.09)7.37 ( 0.13).64 hco3 ( mmol / l)19.9 ( 4.9)17.7 ( 6.6).026721.8 ( 3.1)19.7 ( 5.7).17 base excess ( mmol / l)5 ( 6)7 ( 8).04613 ( 4)6 ( 8).43 slide pf positive547 ( 99%)72 ( 99%)1.018 ( 100%)45 ( 96%)1.0parasitemia ( parasites/l ) geometric mean ( 95% ci)47 141 ( 38 00558 474)68 320 ( 37 680123 874).25133 653 ( 59 082302 343)61 525 ( 24 628153 704).36pfhrp2 ( ng / ml ) geometric mean ( 95% ci)834 ( 712977)1452 ( 9832145).0197457 ( 932236)2471 ( 15094047).0072cd4 percentage 21 ( 1227) 13 ( 626) cd4 absolute cell count 235 ( 118429) 199 ( 76405) hiv - associated immunodeficiency not significant 16 ( 23%) 6 ( 14% ) mild 13 ( 19%) 7 ( 16% ) advanced 12 ( 18%) 8 ( 19% ) severe 27 ( 40%) 22 ( 51%)data are no . ( % ) , mean ( sd ) , or median ( iqr ) , unless otherwise indicated.abbreviations : bcs , blantyre coma score ; bun , blood urea nitrogen ; ci , confidence interval ; gcs , glasgow coma score ; hiv , human immunodeficiency virus ; iqr , interquartile range ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 . for children 10 years ( hiv - negative children , n = 563 ; hiv - positive children , n = 67 ) . bun was available in n = 487 and n = 67 children , n = 15 and n = 45 adults , respectively . hemoglobin was available in n = 551 and n = 70 children , n = 19 and n = 48 adults , respectively . ph was available in n = 489 and n = 68 children , n = 13 and n = 45 adults , respectively . hco3 was available in n = 490 and n = 68 children , n = 13 and n = 44 adults , respectively . be was available in n = 488 and n = 68 children , n = 13 and n = 45 adults , respectively . parasitemia reported from all patients with peripheral blood slide count , n = 511 and n = 66 children , n = 14 and n = 44 adults , respectively . pfhrp2 reported for all patients with pfhrp2 sample n = 580 and n = 73 children , n = 18 and n = 48 adults , respectively . cd4 percentage reported for hiv - infected children < 5 years ( n = 34 ) and adults ( n = 42 ) . cd4 absolute cell counts reported for hiv - infected children 5 years ( n = 34 ) and adults ( n = 43 ) . clinical examination and laboratory assessments in patients with severe malaria according to hiv status data are no . ( % ) , mean ( sd ) , or median ( iqr ) , unless otherwise indicated . abbreviations : bcs , blantyre coma score ; bun , blood urea nitrogen ; ci , confidence interval ; gcs , glasgow coma score ; hiv , human immunodeficiency virus ; iqr , interquartile range ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 . for children 10 years ( hiv - negative children , n = 563 ; hiv - positive children , n = 67 ) . bun was available in n = 487 and n = 67 children , n = 15 and n = 45 adults , respectively . hemoglobin was available in n = 551 and n = 70 children , n = 19 and n = 48 adults , respectively . ph was available in n = 489 and n = 68 children , n = 13 and n = 45 adults , respectively . hco3 was available in n = 490 and n = 68 children , n = 13 and n = 44 adults , respectively . be was available in n = 488 and n = 68 children , n = 13 and n = 45 adults , respectively . parasitemia reported from all patients with peripheral blood slide count , n = 511 and n = 66 children , n = 14 and n = 44 adults , respectively . pfhrp2 reported for all patients with pfhrp2 sample n = 580 and n = 73 children , n = 18 and n = 48 adults , respectively . cd4 percentage reported for hiv - infected children < 5 years ( n = 34 ) and adults ( n = 42 ) . cd4 absolute cell counts reported for hiv - infected children 5 years ( n = 34 ) and adults ( n = 43 ) . cd4 percentages and/or absolute counts were available for 68/74 ( 92% ) of hiv - positive children . increasing hiv - associated immunodeficiency according to the who classification was associated with increasing plasma pfhrp2 concentration ( nonparametric test for trend ; p = .022 ) but not with mortality ( p = .23 ) . comorbidities during hospitalization were more common in hiv - coinfected children ( 15/74 [ 20% ] ) compared with hiv - negative children ( 51/581 [ 9% ] ; p = .002 ) . pneumonia was suspected clinically in 19 ( 3% ) of hiv - negative children versus 9 ( 12% ) in hiv - positive children ( p < .001 ) . chest x - rays were sparsely available , and only a minority of cases had a radiologically confirmed diagnosis of pneumonia . although culture facilities were lacking , clinical sepsis was more common in hiv - coinfected children as well as a variety of other comorbidities ( table 3 ) . after admission , hiv - coinfected children developed more severe malaria related complications compared to hiv - negative children ( table 3 ) . hiv - coinfected children received more blood transfusions ( 38/74 [ 51% ] vs 224/581 [ 39% ] ; p = .034 ) and antibiotics ( 65/74 [ 88% ] vs 447/581 [ 77% ] ; p = .033 ) in hiv - negative children . table 3.comorbidity , complications , and outcome according to hiv statuschildren < 15 yearsadults 15 yearshiv negative ( n = 581)hiv positive ( n = 74)p valuehiv negative ( n = 19)hiv positive ( n = 49)p valuecomorbidity suspected pneumonia19 ( 3%)9 ( 12%)<.0011 ( 5%)2 ( 4%)1.0 confirmed by cxr5 ( 26%)4 ( 44%).411 ( 100%)0 ( 0%).3 clinical sepsis9 ( 2%)4 ( 5%).0490 ( 0%)1 ( 2%)1.0 suspected meningitis3 ( < 1%)1 ( 1%).381 ( 5%)01.0 gastroenteritis7 ( 1%)2 ( 3%).2700 other significant comorbidities22 ( 4%)7 ( 9%).02500 complications ( not present on admission ) development of coma3 ( < 1%)2 ( 3%).1011 ( 5%)2 ( 4%)1.0 deterioration coma score22 ( 4%)9 ( 12%).0012 ( 11%)6 ( 12%)1.0 convulsions developing or persisting > 6 hours after admission61 ( 11%)18 ( 24%).0012 ( 11%)3 ( 6%).61 respiratory distress6 ( 1%)7 ( 9%)<.0014 ( 21%)5 ( 10%).25 severe anemia ( < 5 g / dl)12 ( 2%)2 ( 3%).6701 ( 2%)1.0 black water fever13 ( 2%)4 ( 5%).1102 ( 4%)1.0 renal failure3 ( < 1%)2 ( 3%).103 ( 16%)8 ( 16%)1.0outcome mortality53 ( 9%)19 ( 26%)<.0014 ( 21%)17 ( 35%).38 mortality in strictly defined severe malaria53/533 ( 10%)19/66 ( 29%)<.0014/16 ( 25%)17/41 ( 41%).36 neurological sequelae at 28 days6 ( 1%)2 ( 3%).2300 abbreviations : cxr , chest x - ray ; hiv , human immunodeficiency virus . no culture facilities available . defined as > 6 loose stools/24 h. other significant comorbidities in hiv - negative children included : suspected intoxication with traditional medicine , suspected hepatitis , burn of hand ( 1% ) , asthma / bronchitis , undefined skin rash , reactive arthritis , parasitosis ( tungiasis , ascaris ) , herpes simplex virus labial ulcera , acute otitis media , tonsillitis , conjunctivitis , fever of unknown origin , suspected encephalitis ; in hiv - positive children : suspected intoxication with traditional medicine , asthma / bronchitis , impetigo , submandibular abscess , keratitis , suspected encephalitis . defined as urine output < 0.5 ml / kg / h for > 24 h or blood urea nitrogen > 60 mg / dl . comorbidity , complications , and outcome according to hiv status abbreviations : cxr , chest x - ray ; hiv , human immunodeficiency virus . loose stools/24 h. other significant comorbidities in hiv - negative children included : suspected intoxication with traditional medicine , suspected hepatitis , burn of hand ( 1% ) , asthma / bronchitis , undefined skin rash , reactive arthritis , parasitosis ( tungiasis , ascaris ) , herpes simplex virus labial ulcera , acute otitis media , tonsillitis , conjunctivitis , fever of unknown origin , suspected encephalitis ; in hiv - positive children : suspected intoxication with traditional medicine , asthma / bronchitis , impetigo , submandibular abscess , keratitis , suspected encephalitis . defined as urine output < 0.5 ml / kg / h for > 24 h or blood urea nitrogen > 60 mg / dl . in surviving children without neurological sequelae , median hospitalization time ( interquartile range [ iqr ] ) was 5 ( 47 ) days in hiv - coinfected versus 4 days ( 35 ) in hiv - negative children ( p = .0012 ) . the mortality in hiv - coinfected children was 19/74 ( 26% ) versus 53/581 ( 9% ) in hiv - uninfected children ( p < .001 ) . strictly defined severe malaria ( see the methods section ) were included in the analysis . although this substudy was not powered to look at the treatment effect of artesunate versus quinine , the mortality in hiv - positive children treated with artesunate was 22.2% versus 31% with quinine ( odds ratio [ or ] ( 95% ci ) 0.63 [ .221.85 ] ; p = .40 ) and in hiv - negative children 8.1% versus 10.1% ( or for artesunate 0.78 [ 0.441.38 ] ; p = .39 ) . stratification according to concomitant antiretroviral treatment ( n = 5 ) or antituberculosis treatment ( n = 5 ) did not alter any of the results ( data not shown ) . in a logistic regression model adjusted for age and antimalarial treatment , with death as dependent variable and established predictors of severe malaria as independent variables , significant predictors in the final model ( n = 482 ) were renal impairment ( log bun ) , acidosis ( be ) , parasitemia ( log parasitemia ; but an inverse correlation ) , and plasma pfhrp2 concentration ( table 4 ) . hiv infection was correlated with increased mortality ( unadjusted or 3.44 ; 95% ci 1.886.28 ) , but was not an independent prognosticator when plasma pfhrp2 was introduced into the model , which was related to the correlation between hiv status and plasma pfhrp2 ( test for trend across ordered groups ; p < the independent predictors were identical in a model including categorical hiv immunological stages rather than presence of hiv infection as a binary variable . also , hiv status did not contribute significantly to the final model when introduced as an interaction term with plasma pfhrp2 concentration . table 4.logistic regression analysis for children , adjusted for age , showing the prognostic value of significant risk factors assessed on admission for in - hospital survival of children with severe falciparum malariavariableodds ratio ( 95% ci)p valueplasma base excess0.89 ( .84.94)<.001[log ] blood urea nitrogen3.81 ( 1.907.65)<.001[log ] parasitemia0.66 ( .47.89).007[log ] plasma pfhrp2 ( squared)1.47 ( 1.101.95).008[log ] plasma pfhrp20.12 ( .03.48).003antimalarial drug ( artesunate vs quinine)0.37 ( .18.78).009the association between death and [ log ] plasma pfhrp2 was u - shaped and best described using a quadratic function . the patients with low pfhrp2 concentrations signify children with a low parasite burden where severe illness is likely caused by an alternative diagnosis than severe malaria ( hendriksen et al . introduction of an interaction term ( hiv-1 status plasma pfhrp2 ) did not improve the model ( p = .88 ) . r= 0.29 for the final model ( n = 482).abbreviations : ci , confidence interval ; hiv , human immunodeficiency virus ; pfhrp2 , plasmodium falciparumhistidine - rich protein-2 . logistic regression analysis for children , adjusted for age , showing the prognostic value of significant risk factors assessed on admission for in - hospital survival of children with severe falciparum malaria the association between death and [ log ] plasma pfhrp2 was u - shaped and best described using a quadratic function . the patients with low pfhrp2 concentrations signify children with a low parasite burden where severe illness is likely caused by an alternative diagnosis than severe malaria ( hendriksen et al . introduction of an interaction term ( hiv-1 status plasma pfhrp2 ) did not improve the model ( p = .88 ) . abbreviations : ci , confidence interval ; hiv , human immunodeficiency virus ; pfhrp2 , plasmodium falciparumhistidine - rich protein-2 . expert slide readings with quantitative results were available from 575/655 ( 88% ) of enrolled children with known hiv status . geometric mean ( 95% ci ) parasitemia was 47 140 ( 37 98858 498 ) in hiv - negative ( n = 510 ) and 67 977 ( 37 143124 408 ) in hiv - positive children ( geometric mean ( 95% ci ) plasma pfhrp2 concentration assessed in 653/655 ( > 99% ) children was 831 ( 707975 ) ng / ml in hiv - negative ( n = 510 ) and 1395 ( 9112136 ) ng / ml in coinfected children ( n = 65 ; p = .0321 ; figure 2 ) . figure 2.comparison of the circulating peripheral blood parasitemia ( left y - axis ) and plasma pfhrp2 concentration as a measure of the total body parasite burden ( right y - axis ) , between human immunodeficiency virus ( hiv)negative ( , n = 510 ) and hiv - positive ( , n = 65 ) children with quantified plasma pfhrp2 and peripheral blood parasitemia . abbreviations : ci , confidence interval ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 . comparison of the circulating peripheral blood parasitemia ( left y - axis ) and plasma pfhrp2 concentration as a measure of the total body parasite burden ( right y - axis ) , between human immunodeficiency virus ( hiv)negative ( , n = 510 ) and hiv - positive ( , n = 65 ) children with quantified plasma pfhrp2 and peripheral blood parasitemia . abbreviations : ci , confidence interval ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 . hiv - coinfected children were older and differed in their clinical presentation from uninfected children with an increased frequency of severe acidosis and severe anemia ( both clinically and laboratory assessed ) and respiratory distress ( tables 1 and 2 ) . blood urea nitrogen ( bun ) concentrations were also higher in hiv - infected children . on physical examination , hiv - positive children had significantly lower weight - for - age z scores and more frequent oral candidiasis and lymphadenopathy . in the adult patients , table 2.clinical examination and laboratory assessments in patients with severe malaria according to hiv statusassessmentchildren < 15 yearsadults 15 yearshiv negative ( n = 581)hiv positive ( n = 74)p valuehiv negative ( n = 19)hiv positive ( n = 49)p valueclinical examinationweight - for - age z score1.0 ( 1.3)1.6 ( 1.1).0001 temperature ( c)38.2 ( 0.9)38.3 ( 1.0).7638.1 ( 1.2)38.2 ( 1.3).76blood pressure ( mmhg ) systolic103 ( 16)104 ( 16).48112 ( 16)113 ( 22).82 diastolic64 ( 13)65 ( 16).6569 ( 14)69 ( 13).89respiratory rate ( breaths / min)39 ( 10)39 ( 9).9826 ( 5)29 ( 8).14coma depth ( n , median , range ) bcs70 , 2 ( 05)7 , 2 ( 23).29 gcs511 , 8 ( 315)67 , 9 ( 315).28718,10 ( 314)48 , 8 ( 315).54oral candidiasis1 ( < 1%)9 ( 12%)<.00104 ( 9%).57lymphadenopathy16 ( 3%)20 ( 27%)<.0011 ( 6%)5 ( 10%).54laboratory assessments bun ( mg / dl)12 ( 1112)16 ( 1319)<.000123 ( 1535)32 ( 2540).18 hemoglobin ( g / dl)8.2 ( 2.7)7.5 ( 2.7).03311.3 ( 2.6)10.2 ( 3.2).16 ph7.40 ( 0.11)7.38 ( 0.14).267.39 ( 0.09)7.37 ( 0.13).64 hco3 ( mmol / l)19.9 ( 4.9)17.7 ( 6.6).026721.8 ( 3.1)19.7 ( 5.7).17 base excess ( mmol / l)5 ( 6)7 ( 8).04613 ( 4)6 ( 8).43 slide pf positive547 ( 99%)72 ( 99%)1.018 ( 100%)45 ( 96%)1.0parasitemia ( parasites/l ) geometric mean ( 95% ci)47 141 ( 38 00558 474)68 320 ( 37 680123 874).25133 653 ( 59 082302 343)61 525 ( 24 628153 704).36pfhrp2 ( ng / ml ) geometric mean ( 95% ci)834 ( 712977)1452 ( 9832145).0197457 ( 932236)2471 ( 15094047).0072cd4 percentage 21 ( 1227) 13 ( 626) cd4 absolute cell count 235 ( 118429) 199 ( 76405) hiv - associated immunodeficiency not significant 16 ( 23%) 6 ( 14% ) mild 13 ( 19%) 7 ( 16% ) advanced 12 ( 18%) 8 ( 19% ) severe 27 ( 40%) 22 ( 51%)data are no . ( % ) , mean ( sd ) , or median ( iqr ) , unless otherwise indicated.abbreviations : bcs , blantyre coma score ; bun , blood urea nitrogen ; ci , confidence interval ; gcs , glasgow coma score ; hiv , human immunodeficiency virus ; iqr , interquartile range ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 . for children 10 years ( hiv - negative children , n = 563 ; hiv - positive children , n = 67 ) . bun was available in n = 487 and n = 67 children , n = 15 and n = 45 adults , respectively . hemoglobin was available in n = 551 and n = 70 children , n = 19 and n = 48 adults , respectively . ph was available in n = 489 and n = 68 children , n = 13 and n = 45 adults , respectively . hco3 was available in n = 490 and n = 68 children , be was available in n = 488 and n = 68 children , n = 13 and n = 45 adults , respectively . parasitemia reported from all patients with peripheral blood slide count , n = 511 and n = 66 children , n = 14 and n = 44 adults , respectively . pfhrp2 reported for all patients with pfhrp2 sample n = 580 and n = 73 children , n = 18 and n = 48 adults , respectively . cd4 percentage reported for hiv - infected children < 5 years ( n = 34 ) and adults ( n = 42 ) . cd4 absolute cell counts reported for hiv - infected children 5 years ( n = 34 ) and adults ( n = 43 ) . clinical examination and laboratory assessments in patients with severe malaria according to hiv status data are no . ( % ) , mean ( sd ) , or median ( iqr ) , unless otherwise indicated . abbreviations : bcs , blantyre coma score ; bun , blood urea nitrogen ; ci , confidence interval ; gcs , glasgow coma score ; hiv , human immunodeficiency virus ; iqr , interquartile range ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 . for children 10 years ( hiv - negative children , n = 563 ; hiv - positive children , n = 67 ) . bun was available in n = 487 and n = 67 children , n = 15 and n = 45 adults , respectively . hemoglobin was available in n = 551 and n = 70 children , n = 19 and n = 48 adults , respectively . ph was available in n = 489 and n = 68 children , n = 13 and n = 45 adults , respectively . hco3 was available in n = 490 and n = 68 children , n = 13 and n = 44 adults , respectively . be was available in n = 488 and n = 68 children , n = 13 and n = 45 adults , respectively . parasitemia reported from all patients with peripheral blood slide count , n = 511 and n = 66 children , n = 14 and n = 44 adults , respectively . pfhrp2 reported for all patients with pfhrp2 sample n = 580 and n = 73 children , n = 18 and n = 48 adults , respectively . cd4 percentage reported for hiv - infected children < 5 years ( n = 34 ) and adults ( n = 42 ) . cd4 absolute cell counts reported for hiv - infected children 5 years ( n = 34 ) and adults ( n = 43 ) . based on who criteria . cd4 percentages and/or absolute counts were available for 68/74 ( 92% ) of hiv - positive children . increasing hiv - associated immunodeficiency according to the who classification was associated with increasing plasma pfhrp2 concentration ( nonparametric test for trend ; p = .022 ) but not with mortality ( p = .23 ) . comorbidities during hospitalization were more common in hiv - coinfected children ( 15/74 [ 20% ] ) compared with hiv - negative children ( 51/581 [ 9% ] ; p = .002 ) . pneumonia was suspected clinically in 19 ( 3% ) of hiv - negative children versus 9 ( 12% ) in hiv - positive children ( p < .001 ) . chest x - rays were sparsely available , and only a minority of cases had a radiologically confirmed diagnosis of pneumonia . although culture facilities were lacking , clinical sepsis was more common in hiv - coinfected children as well as a variety of other comorbidities ( table 3 ) . after admission , hiv - coinfected children developed more severe malaria related complications compared to hiv - negative children ( table 3 ) . hiv - coinfected children received more blood transfusions ( 38/74 [ 51% ] vs 224/581 [ 39% ] ; p = .034 ) and antibiotics ( 65/74 [ 88% ] vs 447/581 [ 77% ] ; p table 3.comorbidity , complications , and outcome according to hiv statuschildren < 15 yearsadults 15 yearshiv negative ( n = 581)hiv positive ( n = 74)p valuehiv negative ( n = 19)hiv positive ( n = 49)p valuecomorbidity suspected pneumonia19 ( 3%)9 ( 12%)<.0011 ( 5%)2 ( 4%)1.0 confirmed by cxr5 ( 26%)4 ( 44%).411 ( 100%)0 ( 0%).3 clinical sepsis9 ( 2%)4 ( 5%).0490 ( 0%)1 ( 2%)1.0 suspected meningitis3 ( < 1%)1 ( 1%).381 ( 5%)01.0 gastroenteritis7 ( 1%)2 ( 3%).2700 other significant comorbidities22 ( 4%)7 ( 9%).02500 complications ( not present on admission ) development of coma3 ( < 1%)2 ( 3%).1011 ( 5%)2 ( 4%)1.0 deterioration coma score22 ( 4%)9 ( 12%).0012 ( 11%)6 ( 12%)1.0 convulsions developing or persisting > 6 hours after admission61 ( 11%)18 ( 24%).0012 ( 11%)3 ( 6%).61 respiratory distress6 ( 1%)7 ( 9%)<.0014 ( 21%)5 ( 10%).25 severe anemia ( < 5 g / dl)12 ( 2%)2 ( 3%).6701 ( 2%)1.0 black water fever13 ( 2%)4 ( 5%).1102 ( 4%)1.0 renal failure3 ( < 1%)2 ( 3%).103 ( 16%)8 ( 16%)1.0outcome mortality53 ( 9%)19 ( 26%)<.0014 ( 21%)17 ( 35%).38 mortality in strictly defined severe malaria53/533 ( 10%)19/66 ( 29%)<.0014/16 ( 25%)17/41 ( 41%).36 neurological sequelae at 28 days6 ( 1%)2 ( 3%).2300 abbreviations : cxr , chest x - ray ; hiv , human immunodeficiency virus . no culture facilities available . defined as > 6 loose stools/24 h. other significant comorbidities in hiv - negative children included : suspected intoxication with traditional medicine , suspected hepatitis , burn of hand ( 1% ) , asthma / bronchitis , undefined skin rash , reactive arthritis , parasitosis ( tungiasis , ascaris ) , herpes simplex virus labial ulcera , acute otitis media , tonsillitis , conjunctivitis , fever of unknown origin , suspected encephalitis ; in hiv - positive children : suspected intoxication with traditional medicine , asthma / bronchitis , impetigo , submandibular abscess , keratitis , suspected encephalitis . defined as urine output < 0.5 ml / kg / h for > 24 h or blood urea nitrogen > 60 mg / dl . comorbidity , complications , and outcome according to hiv status abbreviations : cxr , chest x - ray ; hiv , human immunodeficiency virus . no culture facilities available . defined as > 6 loose stools/24 h. other significant comorbidities in hiv - negative children included : suspected intoxication with traditional medicine , suspected hepatitis , burn of hand ( 1% ) , asthma / bronchitis , undefined skin rash , reactive arthritis , parasitosis ( tungiasis , ascaris ) , herpes simplex virus labial ulcera , acute otitis media , tonsillitis , conjunctivitis , fever of unknown origin , suspected encephalitis ; in hiv - positive children : suspected intoxication with traditional medicine , asthma / bronchitis , impetigo , submandibular abscess , keratitis , suspected encephalitis . defined as urine output < 0.5 ml / kg / h for > 24 h or blood urea nitrogen > 60 mg / dl . in surviving children without neurological sequelae , median hospitalization time ( interquartile range [ iqr ] ) was 5 ( 47 ) days in hiv - coinfected versus 4 days ( 35 ) in hiv - negative children ( p = .0012 ) . the mortality in hiv - coinfected children was 19/74 ( 26% ) versus 53/581 ( 9% ) in hiv - uninfected children ( p < .001 ) . strictly defined severe malaria ( see the methods section ) were included in the analysis . although this substudy was not powered to look at the treatment effect of artesunate versus quinine , the mortality in hiv - positive children treated with artesunate was 22.2% versus 31% with quinine ( odds ratio [ or ] ( 95% ci ) 0.63 [ .221.85 ] ; p = .40 ) and in hiv - negative children 8.1% versus 10.1% ( or for artesunate 0.78 [ 0.441.38 ] ; p = .39 ) . stratification according to concomitant antiretroviral treatment ( n = 5 ) or antituberculosis treatment ( n = 5 ) did not alter any of the results ( data not shown ) . in a logistic regression model adjusted for age and antimalarial treatment , with death as dependent variable and established predictors of severe malaria as independent variables , significant predictors in the final model ( n = 482 ) were renal impairment ( log bun ) , acidosis ( be ) , parasitemia ( log parasitemia ; but an inverse correlation ) , and plasma pfhrp2 concentration ( table 4 ) . hiv infection was correlated with increased mortality ( unadjusted or 3.44 ; 95% ci 1.886.28 ) , but was not an independent prognosticator when plasma pfhrp2 was introduced into the model , which was related to the correlation between hiv status and plasma pfhrp2 ( test for trend across ordered groups ; p < the independent predictors were identical in a model including categorical hiv immunological stages rather than presence of hiv infection as a binary variable . also , hiv status did not contribute significantly to the final model when introduced as an interaction term with plasma pfhrp2 concentration . table 4.logistic regression analysis for children , adjusted for age , showing the prognostic value of significant risk factors assessed on admission for in - hospital survival of children with severe falciparum malariavariableodds ratio ( 95% ci)p valueplasma base excess0.89 ( .84.94)<.001[log ] blood urea nitrogen3.81 ( 1.907.65)<.001[log ] parasitemia0.66 ( .47.89).007[log ] plasma pfhrp2 ( squared)1.47 ( 1.101.95).008[log ] plasma pfhrp20.12 ( .03.48).003antimalarial drug ( artesunate vs quinine)0.37 ( .18.78).009the association between death and [ log ] plasma pfhrp2 was u - shaped and best described using a quadratic function . the patients with low pfhrp2 concentrations signify children with a low parasite burden where severe illness is likely caused by an alternative diagnosis than severe malaria ( hendriksen et al . introduction of an interaction term ( hiv-1 status plasma pfhrp2 ) did not improve the model ( p = .88 ) . = 482).abbreviations : ci , confidence interval ; hiv , human immunodeficiency virus ; pfhrp2 , plasmodium falciparumhistidine - rich protein-2 . logistic regression analysis for children , adjusted for age , showing the prognostic value of significant risk factors assessed on admission for in - hospital survival of children with severe falciparum malaria the association between death and [ log ] plasma pfhrp2 was u - shaped and best described using a quadratic function . the patients with low pfhrp2 concentrations signify children with a low parasite burden where severe illness is likely caused by an alternative diagnosis than severe malaria ( hendriksen et al . introduction of an interaction term ( hiv-1 status plasma pfhrp2 ) did not improve the model ( p = .88 ) . abbreviations : ci , confidence interval ; hiv , human immunodeficiency virus ; pfhrp2 , plasmodium falciparumhistidine - rich protein-2 . this is the first prospective study to report the different clinical presentations of severe malaria , parasite burden , and mortality in hiv - coinfected patients . it was shown that hiv - coinfected children with severe malaria were more undernourished and presented more frequently with severe acidosis , severe anemia , respiratory distress , and elevated bun concentrations , and similar ( albeit nonsignificant ) trends were found in hiv - infected adults . previous studies carried out in areas of high malaria transmission have reported increased prevalence and severity of severe anemia and a higher 7- , 28- , or 90-day post - admission mortality in hiv - coinfected children without differences in admission parasitemia [ 15 , 16 , 36 ] . in addition to these findings , we established that a metabolic acidosis is more frequent in hiv - coinfected children . acidosis in severe malaria has been associated with severe anemia and respiratory distress or deep breathing and is an established strong predictor of mortality in adult as well as pediatric severe malaria [ 31 , 37 ] . total body parasite burden , measured as plasma pfhrp2 concentrations , was higher in hiv - infected children and positively correlated with the severity of immunosuppression according to who immunological classification . this total parasite burden includes the sequestered parasite burden , which causes impaired microcirculatory flow , an important cause of metabolic acidosis in severe malaria [ 24 , 25 ] . in line with other studies , hiv - coinfected children with severe malaria were older [ 2 , 14 , 16 ] and had higher parasite densities [ 14 , 38 ] . these results suggest that failure of the acquired immunity in hiv infection leads to a decreased ability to control parasitemia , which increases the risk of developing severe malaria with associated high mortality [ 3 , 9 , 10 , 39 ] . the clinical presentation of malaria in hiv - coinfected children depends on malaria specific immunity , which varies according to age and malaria transmission intensity [ 40 , 41 ] as well as hiv - related immunosuppression [ 9 , 11 , 14 , 39 ] . although our study design did not allow such comparison , our findings support the hypothesis that hiv - related immunosuppression increases the risk of severe malaria in adults , since the hiv-1 seroprevalence in severe malaria was more than twice the reported the hiv-1 seroprevalence in the adult population . elevated bun concentrations have been reported in hiv - coinfected adults with severe malaria [ 11 , 43 ] , and renal failure due to malaria - induced acute tubular necrosis has been described as a common complication and cause of death in asian adults [ 44 , 45 ] . it has been hypothesized that hiv - coinfected adults are more likely to have an hiv - mediated impaired renal function , although normalization of renal function following resolution of the malaria episode has also been described . the significance of elevated bun concentrations in hiv - positive patients , particularly in children , and its association with mortality as also observed in other studies [ 32 , 33 ] requires further investigation . in the adults , sensitivity of the rdts for diagnosing malaria was remarkably low ( < 50% ) . this was explained by low parasite densities on the peripheral blood slide , below the level of detection of the tests . patients with a negative pfhrp2-based rdt and low peripheral blood parasitemia include those with very low total parasite burdens , where an alternative diagnosis other than malaria is more likely . unfortunately , in the setting of the study , possibilities for establishing accurate alternative diagnoses other than malaria were limited . the mozambican national malaria control program recommends parasitological diagnosis of malaria in adults by rapid diagnostic test , although in referral hospitals , severe malaria is commonly diagnosed by peripheral blood slide . slide - positive rdt - negative severe illness represents patients with low parasitemia warrants diagnosis and treatment of other , possibly hiv - related , alternative illnesses [ 14 , 46 , 47 ] . further studies to assess the diagnostic work - up and management of this patient group are needed . comorbidities were more frequent in hiv - coinfected children than in hiv - uninfected children ; notably suspected pneumonia and sepsis . in the aquamat trial site in muheza , tanzania , where blood cultures and confidential hiv testing were performed , 8/38 ( 21% ) of hiv - coinfected children had a positive blood culture versus 45/855 ( 5% ) in hiv - negative children ( p < .001 ) with an almost 3-fold increased case fatality rate ( 14/38 ( 37% ) in hiv - coinfected children versus 112/855 ( 13% ) in hiv - uninfected children ( p < .001 ) [ unpublished data ; personal communication , ilse hendriksen ] . pediatric severe malaria with hiv coinfection has been associated with an increased risk of non typhi salmonella ( nts ) and/or gram - negative bacteremia , both leading to an increased risk of death . a study in southern mozambique ( an area with similar high hiv prevalence ) reported an incidence of 5.4% of concomitant bacteremia in pediatric severe malaria with streptococcus pneumoniae being the most frequently identified organism , especially in children with respiratory distress , and was associated with a higher case fatality . case fatality with hiv coinfection was 282% higher in children ( p < .001 ) and 64% higher in adults with severe malaria ( p = .28 ) . however , in a logistic regression model , hiv infection was not an independent predictor of death when plasma pfhrp2 was included in the regression model , whereas there was a clear correlation between hiv status and plasma pfhrp2 concentration , which is a measure of the total body parasite burden , including the sequestered parasites [ 24 , 25 ] . this again suggests that hiv - induced immune incompetence compromises control of the malaria parasite burden and thus severity of the infection . it also suggests that this mechanism is more important than hiv - related comorbidity , and underscores the importance of potent antimalarial treatment in these children with parenteral artesunate . in accordance with data from asian settings , convulsions , hypoglycemia , and symptomatic severe anemia were more frequent in children , whereas renal impairment and severe jaundice were more common in adult patients in the current african study . the main limitations of this study include the lack of diagnostic information to assess the additional pathology responsible for increased severe malaria mortality in hiv - coinfected patients . chest x - rays were not routinely performed and blood culture facilities were unavailable at the time of this study . in addition , clinical malaria may lower the cd4 lymphocyte count , which may therefore underestimate the patient 's immunological status . in summary , severe malaria in hiv - coinfected children presents with more severe acidosis , anemia and respiratory distress , more complications , and comorbidity , causing higher mortality and prolonged hospitalization in survivors . hiv coinfection is associated with a higher estimated total parasite burden , which is strongly associated with the observed increased severity . higher case fatality and more frequent complications warrant more intense monitoring and a low threshold for additional investigations to diagnose concomitant invasive bacterial infections , including chest x - ray , blood culture , and lumbar puncture with csf examination . since concomitant pneumonia , sepsis , and severe anemia are common , prompt parenteral antimalarial and antibiotic treatment , and availability of supportive treatments ( including oxygen therapy and blood transfusion ) are of extra importance in this group .

background . severe falciparum malaria with human immunodeficiency virus ( hiv ) coinfection is common in settings with a high prevalence of both diseases , but there is little information on whether hiv affects the clinical presentation and outcome of severe malaria.methods . hiv status was assessed prospectively in hospitalized parasitemic adults and children with severe malaria in beira , mozambique , as part of a clinical trial comparing parenteral artesunate versus quinine ( isrctn50258054 ) . clinical signs , comorbidity , complications , and disease outcome were compared according to hiv status.results . hiv-1 seroprevalence was 11% ( 74/655 ) in children under 15 years and 72% ( 49/68 ) in adults with severe malaria . children with hiv coinfection presented with more severe acidosis , anemia , and respiratory distress , and higher peripheral blood parasitemia and plasma plasmodium falciparum histidine - rich protein-2 ( pfhrp2 ) . during hospitalization , deterioration in coma score , convulsions , respiratory distress , and pneumonia were more common in hiv - coinfected children , and mortality was 26% ( 19/74 ) versus 9% ( 53/581 ) in uninfected children ( p < .001 ) . in an age- and antimalarial treatment adjusted logistic regression model , significant , independent predictors for death were renal impairment , acidosis , parasitemia , and plasma pfhrp2 concentration.conclusions . severe malaria in hiv - coinfected patients presents with higher parasite burden , more complications , and comorbidity , and carries a higher case fatality rate . early identification of hiv coinfection is important for the clinical management of severe malaria .

METHODS Statistical Analysis RESULTS Parasitological Markers in Pediatric Cases Clinical Manifestations of Severe Malaria According to HIV Status Comorbidities, Complications, and Outcome in Pediatric Cases DISCUSSION

this study was part of a large multinational trial comparing artesunate versus quinine in the treatment of severe falciparum malaria in african children ( aquamat , registration number isrctn50258054 ) , the results of which have been published elsewhere . children ( < 15 years ) and adults ( 15 years ) presenting with suspected severe malaria according to modified world health organization ( who ) clinical criteria were screened using a plasmodium lactate dehydrogenase ( pldh)based and plasmodium falciparum histidine - rich protein-2 ( pfhrp2)based rapid diagnostic test ( rdt ) and a peripheral blood slide [ 22 , 23 ] . to determine the prognostic significance of hiv coinfection or the who hiv immunological stage classification , a logistic regression model was constructed with death as the dependant variable and hiv infection in addition to established predictors of death as the independent variables , including coma , prostration , convulsions , hypoglycemia , respiratory distress , shock , hemoglobin ( g / dl ) , base excess ( be ; mmol / l ) , log blood urea nitrogen ( mg / dl ) , log parasitemia ( parasites/l ) , plasma pfhrp2 [ as log and ( log ) , ng / ml ] [ 19 , 24 , 25 , 3133 ] and weight - for - age z scores . since the case fatality rates between children and adults differed significantly ( p < .0001 ) and the number of adult participants was limited , only children were included in the logistic regression model , which was adjusted for age and antimalarial treatment ( artesunate or quinine ) [ 19 , 35 ] . to determine the prognostic significance of hiv coinfection or the who hiv immunological stage classification , a logistic regression model was constructed with death as the dependant variable and hiv infection in addition to established predictors of death as the independent variables , including coma , prostration , convulsions , hypoglycemia , respiratory distress , shock , hemoglobin ( g / dl ) , base excess ( be ; mmol / l ) , log blood urea nitrogen ( mg / dl ) , log parasitemia ( parasites/l ) , plasma pfhrp2 [ as log and ( log ) , ng / ml ] [ 19 , 24 , 25 , 3133 ] and weight - for - age z scores . since the case fatality rates between children and adults differed significantly ( p < .0001 ) and the number of adult participants was limited , only children were included in the logistic regression model , which was adjusted for age and antimalarial treatment ( artesunate or quinine ) [ 19 , 35 ] . by history of the carer , 10/74 ( 14% ) of hiv - coinfected children had a prior chronic illness ( 6 tuberculosis , 2 tuberculosis with chronic otitis media , 2 chronic otitis media ) , versus 20/579 ( 3% ) of hiv - uninfected children ( 8 tuberculosis , 1 sequelae of previous cerebral malaria , 4 congenital heart disease , 1 chronic otitis media , 6 bronchial asthma , 2 no information ; p < .001 ) . baseline characteristics of children and adults with severe malaria according to hiv status abbreviations : be , base excess ; hiv , human immunodeficiency virus ; iqr , interquartile range . geometric mean ( 95% ci ) parasitemia was 47 140 ( 37 98858 498 ) in hiv - negative ( n = 510 ) and 67 977 ( 37 143124 408 ) in hiv - positive children ( geometric mean ( 95% ci ) plasma pfhrp2 concentration assessed in 653/655 ( > 99% ) children was 831 ( 707975 ) ng / ml in hiv - negative ( n = 510 ) and 1395 ( 9112136 ) ng / ml in coinfected children ( n = 65 ; p = .0321 ; figure 2 ) . figure 2.comparison of the circulating peripheral blood parasitemia ( left y - axis ) and plasma pfhrp2 concentration as a measure of the total body parasite burden ( right y - axis ) , between human immunodeficiency virus ( hiv)negative ( , n = 510 ) and hiv - positive ( , n = 65 ) children with quantified plasma pfhrp2 and peripheral blood parasitemia . comparison of the circulating peripheral blood parasitemia ( left y - axis ) and plasma pfhrp2 concentration as a measure of the total body parasite burden ( right y - axis ) , between human immunodeficiency virus ( hiv)negative ( , n = 510 ) and hiv - positive ( , n = 65 ) children with quantified plasma pfhrp2 and peripheral blood parasitemia . hiv - coinfected children were older and differed in their clinical presentation from uninfected children with an increased frequency of severe acidosis and severe anemia ( both clinically and laboratory assessed ) and respiratory distress ( tables 1 and 2 ) . ( % ) , mean ( sd ) , or median ( iqr ) , unless otherwise indicated.abbreviations : bcs , blantyre coma score ; bun , blood urea nitrogen ; ci , confidence interval ; gcs , glasgow coma score ; hiv , human immunodeficiency virus ; iqr , interquartile range ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 . abbreviations : bcs , blantyre coma score ; bun , blood urea nitrogen ; ci , confidence interval ; gcs , glasgow coma score ; hiv , human immunodeficiency virus ; iqr , interquartile range ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 . comorbidities during hospitalization were more common in hiv - coinfected children ( 15/74 [ 20% ] ) compared with hiv - negative children ( 51/581 [ 9% ] ; p = .002 ) . pneumonia was suspected clinically in 19 ( 3% ) of hiv - negative children versus 9 ( 12% ) in hiv - positive children ( p < .001 ) . table 3.comorbidity , complications , and outcome according to hiv statuschildren < 15 yearsadults 15 yearshiv negative ( n = 581)hiv positive ( n = 74)p valuehiv negative ( n = 19)hiv positive ( n = 49)p valuecomorbidity suspected pneumonia19 ( 3%)9 ( 12%)<.0011 ( 5%)2 ( 4%)1.0 confirmed by cxr5 ( 26%)4 ( 44%).411 ( 100%)0 ( 0%).3 clinical sepsis9 ( 2%)4 ( 5%).0490 ( 0%)1 ( 2%)1.0 suspected meningitis3 ( < 1%)1 ( 1%).381 ( 5%)01.0 gastroenteritis7 ( 1%)2 ( 3%).2700 other significant comorbidities22 ( 4%)7 ( 9%).02500 complications ( not present on admission ) development of coma3 ( < 1%)2 ( 3%).1011 ( 5%)2 ( 4%)1.0 deterioration coma score22 ( 4%)9 ( 12%).0012 ( 11%)6 ( 12%)1.0 convulsions developing or persisting > 6 hours after admission61 ( 11%)18 ( 24%).0012 ( 11%)3 ( 6%).61 respiratory distress6 ( 1%)7 ( 9%)<.0014 ( 21%)5 ( 10%).25 severe anemia ( < 5 g / dl)12 ( 2%)2 ( 3%).6701 ( 2%)1.0 black water fever13 ( 2%)4 ( 5%).1102 ( 4%)1.0 renal failure3 ( < 1%)2 ( 3%).103 ( 16%)8 ( 16%)1.0outcome mortality53 ( 9%)19 ( 26%)<.0014 ( 21%)17 ( 35%).38 mortality in strictly defined severe malaria53/533 ( 10%)19/66 ( 29%)<.0014/16 ( 25%)17/41 ( 41%).36 neurological sequelae at 28 days6 ( 1%)2 ( 3%).2300 abbreviations : cxr , chest x - ray ; hiv , human immunodeficiency virus . comorbidity , complications , and outcome according to hiv status abbreviations : cxr , chest x - ray ; hiv , human immunodeficiency virus . the mortality in hiv - coinfected children was 19/74 ( 26% ) versus 53/581 ( 9% ) in hiv - uninfected children ( p < .001 ) . in a logistic regression model adjusted for age and antimalarial treatment , with death as dependent variable and established predictors of severe malaria as independent variables , significant predictors in the final model ( n = 482 ) were renal impairment ( log bun ) , acidosis ( be ) , parasitemia ( log parasitemia ; but an inverse correlation ) , and plasma pfhrp2 concentration ( table 4 ) . hiv infection was correlated with increased mortality ( unadjusted or 3.44 ; 95% ci 1.886.28 ) , but was not an independent prognosticator when plasma pfhrp2 was introduced into the model , which was related to the correlation between hiv status and plasma pfhrp2 ( test for trend across ordered groups ; p < the independent predictors were identical in a model including categorical hiv immunological stages rather than presence of hiv infection as a binary variable . geometric mean ( 95% ci ) parasitemia was 47 140 ( 37 98858 498 ) in hiv - negative ( n = 510 ) and 67 977 ( 37 143124 408 ) in hiv - positive children ( geometric mean ( 95% ci ) plasma pfhrp2 concentration assessed in 653/655 ( > 99% ) children was 831 ( 707975 ) ng / ml in hiv - negative ( n = 510 ) and 1395 ( 9112136 ) ng / ml in coinfected children ( n = 65 ; p = .0321 ; figure 2 ) . figure 2.comparison of the circulating peripheral blood parasitemia ( left y - axis ) and plasma pfhrp2 concentration as a measure of the total body parasite burden ( right y - axis ) , between human immunodeficiency virus ( hiv)negative ( , n = 510 ) and hiv - positive ( , n = 65 ) children with quantified plasma pfhrp2 and peripheral blood parasitemia . comparison of the circulating peripheral blood parasitemia ( left y - axis ) and plasma pfhrp2 concentration as a measure of the total body parasite burden ( right y - axis ) , between human immunodeficiency virus ( hiv)negative ( , n = 510 ) and hiv - positive ( , n = 65 ) children with quantified plasma pfhrp2 and peripheral blood parasitemia . hiv - coinfected children were older and differed in their clinical presentation from uninfected children with an increased frequency of severe acidosis and severe anemia ( both clinically and laboratory assessed ) and respiratory distress ( tables 1 and 2 ) . ( % ) , mean ( sd ) , or median ( iqr ) , unless otherwise indicated.abbreviations : bcs , blantyre coma score ; bun , blood urea nitrogen ; ci , confidence interval ; gcs , glasgow coma score ; hiv , human immunodeficiency virus ; iqr , interquartile range ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 . abbreviations : bcs , blantyre coma score ; bun , blood urea nitrogen ; ci , confidence interval ; gcs , glasgow coma score ; hiv , human immunodeficiency virus ; iqr , interquartile range ; pfhrp2 , plasmodium falciparum histidine - rich protein-2 . comorbidities during hospitalization were more common in hiv - coinfected children ( 15/74 [ 20% ] ) compared with hiv - negative children ( 51/581 [ 9% ] ; p = .002 ) . pneumonia was suspected clinically in 19 ( 3% ) of hiv - negative children versus 9 ( 12% ) in hiv - positive children ( p < .001 ) . hiv - coinfected children received more blood transfusions ( 38/74 [ 51% ] vs 224/581 [ 39% ] ; p = .034 ) and antibiotics ( 65/74 [ 88% ] vs 447/581 [ 77% ] ; p table 3.comorbidity , complications , and outcome according to hiv statuschildren < 15 yearsadults 15 yearshiv negative ( n = 581)hiv positive ( n = 74)p valuehiv negative ( n = 19)hiv positive ( n = 49)p valuecomorbidity suspected pneumonia19 ( 3%)9 ( 12%)<.0011 ( 5%)2 ( 4%)1.0 confirmed by cxr5 ( 26%)4 ( 44%).411 ( 100%)0 ( 0%).3 clinical sepsis9 ( 2%)4 ( 5%).0490 ( 0%)1 ( 2%)1.0 suspected meningitis3 ( < 1%)1 ( 1%).381 ( 5%)01.0 gastroenteritis7 ( 1%)2 ( 3%).2700 other significant comorbidities22 ( 4%)7 ( 9%).02500 complications ( not present on admission ) development of coma3 ( < 1%)2 ( 3%).1011 ( 5%)2 ( 4%)1.0 deterioration coma score22 ( 4%)9 ( 12%).0012 ( 11%)6 ( 12%)1.0 convulsions developing or persisting > 6 hours after admission61 ( 11%)18 ( 24%).0012 ( 11%)3 ( 6%).61 respiratory distress6 ( 1%)7 ( 9%)<.0014 ( 21%)5 ( 10%).25 severe anemia ( < 5 g / dl)12 ( 2%)2 ( 3%).6701 ( 2%)1.0 black water fever13 ( 2%)4 ( 5%).1102 ( 4%)1.0 renal failure3 ( < 1%)2 ( 3%).103 ( 16%)8 ( 16%)1.0outcome mortality53 ( 9%)19 ( 26%)<.0014 ( 21%)17 ( 35%).38 mortality in strictly defined severe malaria53/533 ( 10%)19/66 ( 29%)<.0014/16 ( 25%)17/41 ( 41%).36 neurological sequelae at 28 days6 ( 1%)2 ( 3%).2300 abbreviations : cxr , chest x - ray ; hiv , human immunodeficiency virus . comorbidity , complications , and outcome according to hiv status abbreviations : cxr , chest x - ray ; hiv , human immunodeficiency virus . the mortality in hiv - coinfected children was 19/74 ( 26% ) versus 53/581 ( 9% ) in hiv - uninfected children ( p < .001 ) . in a logistic regression model adjusted for age and antimalarial treatment , with death as dependent variable and established predictors of severe malaria as independent variables , significant predictors in the final model ( n = 482 ) were renal impairment ( log bun ) , acidosis ( be ) , parasitemia ( log parasitemia ; but an inverse correlation ) , and plasma pfhrp2 concentration ( table 4 ) . hiv infection was correlated with increased mortality ( unadjusted or 3.44 ; 95% ci 1.886.28 ) , but was not an independent prognosticator when plasma pfhrp2 was introduced into the model , which was related to the correlation between hiv status and plasma pfhrp2 ( test for trend across ordered groups ; p < the independent predictors were identical in a model including categorical hiv immunological stages rather than presence of hiv infection as a binary variable . this is the first prospective study to report the different clinical presentations of severe malaria , parasite burden , and mortality in hiv - coinfected patients . it was shown that hiv - coinfected children with severe malaria were more undernourished and presented more frequently with severe acidosis , severe anemia , respiratory distress , and elevated bun concentrations , and similar ( albeit nonsignificant ) trends were found in hiv - infected adults . the clinical presentation of malaria in hiv - coinfected children depends on malaria specific immunity , which varies according to age and malaria transmission intensity [ 40 , 41 ] as well as hiv - related immunosuppression [ 9 , 11 , 14 , 39 ] . in the aquamat trial site in muheza , tanzania , where blood cultures and confidential hiv testing were performed , 8/38 ( 21% ) of hiv - coinfected children had a positive blood culture versus 45/855 ( 5% ) in hiv - negative children ( p < .001 ) with an almost 3-fold increased case fatality rate ( 14/38 ( 37% ) in hiv - coinfected children versus 112/855 ( 13% ) in hiv - uninfected children ( p < .001 ) [ unpublished data ; personal communication , ilse hendriksen ] . a study in southern mozambique ( an area with similar high hiv prevalence ) reported an incidence of 5.4% of concomitant bacteremia in pediatric severe malaria with streptococcus pneumoniae being the most frequently identified organism , especially in children with respiratory distress , and was associated with a higher case fatality . case fatality with hiv coinfection was 282% higher in children ( p < .001 ) and 64% higher in adults with severe malaria ( p = .28 ) . however , in a logistic regression model , hiv infection was not an independent predictor of death when plasma pfhrp2 was included in the regression model , whereas there was a clear correlation between hiv status and plasma pfhrp2 concentration , which is a measure of the total body parasite burden , including the sequestered parasites [ 24 , 25 ] . in accordance with data from asian settings , convulsions , hypoglycemia , and symptomatic severe anemia were more frequent in children , whereas renal impairment and severe jaundice were more common in adult patients in the current african study . in summary , severe malaria in hiv - coinfected children presents with more severe acidosis , anemia and respiratory distress , more complications , and comorbidity , causing higher mortality and prolonged hospitalization in survivors .

the result of this trend is the rising proportions of older persons in the total population that increased from 9.2 percent in 1990 to 11.7 percent in 2013 and will continue to grow as a proportion of the world population , reaching 21.1 percent by 2050 . it has been estimated that older persons exceed the number of children for the first time in 2047 . although patients 65 years and older comprise less than 13 percent of the population , they represent 40 percent of hospitalized adults and nearly half of all healthcare dollars spent on hospitalization . it has been estimated that annual healthcare costs for the elderly are approximately four to five times those of people in their early teens . with the continuing trend of population aging , hospitalization and healthcare spending for older adults are expected to be rising . despite the disproportionate prevalence of hospitalized patients who are in the older age range , hospitalist programs often do not emphasize the need for geriatric skills and most hospital - based clinicians are not trained to treat older adult patients . the term geriatric syndrome is used to capture those clinical conditions in older persons that do not fit into discrete disease categories , including delirium , falls , frailty , dizziness , syncope , and urinary incontinence . although the concept of the geriatric syndrome remains poorly defined , four underlying risk factors have been identified for this syndrome , including older age , cognitive impairment , functional impairment , and impaired mobility . when an older adult with several chronic medical conditions develops an acute illness , one or more organ systems may fail . in addition , because of age - related diminution of physiologic reserves and greater vulnerability of elderly to acute stress , other organ systems that are seemingly unrelated to the presenting problem may lack the reserve to withstand the stresses of the acute illness . as a result , patient presents with failure of the organs that appear apart from the original complaint for which the patient is hospitalized . therefore , the chief complaint frequently does not represent the specific pathologic condition underlying the change in health status . in some cases , the two processes may involve distinct and distant organs with a disconnect between the site of the underlying physiologic insult and the resulting clinical symptom . the fact that these syndromes cross organ systems along with their multifactorial nature challenges traditional ways of viewing clinical care and research . for example , changes in mental status from baseline are commonly seen even when the main process does not involve the central nervous system ( cns ) ; this is the consequence of altered neural function in the form of cognitive and behavioral changes which permits the diagnosis of delirium . delirium is one of the most frequent presentations in the elderly that result from diminished cognitive reserve and brain dysfunction . it is an under recognized public health problem that affects significant number of older emergency department ( ed ) patients . with the elderly population expected to grow exponentially over the next several decades , delirium 's burden on eds will intensify . delirium is defined as an acute change in cognition that can not be better accounted for by preexisting or evolving dementia . this change in cognition is rapid , occurring over a period of hours or days , and is classically described as reversible . delirium in hospitalized patients is most closely associated with factors already present on admission such as prior cognitive impairment and advanced age . the incidence of delirium increases progressively after the forth decade of life . in elderly patients although confusion can be a presentation of dementia or psychological disorders , until other causes of confusion are identified , the confused patient should be assumed to have delirium , which is often reversible with treatment of the underlying disorder . delirium may result from a number of general medical and neurologic conditions ; the most common causes are medical conditions such as infections . aging causes the increased susceptibility to infection that is most likely a reflection of the age - associated decline in the competence of the immune system . moreover , both morbidity and mortality from any infections may be severalfold higher in the elderly [ 10 , 11 ] . infection is the primary cause of death in one - third of individuals aged 65 years and older and is a contributor to death for many others . a change in mental status or decline in function may be the only presenting problem in an older patient with an infection . in this situation one should distinguish between infections , especially those involving cns , versus noninfectious causes of altered mental status . in fact , one of the common problems encountered by the physicians in ed is identification of cause and treatment of patients who present with acute onset of fever and altered mental status , not only to ensure survival but also to prevent long - term neurological sequelae . this is the fact especially in the management of older adult patients with acute febrile encephalopathy ( afe ) and several chronic medical conditions . afe is used to describe patients with a condition in which altered mental status either accompanies or follows a short febrile illness . recently , it has been suggested that the term acute encephalitis syndrome ( aes ) be used instead of afe . a case of aes is defined as a person of any age , at any time of year , with the acute onset of fever and a change in mental status and/or new onset of seizure ( excluding simple febrile seizures ) . although the clinical definition of aes was introduced to facilitate surveillance for japanese encephalitis , this definition is broad and includes illnesses caused by many infectious as well as noninfectious etiologies . considering the fact that confusion ( altered mental state ) is the hallmark of encephalopathy , it would be present in up to 50 percent of elderly hospitalized patients , 10 percent of all hospitalized patients , and 2 percent of ed patients . according to han et al it has been noted that cns infections are the most common causes of altered mental status in patients with nontraumatic coma . here , a question comes to mind : is it true for elderly patients who present with altered mental status to the ed ? many physicians face great number of febrile encephalopathic older adults in the ed most of whom do not have cns infections . in fact , although one of the most important differential diagnoses in a confused elderly , who present to ed , especially when altered mental status is accompanied with fever or sepsis syndrome , is cns infection , this type of infection does not seem to be the most common underlying cause . in this regard , is it reasonable to perform lumbar puncture ( lp ) on all febrile older patients who admitted with acute alteration in mental status after normal neuroimaging results ? despite the growing number of elderly population , especially those who require hospitalization as well as the high burden of altered mental status among older ed patients , there are several unresolved questions about the optimal management of elderly patients with afe that require hospitalization . therefore , we decided to answer these questions by searching in the literature in the form of a systematic review . the questions posed in this review concern the need of early lp in all afe elderly patients in the routine management of afe . the questions are as follows : what is the etiologic distribution of afe in elderly patients?is higher prevalence of cns infection among the elderly responsible for higher frequency of afe in this age group ? what is the real frequency of cns infection among elderly patients who present with afe?does sepsis - associated encephalopathy ( sae ) explain the higher prevalence of afe among elderly patients that require hospitalization?is the classic triad of meningitis a sensitive indicator of cns infection among elderly patients ? is the combination of nuchal rigidity and encephalopathy a specific indicator of cns infection in elderly patients with afe?is a lp necessary when evaluating an older patient with afe ? how many elderly patients with afe undergo lp ? too many or too few?finally , we discuss the necessity for developing an evidence - based guideline for optimal management of afe in the elderly . what is the etiologic distribution of afe in elderly patients ? is higher prevalence of cns infection among the elderly responsible for higher frequency of afe in this age group ? what is the real frequency of cns infection among elderly patients who present with afe ? does sepsis - associated encephalopathy ( sae ) explain the higher prevalence of afe among elderly patients that require hospitalization ? is the classic triad of meningitis a sensitive indicator of cns infection among elderly patients ? is the combination of nuchal rigidity and encephalopathy a specific indicator of cns infection in elderly patients with afe ? is a lp necessary when evaluating an older patient with afe ? we sought to identify all potentially relevant articles using searches of web - based databases ( google scholar , medline , pubmed , scopus , and researchgate ) with no language restriction . aged adult , older adult , confusion , delirium , febrile , fever , encephalopathy , altered mental status , acute encephalitis syndrome , febrile encephalopathy , lumbar puncture , and lp . disagreements were resolved by discussion between the two review authors ; if no agreement could be reached , it was planned that a third author would decide . the references of the retrieved articles were examined for pertinent studies . by reviewing the titles of articles the reasons for exclusion were as follows : 784 because they were related to children , 341 because they were case reports or letter to editors , and 273 because they exclusively dealt with a specific pathogen(s ) of cns infection in elderly . the remaining 93 articles underwent full text review , which eliminated another 79 that were not relevant to the study questions . out of the finally selected articles , 4 were relevant to lumbar puncture and frequency of cns infection in older patients with encephalopathy , 6 addressed meningeal signs in elderly , and 4 were relevant to etiologic diagnosis of afe in adults ( figure 1 ) . the most frequent disorders causing altered mental status are common systemic disorders , such as urinary tract infections or pneumonia . the elderly , particularly those with some chronic cognitive impairment , are the most vulnerable group . patients with dementia who develop a systemic illness can present with an acute change in mental status . the first challenge facing the emergency clinician is to define what is meant by altered mental status or confusion and to ascertain why it led to the emergency department ( ed ) visit . when confusion is accompanied by fever or sepsis syndrome , the possibility of infection especially central nervous system ( cns ) infection as the etiologic cause of alteration in mental status comes to mind . in this part , we try to determine which underlying etiologies are more frequently presented as afe in elderly patients admitted to the ed there are numerous studies performed to address the etiology of afe in children ; however , we found only 4 studies in the literature that evaluated the etiologic diagnosis of afe in adult patients [ 14 , 22 , 27 , 28 ] . the mean age of participants of the 3 other studies was between 30 and 40 years . therefore , older adults had not a significant role in these studies . in 3 of these studies the most frequent etiologic diagnoses of afe in adult patients were pyogenic meningitis , viral encephalitis , and sepsis - associated encephalopathy ( sae ) , and less frequently tuberculous meningitis ( tm ) , cerebral malaria , leptospirosis , and brain abscess . in forth study that included patients with acute encephalitis syndrome ( fever , headache , altered mental status , vomiting , seizure , and neurodeficit ) , 25 ( 13.1 percent ) out of 190 patients were elderly cases . the results of this study showed 99 ( 52 percent ) patients with meningitis including 7 patients with confirmed bacterial meningitis ( bm ) and 13 with csf neutrophilic pleocytosis . others finally were diagnosed as acute hepatic encephalopathy , metabolic encephalopathy , alcoholic encephalopathy , cerebral malaria , brain abscess , sae , and so forth . there would be substantial variation in the distribution of etiologic diagnosis of afe among different studies on the basis of the age range of the participants , as well as the population studied . all of the above - mentioned studies were performed in india that is a tropical area with high endemicity for tuberculosis and malaria . the conclusion is that there is no enough information about the etiologic distribution of afe in adults including elderly population except few studies from limited parts of the world . bacterial meningitis ( bm ) remains one of the most feared infectious diseases because of its subtle onset and high mortality rate . although the incidence of meningitis is the highest among infants during the first month of life , several large studies have documented a later peak of incidence among persons aged 60 and over . estimates of the incidence of meningitis in these patients range from 2 to 9 per 10 per year . in recent years , bm has radically changed to become a disease largely of adults in particular , of older adults . the introduction of conjugate vaccines and preventive treatment of colonized pregnant women have had a major impact on the epidemiology and characteristics of bm . this circumstance highlights key problem areas in its management including recognition of the disease in older patients who present with fewer of the classic symptoms of meningitis or for whom there are other explanations for these symptoms . in the prospective study conducted by domingo et al . on 635 episodes of acute bacterial meningitis ( abm ) in adult patients in barcelona , the corresponding incidence was 4.03 per 10 and 7.40 per 10 inhabitants / year for patients aged 15 to 64 years and patients aged 65 years , respectively . the higher prevalence of meningitis among the elderly compared with the general adult population has been also documented in developing countries . although older age is a known risk factor for cns infection , estimation of the true prevalence of cns infection in febrile aged patients with acute alteration of mental status has received little attention . in the study conducted by cagatay et al . 25 out of 135 ( 18.5 percent ) acutely febrile aged patients with final diagnosis of infectious disease had confusion at the time of hospital admission , whereas only 10 ( 7.4 percent ) patients were documented to have cns infection . given the aim of the study that was the evaluation of the etiologic distribution of acute fever in the elderly , small number of aged adults with fever and confusion were included . in this part , we try to estimate the frequency of cns infection among afe elderly patients . a thorough search in the literature revealed only that few studies provided data about the frequency of cns infection in elderly patients with afe to be included ( table 1 ) . we excluded articles in which the field of interest was restricted to a few specific pathogens and articles that only investigate the etiologic diagnosis of bm in elderly patients . all , except one that did not mention the design of study , used a retrospective design . the results showed the frequency of 2.4 to 24 percent for cns infection among afe elderly patients . however , the small sample size , retrospective design , and selection bias of these studies that only included patients who underwent lumbar puncture ( lp ) make an estimation of true prevalence of cns infection in aged adults with afe impossible . the incidence of sepsis among individuals 85 years of age and older has been estimated to be 26.2 cases per 1000 populations , which is more than 100 times greater than the incidence noted among individuals 5 to 14 years of age . according to marco et al . the most common final diagnoses among geriatric emergency department ( ed ) patients who presented with acute fever were pneumonia , urinary tract infection , and sepsis . among the myriad of conditions that can induce delirium in critical illness , sepsis in the form of sae represents the most frequent and severe cause . up to 70 percent of patients with bacteremia have wide spectrum of neurological symptoms that include fluctuating mental status changes , inattention , and disorganized thinking and therefore match with current criteria for delirium [ 36 , 37 ] . the cardinal feature of sae is a diffuse disturbance in cerebral function without any lateralizing signs . two key prerequisites for making a diagnosis of sae are presence of extracranial infection and impaired mental status . diagnosis of brain dysfunction in a patient with sepsis implies a systematic diagnostic approach of all potential factors , in addition to sepsis , that can contribute to aggravate or prolong brain dysfunction . in addition to the frequent lack of fever , infections in older adults may be associated with a nonspecific decline in baseline functional status such as increased confusion and falling . cognitive impairment further contributes to the atypical presentation of infections in older adults , reducing the capacity to communicate symptoms . severe sepsis in older population is associated with substantial and persistent new cognitive impairment and functional disability among survivors . the magnitude of these new deficits is large , likely resulting in a pivotal downturn in patients ' ability to live independently . in fact , sepsis is often a sentinel event in the lives of older patients , initiating major and enduring cognitive and functional declines . studied the cognitive impairment of older patients with severe sepsis ( 1194 patients , mean age of survivors 76.9 years ) and found that prevalence of moderate to severe cognitive impairment increases by 10.6 percentage points among patients who survived severe sepsis . although older age is a known risk factor for sae , it does not seem to fully explain the frequent presentation of acutely ill febrile older adults with encephalopathy syndrome . on the other hand , it has been noted that sae is a diagnosis of exclusion : there should be no clinical or laboratory evidence of direct central nervous system ( cns ) infection ( e.g. , meningitis , macroscopic intracranial abscess , or empyema ) , head trauma , fat embolism , adverse reactions to medications , or sedative or paralyzing drug effects . it has been suggested that occurrence of sudden fluctuation in mental status , occurrence of focal neurological sign , seizure(s ) , and/or neck stiffness should prompt the physician to consider neuroimaging , electroencephalogram ( eeg ) , and/or lumbar puncture ( lp ) to rule out a direct cns infection . it has been recommended to perform a lp on obtunded patients with systemic inflammatory response syndrome ( sirs ) , to rule out meningitis . considering the high frequency of altered mental status in elderly patients with infectious syndromes outside the cns , is it reasonable to perform early lp on every confused aged adult with fever or sepsis syndrome ? answering to this question requires the knowledge about the frequency of cns infection and sae in elderly patients with afe . systematic review . on the basis of literature review , we could not find any relevant prospective study to the frequency of sae in elderly patients with afe . however , the role of older age as a risk factor for sae has been investigated in several studies . in few retrospective studies , the frequency of abnormal csf in elderly patients with afe varied between 2.4 to 24 percent ( table 1 ) . nonetheless , because of the retrospective design of these studies it can not be interpreted as an estimation of sae in elderly patients with afe . common clinical practice relies on the absence of neck stiffness or other meningeal signs to rule out meningitis in the previously healthy adult . meningeal signs have been assumed to be reliable and usually present in awaked adults with meningitis , except infants , the elderly , and the immunesuppressed . according to van de beek et al . in adults presenting with community - acquired bacterial meningitis ( bm ) , the sensitivity of the classic triad of fever , neck stiffness , and altered mental status is low ( 44 percent ) , but almost all ( 95 percent ) present with at least two of the four symptoms of headache , fever , neck stiffness , and altered mental status . it has been noted that among adults with a clinical presentation that is low risk for meningitis , the clinical examination aids in excluding the diagnosis . nevertheless , in the study of waghdhare et al . physical signs of meningeal inflammation were not helpful for ruling in or ruling out meningitis accurately ( age range of participants : 1381 ; mean : 38 18 ) similarly , brouwer et al . reported low diagnostic accuracy of signs of meningeal irritation for prediction of cerebrospinal fluid ( csf ) pleocytosis , suggesting that clinical assessment alone is insufficient to exclude bm . in addition , stockdale et al . found that , in patients with abm , the classical clinical features are uncommon on arrival to hospital and frequently evolve following admission . recommended patients suspected to have meningitis to undergo a lp regardless of the presence or absence of physical signs . bm in elderly patients is associated with greater diagnostic difficulties and more complications , as well as with increased mortality . according to choi in the clinical assessment for possible bm , older adults who present without fever , neck stiffness , or altered mental function those individuals with 2 or 3 of the 3 classic findings are more likely to have meningitis , but even the presence of all 3 findings is not entirely specific . since febrile responses are often blunted or absent in older adults , it is not surprising that fever is not a universal symptom , varying in occurrence from 59 to 100 percent in different studies . similarly , headache has been noted in only some older adults with meningitis , and depressed levels of consciousness such as stupor or coma are often but not universally present [ 29 , 45 ] . diagnostic difficulty of abm in the elderly is attributed to its atypical and more subtle presentation [ 10 , 46 ] . it has been shown that the time from arrival to starting antibiotic is longer among those patients with suspected meningitis who had atypical presentation or complex medical histories . another major problem in attaining a correct diagnosis in the elderly is the presence of multiple pathologies . although the majority of elderly patients present with fever , confusion , and stiff neck , confusion may be assumed to be secondary to senility , and the stiff neck to cervical osteoarthritis . in such circumstances one would recommend more frequent spinal tap as a prerequisite to exclude meningitis in the elderly . it seems that nuchal rigidity is neither sensitive nor specific sign compared with younger patients . nuchal rigidity is often found on examination of elderly patients who do not have bm and usually have coexistent neurologic deficits . the issue to be discussed here is how one can be sure if the neck stiffness or rigidity in an elderly patient is of new onset . nuchal rigidity , which may be a sign of meningitis , was found in 35 percent of geriatric patients on acute - care and rehabilitation wards and was significantly associated with cerebrovascular disease ( cva ) , confusion , abnormal plantar responses , and primitive reflexes . accordingly , they suggested that elderly patients who have nuchal rigidity with no history of neurologic or cognitive disorders should be investigated for meningitis . neck stiffness in older adults without meningitis may be caused by prior cva , cervical osteoarthritis , parkinson 's diseases , or certain drugs . considering the high prevalence of these underlying conditions among the elderly , interpretation of clinical tests evoking meningeal irritation may be inconclusive in significant number of hospitalized aged adults . for example , it has been estimated that overall prevalence of parkinsonism is more than 15 percent for people 65 years and older and its prevalence increases markedly with age . the prevalence of mild parkinsonian signs is even higher and has been reported to exceed 30 percent among community - dwelling older people [ 50 , 51 ] . although it has been suggested that hypotonicity of the neck muscle resulting from diseases of basal ganglia , such as parkinsonism , can be distinguished from true nuchal rigidity , in practice this differentiation is difficult . resistance to passive movement of the neck is a common physical finding in elderly patients because of the presence of cervical spondylosis . it has been recommended in the reference books that elderly patients who have nuchal rigidity in the absence of other neurologic problems should not be dismissed as having osteoarthritis of the cervical spine but should be intensively investigated for possible meningitis . although it is practically difficult to distinguish between the cervical spondylosis and nuchal rigidity resulting from meningitis , some clinical clues have been proposed . for example , it has been noted that , in nuchal rigidity , the neck resists flexion but in spinal disease , lateral rotation , extension , and flexion of the neck are all associated with resistance . it has also been suggested that a somewhat helpful clinical sign is that with cervical osteoarthritis , in particular , passive flexion of the neck may elicit resistance more at the extremes of range of motion , whereas with meningeal irritation , resistance may be felt more immediately . not only test of passive flexion of the neck is unreliable in elderly patients , but also kernig and brudzinski signs are probably of little or no diagnostic value . although it has been reported that more than 80 percent of elderly patients with bm have nuchal rigidity [ 54 , 55 ] , several other studies have noted that the diagnosis of bm is more difficult in the elderly because of the absence of characteristic meningeal signs . in a series of cns infections in aged patients , found that elderly patients were less likely to have neck stiffness than younger adults but more likely to have impairment of consciousness . showed that the most common symptoms of bm in elderly patients were fever 79 percent , change in mental status 69 percent , and meningismus 54 percent . according to the study of domingo et al . , elderly patients had comorbid conditions more frequently and more frequently lacked fever and neck stiffness but had an altered level of consciousness more often . in a review that compared the clinical presentation of bm in elderly patients with that in younger patients , the incidence of more severe abnormalities of mental status in the two groups was significantly different . in this study that included 54 cases of bm in the elderly , confusion was presented in 92 percent of the patients with pneumococcal meningitis and in 78 percent of those with gram - negative meningitis on initial presentation . data from studies addressing the nontraumatic , spontaneous gram - negative bacillary meningitis in the elderly or debilitated patients showed that the classic signs and symptoms of meningitis may be subtle at initial presentation . these are assumed as a distinct group of elderly patients who may have only low - grade fever and altered mental status without headache or nuchal rigidity ; however , patients with spontaneous gram - negative bacillary meningitis tend to have a rapidly progressive fulminant course associated with bacteremia , shock , and coma after presenting what at first appeared to be a minor illness . the contemporary recommendation is that meningitis should be suspected in every elderly patient who is febrile and either disoriented , stuporous , or comatose . but what is the true prevalence of cns infection among these patients ? is encephalopathy a specific sign for cns infection in elderly patients ? when febrile responses and systemic inflammatory syndromes are often blunted in older adults , is it necessary to manage any elderly patient with acute alteration in mental status as the patient with cns infection ? systematic review . on the basis of literature review , we could not find any prospective study about the frequency of meningeal signs in elderly patients who present with afe and their relations to the etiologic diagnosis . the frequency of various signs and symptoms in afe elderly patients varies among different studies on the basis of the design of the studies , as well as the variables studied . according to the few studies in elderly patients ( table 2 ) , meningeal signs are not universal finding in older adults , varying in occurrence from 54 to 82 percent in different studies . it seems that meningeal signs are insensitive and nonspecific for cns infection among elderly patients . most elderly patients with meningitis present only with fever and altered mental status . even when clinical findings suggestive of neck stiffness or other meningeal signs existed in an older patient , the high frequency of underlying diseases such as cervical spondylosis and parkinson 's disease makes these findings difficult to interpret . comparing with adult patients younger than 65 years of age , the older patients present more often ( up to one third ) with neurologic deficits and show greater neurologic severity with a high number presenting with coma on admission , seizures , and hemiparesis [ 44 , 55 ] . early recognition and treatment of acute community - acquired bacterial meningitis ( bm ) although textbooks of infectious diseases recommend appropriate antibiotic administration within 30 minutes of presentation , recent studies consistently report substantial delays in the time to antibiotic administration , ranging from 2 to 4.9 hours . there is an independent incremental association between delays in administrating antibiotics and mortality from adult acute bacterial meningitis ( abm ) . inappropriate diagnostic - treatment sequences are significant predictors of such treatment delays . because of the excessive morbidity and mortality associated with delays in treatment of bm and insufficiency of clinical assessment to exclude central nervous system ( cns ) infection , it is common practice to perform an immediate lp upon any patient with suggestive symptoms , no matter how unlikely the diagnosis is thought to be . statements such as if you think of doing a spinal tap , do one are seen in standard medical textbooks . as mentioned before , nonclassical presentations of acute illnesses occur frequently in the frail elderly and acute infections including cns infections are no exception . none of us is accurate enough with a physical exam to reliably determine if a febrile elderly patient with confusion has an underlying cns infection or not . more than 50 percent of all deaths from meningitis occur in persons aged 60 and older . first , elderly patients with meningitis more often develop complications than younger adults , which resulted in a higher mortality rate [ 10 , 59 ] . according to weisfelt et al . older people with meningitis tended to die more often from cardiorespiratory failure , whereas younger adults more often died from brain herniation . although they attributed the poorer outcome of the elderly to higher rate of pneumococcal meningitis in this age group , multivariate analysis revealed that older age is an independent risk factor for adverse outcome even after adjustment for the causative pathogen . the second major reason for high mortality rate of meningitis in older patients is delay in diagnosis . in fact , because of the nonspecific presentation of cns infection among older adults , it is likely that a significant proportion of these infections in the elderly goes unrecognized and untreated especially in the presence of stroke or dementia . on the other hand , there are also some findings which appear to suggest that the examination of csf in the emergency department ( ed ) may not always be necessary and that some cerebrospinal fluid ( csf ) tests may be excessive . the standard care is that once there is suspicion for abm , blood cultures must be obtained and a lp is performed immediately to determine whether the csf findings are consistent with the clinical diagnosis . in the circumstances in which the clinician can not emergently perform the diagnostic lp or is concerned that the clinical presentation is consistent with a cns mass lesion or another cause of increased intracranial pressure and wants to obtain a computed tomography ( ct ) scan of the head prior to lp , blood cultures must be obtained and appropriate antimicrobial and adjunctive therapy given to the patient prior to lp . lp is frequently performed in the ed , mostly for suspicion of cns infection , which is eventually confirmed in one - third of cases . in the study of powers charts of 104 adult patients who underwent lp in a university hospital ed examination of the csf revealed pleocytosis in 24 percent of the patients . according to the preponderance of negative or normal results of csf analysis , authors suggested that extensive testing may not be necessary for all patients . on the other hand , in the study conducted by khasawneh et al . the results of lp led to a change in management in 30 percent of critically ill medical patients who admitted to the intensive care unit ( icu ) . many experts recommend that all older adults with acute onset of fever and confusion should be treated with empiric antibiotics for cns infection including bm and herpetic encephalitis and have lp performed after neuroimaging in the first hours after admission . nevertheless , it has also been suggested that because bm is an uncommon disorder in elderly patients , routine csf evaluation may not be necessary in all febrile or septic appearing older patients with delirium as long as other infectious foci are obvious . given the lack of specificity or sensitivity of symptoms and signs in the elderly , the basis for the diagnosis of meningitis is the lp , with analysis of the csf . suggested that csf should be analyzed in atypical cases of stroke , or when pyrexia develops without an apparent source of infection in an elderly patient with stroke . although for other age groups , a major distinction must often be made between bacterial and viral meningitis , more common clinical problem in the geriatric population is distinguishing between bm and infection at another site as the cause of fever and acute alteration of mental function . considering published literature data , only few surveys studied the use and the diagnostic efficiency of lp in afe adults . lp efficiency is assumed to be modest especially among elderly patients when an infection of the cns is suspected . the question to be answered is whether it is reasonable to recommend early lp as part of routine diagnostic workup of every afe elderly patient who admitted to ed after normal neuroimaging results . according to choi in deciding whether lp is indicated , one must consider the pretest likelihood of meningitis , and since this is a complex clinical analysis , it is difficult to provide a rigid guideline . they noted that some patients with fever , acutely depressed mental function , and infection at a nonmeningeal site may be treated for the infection and closely observed without lp ; however , most patients who develop these symptoms should probably undergo lp if it is safe to do so , particularly if their symptoms began before hospitalization . we try to make a conclusion for determining the threshold of performing lp in elderly patients with afe . the young febrile infant may demonstrate few , if any , interpretable clues to the underlying illness . the limitations of the history and physical examination in neonates and young infants with fever traditionally have led to an aggressive laboratory evaluation , even for patients who were previously healthy , are well - appearing , and have no focal infection . to some extent this is the same for significant number of febrile elderly who are admitted to ed especially those with afe . in the past , it was the standard of care that most young febrile infants including all febrile neonates 28 days of age or younger have blood , urine , and csf cultures performed regardless of clinical appearance . subsequently , criteria have been developed that can identify young infants with fever who are at low risk for serious bacterial illness and can be safely managed as outpatients . however , the available guidelines and approaches to fever in young infants still recommend that all febrile neonates 28 days of age or younger have sepsis workup including lp and treated with empiric antibiotics regardless of clinical appearance . the underlying basis for this recommendation is the relatively high prevalence of serious bacterial infection and high frequency of bm in bacteremic neonates . let us make a comparison : if the frequency of cns infection among elderly patients with afe was high , it would be reasonable to perform early lp for every febrile aged patient with acute alteration in mental status after neuroimaging . in contrast , if the frequency was considerably low , this management would cause unacceptable aggressive diagnostic evaluation and cost . systematic review . we found only 4 studies that evaluated the efficiency of lp in old aged adult patients with afe . study number 1 . in the study conducted by d'amore and nelson among 191 elderly patients presenting with fever and altered mental status who underwent lp , 21 percent of patients had a source identified by csf analysis : 10 ( 6.2 percent ) cases of bm , 21 ( 13 percent ) cases of viral meningitis , and 4 ( 2.5 percent ) cases of viral encephalitis . the authors of the study concluded that patients with fever and altered mental status and a pre - lp source of infection may have a higher rate ( 9.5 versus 2.8 percent ) of bm . the results of the study showed that when a pre - lp source is found , nursing home patients appear less likely to receive lp . despite the retrospective design of the study and inclusion of only those patients who underwent lp , they suggested that ed physicians should perform lp on all elderly patients with fever and altered mental status . shah et al . conducted a retrospective study to compare the diagnostic utility of lp in febrile versus afebrile elderly patients with altered mental status . their null hypothesis was that there is no utility of performing an lp on the afebrile delirious elderly patients . eighteen percent of afebrile patients and 24 percent of 41 febrile patients had abnormal csf . comparing the elderly patient group without fever with the elderly patient group with fever , they could not reject their null hypothesis and suggested to not relying solely on the presence or absence of fever to determine management in the elderly . alavi and moogahi studied 60 elderly patients with confusion and fever in a teaching hospital to determine the causes of confusion and fever and identify the necessity of the csf examination . the results of the study showed that older age was not a statistically significant predictor of bm in patients with acute onset of fever and confusion . they also found that among elderly persons , extra - meningeal infection with or without underlying illness is a more important cause of confusion and fever than bm . they concluded that elderly patients with fever and confusion , without signs of meningeal irritation , may not require a routine lp performance for evaluation of their csf . seventy percent of lps were performed as part of the admitting workup , and the remaining 30 percent during the hospitalization . they found only one case of bm and the authors concluded that most hospitalized elderly patients with febrile delirium have primary causes of the confusion outside the cns and may not require a routine csf analysis . in the retrospective study conducted by majed et al . with the aim of evaluating the frequency of use and the diagnostic efficiency of lp , total of 247 patients , representing 0.5 percent of all ed admissions , underwent a lp . the main assumed lp indications were to search for cns infection 62 percent and for subarachnoid hemorrhage ( sah ) 25 percent . lp was efficient in fewer than 15 percent of cases and confirmed aseptic meningitis 8.5 percent , bm 2.4 percent , guillain - barr syndromes 1.6 percent , sah 0.4 percent , and carcinomatous meningitis 0.4 percent . the principal differential diagnoses were infections outside the cns , noninfectious neurological disorders , and benign headaches accordingly , contributive lp represented 13.4 percent ( 95 percent ci : 9.117.6 ) of the cases and varied significantly according to patients ' age : the proportion of efficient lp decreased from 25 percent among young patients to 14.2 percent among middle - aged patients and to less than 5 percent among elderly patients . in this study , total number of elderly patients who underwent lp with the suspicion of cns infection was 87 of whom 55 ( 82 percent ) cases had confusion and 36 ( 53.7 percent ) were febrile . of the total elderly patients 43.7 percent had infection outside the cns and only 3.4 percent had meningitis . in 4 ( 4.6 percent ) elderly patients , lp was contributive . with regard to answering the question of this part , one should estimate the missing number of cns infections in the elderly patients who present to ed including those who die undiagnosed . because of the retrospective design of most studies about cns infection in elderly patients , the estimation is impossible . on the other hand , nearly all studies with regard to this topic are limited by selection bias of only those patients who undergo lp . a number of the studies described in this section seem to point out that many elderly patients with afe do not require lp . however , because of the insufficient number of the studies performed to address the issue , their heterogeneity , and retrospective design , drawing a conclusion about the proper threshold for performing lp in elderly patients with afe is somewhat difficult . although clinical judgment of individual patients by their physicians remains the most important factor in the diagnosis of acute bacterial meningitis ( abm ) , the use of evidence - based guidelines help standardize care among physicians , as well as various institutions . prognostic classification of disease remains a powerful tool for the bedside clinician in diagnostic and management decision making . accurate and valid prognostic models are difficult to develop , because they require detailed clinical data collection from a large cohort of patients with a clinically relevant outcome assessment . for some of the most common infectious diseases , prognostic classification with validated scoring systems is used . however , because of including heterogeneous adult population and their primary outcomes that are limited to the mortality end - points instead of more relevant outcomes for the elderly such as rate and duration of functional decline , their applicability to the care of older adults is limited [ 42 , 66 ] . although the few existing prediction models can be used to estimate the risk of abm , these models need to be refined and validated further in specific settings and populations such as the elderly . refining and validating prediction models for a specific age group could be helpful in developing practical diagnostic and therapeutic recommendations and evidence - based guidelines in the age group . in this regard , we suggest conducting further prospective well - designed studies with adequate sample size that focus on the following aims : to estimate the true prevalence of central nervous system ( cns ) infection among elderly patients who present with acute febrile encephalopathy ( afe),to define different set of classic clinical criteria for meningitis in elderly patients ( instead of considering their presentation atypical most of the time),to determine the proper threshold for performing lumbar puncture ( lp ) in elderly patients who present with afe , to design a practical evidence - based guideline to help physicians to recognize elderly patients who benefit from early lp , those for whom delaying lp is not harmful , and those who do not require lp at all . to estimate the true prevalence of central nervous system ( cns ) infection among elderly patients who present with acute febrile encephalopathy ( afe ) , to define different set of classic clinical criteria for meningitis in elderly patients ( instead of considering their presentation atypical most of the time ) , to determine the proper threshold for performing lumbar puncture ( lp ) in elderly patients who present with afe , to design a practical evidence - based guideline to help physicians to recognize elderly patients who benefit from early lp , those for whom delaying lp is not harmful , and those who do not require lp at all . unfortunately , our attempt is to develop a recommendation about the optimal threshold of lumbar puncture ( lp ) defeated because of the inadequate number and weak evidence from retrospective studies performed to address the issue in the literature . considering the diagnostic dilemmas in elderly patients with acute febrile encephalopathy ( afe ) , it would be more useful to design guidelines around this topic based on a clinical syndrome , that is , afe not based on disease category , that is , bacterial meningitis ( bm ) . in addition , prospective studies with the aim of revising or validating clinical predictors of central nervous system ( cns ) infection in elderly patients are welcomed . accordingly , it is mandatory to perform prospective well - designed studies with the aim of evaluating the etiologic diagnosis of afe in elderly patients and the proper sequence of diagnostic and therapeutic management .

the elderly comprise less than 13 percent of world population . nonetheless , they represent nearly half of all hospitalized adults . acute change in mental status from baseline is commonly seen among the elderly even when the main process does not involve the central nervous system . the term geriatric syndrome is used to capture those clinical conditions in older people that do not fit into discrete disease categories , including delirium , falls , frailty , dizziness , syncope , and urinary incontinence . despite the growing number of elderly population , especially those who require hospitalization and the high burden of common infections accompanied by encephalopathy among them , there are several unresolved questions regarding the optimal management they deserve . the questions posed in this systematic review concern the need to rule out cns infection in all elderly patients presented with fever and altered mental status in the routine management of febrile encephalopathy . in doing so , we sought to identify all potentially relevant articles using searches of web - based databases with no language restriction . finally , we reviewed 93 research articles that were relevant to each part of our study . no prospective study was found to address how should afe in the aged be optimally managed .

1. Background 2. Methods 3. The Necessity of Developing an Evidence-Based Guideline 4. Conclusion

the result of this trend is the rising proportions of older persons in the total population that increased from 9.2 percent in 1990 to 11.7 percent in 2013 and will continue to grow as a proportion of the world population , reaching 21.1 percent by 2050 . although patients 65 years and older comprise less than 13 percent of the population , they represent 40 percent of hospitalized adults and nearly half of all healthcare dollars spent on hospitalization . despite the disproportionate prevalence of hospitalized patients who are in the older age range , hospitalist programs often do not emphasize the need for geriatric skills and most hospital - based clinicians are not trained to treat older adult patients . the term geriatric syndrome is used to capture those clinical conditions in older persons that do not fit into discrete disease categories , including delirium , falls , frailty , dizziness , syncope , and urinary incontinence . although the concept of the geriatric syndrome remains poorly defined , four underlying risk factors have been identified for this syndrome , including older age , cognitive impairment , functional impairment , and impaired mobility . for example , changes in mental status from baseline are commonly seen even when the main process does not involve the central nervous system ( cns ) ; this is the consequence of altered neural function in the form of cognitive and behavioral changes which permits the diagnosis of delirium . a change in mental status or decline in function may be the only presenting problem in an older patient with an infection . in this situation one should distinguish between infections , especially those involving cns , versus noninfectious causes of altered mental status . in fact , one of the common problems encountered by the physicians in ed is identification of cause and treatment of patients who present with acute onset of fever and altered mental status , not only to ensure survival but also to prevent long - term neurological sequelae . this is the fact especially in the management of older adult patients with acute febrile encephalopathy ( afe ) and several chronic medical conditions . afe is used to describe patients with a condition in which altered mental status either accompanies or follows a short febrile illness . a case of aes is defined as a person of any age , at any time of year , with the acute onset of fever and a change in mental status and/or new onset of seizure ( excluding simple febrile seizures ) . considering the fact that confusion ( altered mental state ) is the hallmark of encephalopathy , it would be present in up to 50 percent of elderly hospitalized patients , 10 percent of all hospitalized patients , and 2 percent of ed patients . according to han et al it has been noted that cns infections are the most common causes of altered mental status in patients with nontraumatic coma . here , a question comes to mind : is it true for elderly patients who present with altered mental status to the ed ? many physicians face great number of febrile encephalopathic older adults in the ed most of whom do not have cns infections . in fact , although one of the most important differential diagnoses in a confused elderly , who present to ed , especially when altered mental status is accompanied with fever or sepsis syndrome , is cns infection , this type of infection does not seem to be the most common underlying cause . in this regard , is it reasonable to perform lumbar puncture ( lp ) on all febrile older patients who admitted with acute alteration in mental status after normal neuroimaging results ? despite the growing number of elderly population , especially those who require hospitalization as well as the high burden of altered mental status among older ed patients , there are several unresolved questions about the optimal management of elderly patients with afe that require hospitalization . therefore , we decided to answer these questions by searching in the literature in the form of a systematic review . the questions posed in this review concern the need of early lp in all afe elderly patients in the routine management of afe . the questions are as follows : what is the etiologic distribution of afe in elderly patients?is higher prevalence of cns infection among the elderly responsible for higher frequency of afe in this age group ? is the combination of nuchal rigidity and encephalopathy a specific indicator of cns infection in elderly patients with afe?is a lp necessary when evaluating an older patient with afe ? too many or too few?finally , we discuss the necessity for developing an evidence - based guideline for optimal management of afe in the elderly . is higher prevalence of cns infection among the elderly responsible for higher frequency of afe in this age group ? is the combination of nuchal rigidity and encephalopathy a specific indicator of cns infection in elderly patients with afe ? we sought to identify all potentially relevant articles using searches of web - based databases ( google scholar , medline , pubmed , scopus , and researchgate ) with no language restriction . aged adult , older adult , confusion , delirium , febrile , fever , encephalopathy , altered mental status , acute encephalitis syndrome , febrile encephalopathy , lumbar puncture , and lp . by reviewing the titles of articles the reasons for exclusion were as follows : 784 because they were related to children , 341 because they were case reports or letter to editors , and 273 because they exclusively dealt with a specific pathogen(s ) of cns infection in elderly . out of the finally selected articles , 4 were relevant to lumbar puncture and frequency of cns infection in older patients with encephalopathy , 6 addressed meningeal signs in elderly , and 4 were relevant to etiologic diagnosis of afe in adults ( figure 1 ) . patients with dementia who develop a systemic illness can present with an acute change in mental status . when confusion is accompanied by fever or sepsis syndrome , the possibility of infection especially central nervous system ( cns ) infection as the etiologic cause of alteration in mental status comes to mind . in this part , we try to determine which underlying etiologies are more frequently presented as afe in elderly patients admitted to the ed there are numerous studies performed to address the etiology of afe in children ; however , we found only 4 studies in the literature that evaluated the etiologic diagnosis of afe in adult patients [ 14 , 22 , 27 , 28 ] . in forth study that included patients with acute encephalitis syndrome ( fever , headache , altered mental status , vomiting , seizure , and neurodeficit ) , 25 ( 13.1 percent ) out of 190 patients were elderly cases . the higher prevalence of meningitis among the elderly compared with the general adult population has been also documented in developing countries . although older age is a known risk factor for cns infection , estimation of the true prevalence of cns infection in febrile aged patients with acute alteration of mental status has received little attention . given the aim of the study that was the evaluation of the etiologic distribution of acute fever in the elderly , small number of aged adults with fever and confusion were included . in this part , we try to estimate the frequency of cns infection among afe elderly patients . a thorough search in the literature revealed only that few studies provided data about the frequency of cns infection in elderly patients with afe to be included ( table 1 ) . however , the small sample size , retrospective design , and selection bias of these studies that only included patients who underwent lumbar puncture ( lp ) make an estimation of true prevalence of cns infection in aged adults with afe impossible . among the myriad of conditions that can induce delirium in critical illness , sepsis in the form of sae represents the most frequent and severe cause . up to 70 percent of patients with bacteremia have wide spectrum of neurological symptoms that include fluctuating mental status changes , inattention , and disorganized thinking and therefore match with current criteria for delirium [ 36 , 37 ] . on the other hand , it has been noted that sae is a diagnosis of exclusion : there should be no clinical or laboratory evidence of direct central nervous system ( cns ) infection ( e.g. it has been suggested that occurrence of sudden fluctuation in mental status , occurrence of focal neurological sign , seizure(s ) , and/or neck stiffness should prompt the physician to consider neuroimaging , electroencephalogram ( eeg ) , and/or lumbar puncture ( lp ) to rule out a direct cns infection . considering the high frequency of altered mental status in elderly patients with infectious syndromes outside the cns , is it reasonable to perform early lp on every confused aged adult with fever or sepsis syndrome ? on the basis of literature review , we could not find any relevant prospective study to the frequency of sae in elderly patients with afe . common clinical practice relies on the absence of neck stiffness or other meningeal signs to rule out meningitis in the previously healthy adult . meningeal signs have been assumed to be reliable and usually present in awaked adults with meningitis , except infants , the elderly , and the immunesuppressed . in adults presenting with community - acquired bacterial meningitis ( bm ) , the sensitivity of the classic triad of fever , neck stiffness , and altered mental status is low ( 44 percent ) , but almost all ( 95 percent ) present with at least two of the four symptoms of headache , fever , neck stiffness , and altered mental status . according to choi in the clinical assessment for possible bm , older adults who present without fever , neck stiffness , or altered mental function those individuals with 2 or 3 of the 3 classic findings are more likely to have meningitis , but even the presence of all 3 findings is not entirely specific . although the majority of elderly patients present with fever , confusion , and stiff neck , confusion may be assumed to be secondary to senility , and the stiff neck to cervical osteoarthritis . nuchal rigidity is often found on examination of elderly patients who do not have bm and usually have coexistent neurologic deficits . nuchal rigidity , which may be a sign of meningitis , was found in 35 percent of geriatric patients on acute - care and rehabilitation wards and was significantly associated with cerebrovascular disease ( cva ) , confusion , abnormal plantar responses , and primitive reflexes . accordingly , they suggested that elderly patients who have nuchal rigidity with no history of neurologic or cognitive disorders should be investigated for meningitis . considering the high prevalence of these underlying conditions among the elderly , interpretation of clinical tests evoking meningeal irritation may be inconclusive in significant number of hospitalized aged adults . although it has been reported that more than 80 percent of elderly patients with bm have nuchal rigidity [ 54 , 55 ] , several other studies have noted that the diagnosis of bm is more difficult in the elderly because of the absence of characteristic meningeal signs . showed that the most common symptoms of bm in elderly patients were fever 79 percent , change in mental status 69 percent , and meningismus 54 percent . , elderly patients had comorbid conditions more frequently and more frequently lacked fever and neck stiffness but had an altered level of consciousness more often . in a review that compared the clinical presentation of bm in elderly patients with that in younger patients , the incidence of more severe abnormalities of mental status in the two groups was significantly different . in this study that included 54 cases of bm in the elderly , confusion was presented in 92 percent of the patients with pneumococcal meningitis and in 78 percent of those with gram - negative meningitis on initial presentation . these are assumed as a distinct group of elderly patients who may have only low - grade fever and altered mental status without headache or nuchal rigidity ; however , patients with spontaneous gram - negative bacillary meningitis tend to have a rapidly progressive fulminant course associated with bacteremia , shock , and coma after presenting what at first appeared to be a minor illness . is encephalopathy a specific sign for cns infection in elderly patients ? when febrile responses and systemic inflammatory syndromes are often blunted in older adults , is it necessary to manage any elderly patient with acute alteration in mental status as the patient with cns infection ? on the basis of literature review , we could not find any prospective study about the frequency of meningeal signs in elderly patients who present with afe and their relations to the etiologic diagnosis . according to the few studies in elderly patients ( table 2 ) , meningeal signs are not universal finding in older adults , varying in occurrence from 54 to 82 percent in different studies . most elderly patients with meningitis present only with fever and altered mental status . although they attributed the poorer outcome of the elderly to higher rate of pneumococcal meningitis in this age group , multivariate analysis revealed that older age is an independent risk factor for adverse outcome even after adjustment for the causative pathogen . in fact , because of the nonspecific presentation of cns infection among older adults , it is likely that a significant proportion of these infections in the elderly goes unrecognized and untreated especially in the presence of stroke or dementia . on the other hand , there are also some findings which appear to suggest that the examination of csf in the emergency department ( ed ) may not always be necessary and that some cerebrospinal fluid ( csf ) tests may be excessive . many experts recommend that all older adults with acute onset of fever and confusion should be treated with empiric antibiotics for cns infection including bm and herpetic encephalitis and have lp performed after neuroimaging in the first hours after admission . nevertheless , it has also been suggested that because bm is an uncommon disorder in elderly patients , routine csf evaluation may not be necessary in all febrile or septic appearing older patients with delirium as long as other infectious foci are obvious . they noted that some patients with fever , acutely depressed mental function , and infection at a nonmeningeal site may be treated for the infection and closely observed without lp ; however , most patients who develop these symptoms should probably undergo lp if it is safe to do so , particularly if their symptoms began before hospitalization . to some extent this is the same for significant number of febrile elderly who are admitted to ed especially those with afe . let us make a comparison : if the frequency of cns infection among elderly patients with afe was high , it would be reasonable to perform early lp for every febrile aged patient with acute alteration in mental status after neuroimaging . in the study conducted by d'amore and nelson among 191 elderly patients presenting with fever and altered mental status who underwent lp , 21 percent of patients had a source identified by csf analysis : 10 ( 6.2 percent ) cases of bm , 21 ( 13 percent ) cases of viral meningitis , and 4 ( 2.5 percent ) cases of viral encephalitis . the authors of the study concluded that patients with fever and altered mental status and a pre - lp source of infection may have a higher rate ( 9.5 versus 2.8 percent ) of bm . despite the retrospective design of the study and inclusion of only those patients who underwent lp , they suggested that ed physicians should perform lp on all elderly patients with fever and altered mental status . conducted a retrospective study to compare the diagnostic utility of lp in febrile versus afebrile elderly patients with altered mental status . comparing the elderly patient group without fever with the elderly patient group with fever , they could not reject their null hypothesis and suggested to not relying solely on the presence or absence of fever to determine management in the elderly . they concluded that elderly patients with fever and confusion , without signs of meningeal irritation , may not require a routine lp performance for evaluation of their csf . seventy percent of lps were performed as part of the admitting workup , and the remaining 30 percent during the hospitalization . with the aim of evaluating the frequency of use and the diagnostic efficiency of lp , total of 247 patients , representing 0.5 percent of all ed admissions , underwent a lp . the principal differential diagnoses were infections outside the cns , noninfectious neurological disorders , and benign headaches accordingly , contributive lp represented 13.4 percent ( 95 percent ci : 9.117.6 ) of the cases and varied significantly according to patients ' age : the proportion of efficient lp decreased from 25 percent among young patients to 14.2 percent among middle - aged patients and to less than 5 percent among elderly patients . in this study , total number of elderly patients who underwent lp with the suspicion of cns infection was 87 of whom 55 ( 82 percent ) cases had confusion and 36 ( 53.7 percent ) were febrile . with regard to answering the question of this part , one should estimate the missing number of cns infections in the elderly patients who present to ed including those who die undiagnosed . because of the retrospective design of most studies about cns infection in elderly patients , the estimation is impossible . a number of the studies described in this section seem to point out that many elderly patients with afe do not require lp . however , because of the insufficient number of the studies performed to address the issue , their heterogeneity , and retrospective design , drawing a conclusion about the proper threshold for performing lp in elderly patients with afe is somewhat difficult . although clinical judgment of individual patients by their physicians remains the most important factor in the diagnosis of acute bacterial meningitis ( abm ) , the use of evidence - based guidelines help standardize care among physicians , as well as various institutions . although the few existing prediction models can be used to estimate the risk of abm , these models need to be refined and validated further in specific settings and populations such as the elderly . in this regard , we suggest conducting further prospective well - designed studies with adequate sample size that focus on the following aims : to estimate the true prevalence of central nervous system ( cns ) infection among elderly patients who present with acute febrile encephalopathy ( afe),to define different set of classic clinical criteria for meningitis in elderly patients ( instead of considering their presentation atypical most of the time),to determine the proper threshold for performing lumbar puncture ( lp ) in elderly patients who present with afe , to design a practical evidence - based guideline to help physicians to recognize elderly patients who benefit from early lp , those for whom delaying lp is not harmful , and those who do not require lp at all . to estimate the true prevalence of central nervous system ( cns ) infection among elderly patients who present with acute febrile encephalopathy ( afe ) , to define different set of classic clinical criteria for meningitis in elderly patients ( instead of considering their presentation atypical most of the time ) , to determine the proper threshold for performing lumbar puncture ( lp ) in elderly patients who present with afe , to design a practical evidence - based guideline to help physicians to recognize elderly patients who benefit from early lp , those for whom delaying lp is not harmful , and those who do not require lp at all . unfortunately , our attempt is to develop a recommendation about the optimal threshold of lumbar puncture ( lp ) defeated because of the inadequate number and weak evidence from retrospective studies performed to address the issue in the literature . in addition , prospective studies with the aim of revising or validating clinical predictors of central nervous system ( cns ) infection in elderly patients are welcomed . accordingly , it is mandatory to perform prospective well - designed studies with the aim of evaluating the etiologic diagnosis of afe in elderly patients and the proper sequence of diagnostic and therapeutic management .

the study was performed within the frame of the verona diabetes study , an observational longitudinal study on chronic complications in type 2 diabetic outpatients attending the diabetes clinic at the university hospital of verona ( 13 ) . briefly , for this analysis we retrospectively analyzed the electronic records of all caucasian type 2 diabetic outpatients , who regularly attended our diabetes clinic during the years 19922007 . of these , we selected all patients with type 2 diabetes who had undergone a first transthoracic echocardiography for clinical reasons ( e.g. , dyspnea , palpitations , chest pain , heart murmur , history of myocardial infarction , electrocardiographic abnormalities , assessment of left ventricular wall thickness , presence of multiple cvd risk factors ) at our institution during the same period of time . on the basis of these criteria , 937 patients with type 2 the informed consent requirement for this study was exempted by the ethics committee because researchers only accessed retrospectively a deidentified database for analysis purposes . bmi was measured as kilograms divided by the square of height in meters . a physician measured blood pressure in duplicate with a mercury sphygmomanometer ( at the right upper arm using an appropriate cuff size ) after the patient had been seated quietly for at least 5 min . subjects were considered to have arterial hypertension if their blood pressure was 140/90 mmhg or if they were taking any antihypertensive drugs . information on medical history and smoking status was obtained from all patients via interviews during medical examinations . serum lipids , creatinine , and other biochemical blood measurements were determined in the same laboratory using standard laboratory procedures ( dax 96 ; bayer diagnostics , milan , italy ) . a1c was measured by an automated high - performance liquid chromatography analyzer ( bio - rad diamat ; bio - rad , milan , italy ) ; the upper limit of normal for our laboratory was 5.6% . glomerular filtration rate was estimated from the four - variable modification of diet in renal disease study equation ( 14 ) . urinary albumin excretion rate was measured from a 24-h urine sample using an immunonephelometric method . the presence of abnormal albuminuria ( defined as albumin excretion rate 30 mg / day ) was confirmed in at least two of three consecutive urine samples ( 14 ) . left ventricular ( lv ) chamber dimensions and wall thickness were measured from m - mode recordings as suggested by the american society of echocardiography , and lv mass was calculated using a necropsy validated equation ( 15 ) . lv ejection fraction ( lv - ef ) was measured from lv diameters or two - dimensional area changes in systole and diastole ( 16 ) . regional lv function was evaluated by means of wall motion score index ( wmsi ) . each segment was analyzed individually and scored on the basis of its motion and systolic thickening . lv segments were scored as follows : normal , 1 ; hypokinetic , 2 ; akinetic , 3 ; and dyskinetic or aneurysmatic , 4 . wmsi was derived as the sum of all scores divided by the number of lv segments visualized ( 15 ) . mac was defined by increased echodensity located at the junction of the atrioventricular groove and posterior mitral leaflet on the parasternal long - axis , short - axis , or apical four - chamber view . avs was defined as focal or diffuse calcification and thickening of the aortic leaflets with or without restriction of leaflet motion on echocardiography ( 17 ) . a transaortic peak instantaneous velocity 2.5 m / s was considered as aortic stenosis . vital status on 30 september 2007 was ascertained for all participants through examination of the electronic databases of the social health unit of the veneto region , which include all records of deaths that occurred within the veneto region as well as the specific causes of death . skewed variables were logarithmically transformed to improve normality prior to analysis ( duration of diabetes , triglycerides , and albuminuria ) . in accord with a previous study ( 5 ) , we calculated a heart valve calcium ( hvc ) score combining the presence of calcium at the level of aortic and mitral valve as follows : hvc-0 , absence of any thickening or calcification ; hvc-1 , presence of either isolated avs or isolated mac ; and hvc-2 , coexistence of avs and mac . comparisons between groups were made using one - way anova ( for continuous variables ) and the test ( for categorical variables ) . univariate survival analysis was performed by the kaplan - meier analysis , and the overall significance was calculated by the log - rank test . cox regression analysis was used to study the association between the hvc score and the risk of all - cause and cvd mortality after adjustment for potential confounders . the model assumptions for the cox proportional hazards regression models were checked by visual inspection of proportional hazard assumption , schoenfeld residuals , and covariance matrix . four forced - entry cox regression models were performed : an unadjusted model ; a model adjusted for age and sex ( model 1 ) ; a model adjusted for age , sex , bmi , duration of diabetes , smoking status , systolic blood pressure , a1c , ldl cholesterol , estimated glomerular filtration rate ( egfr ) , history of myocardial infarction , and use of any antihypertensive and lipid - lowering medications ( model 2 , where the number of subjects with available data for analysis was 792 ) ; and a model adjusted for age , sex , lv mass , lv - ef < 50% , wmsi , and la diameter ( model 3 , where all subjects had available data for analysis ) . the covariates included in these multivariate regression models were chosen as potential confounding factors on the basis of their significance in univariate analysis or on the basis of their biological plausibility . all analyses were performed using statistical package spss 19.0 , and statistical significance was assessed at the two - tailed 0.05 threshold . bmi was measured as kilograms divided by the square of height in meters . a physician measured blood pressure in duplicate with a mercury sphygmomanometer ( at the right upper arm using an appropriate cuff size ) after the patient had been seated quietly for at least 5 min . subjects were considered to have arterial hypertension if their blood pressure was 140/90 mmhg or if they were taking any antihypertensive drugs . information on medical history and smoking status was obtained from all patients via interviews during medical examinations . serum lipids , creatinine , and other biochemical blood measurements were determined in the same laboratory using standard laboratory procedures ( dax 96 ; bayer diagnostics , milan , italy ) . a1c was measured by an automated high - performance liquid chromatography analyzer ( bio - rad diamat ; bio - rad , milan , italy ) ; the upper limit of normal for our laboratory was 5.6% . glomerular filtration rate was estimated from the four - variable modification of diet in renal disease study equation ( 14 ) . urinary albumin excretion rate was measured from a 24-h urine sample using an immunonephelometric method . the presence of abnormal albuminuria ( defined as albumin excretion rate 30 mg / day ) was confirmed in at least two of three consecutive urine samples ( 14 ) . all echocardiographic examinations were performed at our institution by experienced cardiologists ( a.r . and s.b . ) . left ventricular ( lv ) chamber dimensions and wall thickness were measured from m - mode recordings as suggested by the american society of echocardiography , and lv mass was calculated using a necropsy validated equation ( 15 ) . lv ejection fraction ( lv - ef ) was measured from lv diameters or two - dimensional area changes in systole and diastole ( 16 ) . regional lv function was evaluated by means of wall motion score index ( wmsi ) . each segment was analyzed individually and scored on the basis of its motion and systolic thickening . lv segments were scored as follows : normal , 1 ; hypokinetic , 2 ; akinetic , 3 ; and dyskinetic or aneurysmatic , 4 . wmsi was derived as the sum of all scores divided by the number of lv segments visualized ( 15 ) . mac was defined by increased echodensity located at the junction of the atrioventricular groove and posterior mitral leaflet on the parasternal long - axis , short - axis , or apical four - chamber view . avs was defined as focal or diffuse calcification and thickening of the aortic leaflets with or without restriction of leaflet motion on echocardiography ( 17 ) . a transaortic peak instantaneous velocity 2.5 m / s was considered as aortic stenosis . vital status on 30 september 2007 was ascertained for all participants through examination of the electronic databases of the social health unit of the veneto region , which include all records of deaths that occurred within the veneto region as well as the specific causes of death . skewed variables were logarithmically transformed to improve normality prior to analysis ( duration of diabetes , triglycerides , and albuminuria ) . in accord with a previous study ( 5 ) , we calculated a heart valve calcium ( hvc ) score combining the presence of calcium at the level of aortic and mitral valve as follows : hvc-0 , absence of any thickening or calcification ; hvc-1 , presence of either isolated avs or isolated mac ; and hvc-2 , coexistence of avs and mac . comparisons between groups were made using one - way anova ( for continuous variables ) and the test ( for categorical variables ) . univariate survival analysis was performed by the kaplan - meier analysis , and the overall significance was calculated by the log - rank test . cox regression analysis was used to study the association between the hvc score and the risk of all - cause and cvd mortality after adjustment for potential confounders . the model assumptions for the cox proportional hazards regression models were checked by visual inspection of proportional hazard assumption , schoenfeld residuals , and covariance matrix . four forced - entry cox regression models were performed : an unadjusted model ; a model adjusted for age and sex ( model 1 ) ; a model adjusted for age , sex , bmi , duration of diabetes , smoking status , systolic blood pressure , a1c , ldl cholesterol , estimated glomerular filtration rate ( egfr ) , history of myocardial infarction , and use of any antihypertensive and lipid - lowering medications ( model 2 , where the number of subjects with available data for analysis was 792 ) ; and a model adjusted for age , sex , lv mass , lv - ef < 50% , wmsi , and la diameter ( model 3 , where all subjects had available data for analysis ) . the covariates included in these multivariate regression models were chosen as potential confounding factors on the basis of their significance in univariate analysis or on the basis of their biological plausibility . all analyses were performed using statistical package spss 19.0 , and statistical significance was assessed at the two - tailed 0.05 threshold . of the initial sample of 937 type 2 diabetic patients , 35 who had either aortic valve prosthesis or rheumatic or congenital aortic valve disease were excluded from analysis . thus , 902 ( 362 women ) patients with type 2 diabetes represent the final analytical sample . overall , 477 ( 52.9% ) patients were free of any thickening or calcification at the valvular level ( i.e. , hvc-0 ) , and 304 ( 33.7% ) patients had either isolated mac or isolated avs ( hvc-1 ) ; i.e. , 43 ( 4.8% ) patients had isolated mac and 261 ( 28.9% ) patients had avs , and 17 of them had aortic stenosis . the remaining 121 ( 13.4% ) patients had combined avs and mac ( hvc-2 ) , and 39 of them had aortic stenosis . as shown in table 1 , age , systolic blood pressure , ldl cholesterol , albuminuria , and the prevalence of hypertension significantly increased , whereas bmi , triglycerides , and egfr decreased across the hvc score . sex , smoking history , duration of diabetes , frequency of diabetic retinopathy , fasting plasma glucose , a1c , and hdl cholesterol did not significantly change across the hvc score . among the echocardiographic parameters , only la diameter increased across the hvc score , whereas lv mass , lv - ef , and wmsi were not significantly different among the groups . baseline clinical , biochemical , and echocardiographic characteristics of type 2 diabetic patients , stratified by hvc score during a mean follow - up period of 9 years ( median 7.6 years ) , 137 ( 15.2% of total ) patients died ( 78 of these from cvd causes ) . as shown in fig . 1a , the hvc score was strongly associated with an increased risk of death from all causes and from cvd causes . during the follow - up period , patients with hvc-2 had an 20% overall survival rate compared with 80% of those with hvc-0 and with an intermediate survival rate ( 40% ) in those with hvc-1 . the differences among these curves were statistically significant ( p < 0.0001 by the log - rank test ) . kaplan - meier survival analysis for all - cause ( a ) and cardiovascular ( b ) mortality in 902 type 2 diabetic patients , stratified by the hvc score . in cox univariate survival analysis ( see unadjusted models in tables 2 and 3 ) , both hvc-1 and hvc-2 were associated with an increased risk of all - cause and cvd mortality . as also shown in tables 2 and 3 , the results remained essentially unchanged after adjustment for age , sex , bmi , smoking status , systolic blood pressure , duration of diabetes , a1c , ldl cholesterol , egfr , history of myocardial infarction , and current use of any antihypertensive and lipid - lowering medications ( regression models 1 and 2 ) or after adjustment for age , sex , and other baseline echocardiographic parameters , such as lv mass , lv - ef < 50% , wmsi , and la diameter ( model 3 ) . association between hvc score and the risk of all - cause mortality in patients with type 2 diabetes association between hvc score and the risk of cardiovascular mortality in patients with type 2 diabetes notably , other independent predictors of cvd and all - cause mortality ( p < 0.010.001 ) were older age , longer duration of diabetes , smoking status , lower egfr , and greater lv mass ( data not shown ) . the significant , graded association between the hvc score and the risk of both all - cause and cvd mortality was consistent in men versus women ( as shown in supplementary tables 14 ) and in younger versus older individuals ( data not shown ) . almost identical results were also found when separate analyses were performed in those with ( n = 210 ) and those without ( n = 692 ) a previous history of myocardial infarction . in both of these subgroups , increasing hvc score predicted all - cause and cvd mortality , independently of potential confounders . in particular , among those with a history of myocardial infarction , the hvc score was associated with both all - cause mortality ( hr 4.9 [ 95% ci 2.410.4 ] , p < 0.001 for hvc-1 , and 7.5 [ 2.720.6 ] , p < 0.001 for hvc-2 ) and cvd mortality ( 4.2 [ 1.710.9 ] , p < 0.005 , and 9.0 [ 2.631.1 ] , p < 0.001 ) even after adjustment for age , sex , lv mass , lv - ef < 50% , wmsi , and la diameter . the strong association between the hvc score and mortality was further reinforced by the finding of a positive , graded relationship between the extent of mitral and aortic annular calcification and the clinical outcomes . indeed , the hr for all - cause mortality was more than doubled in those with both valves affected ( unadjusted hr 7.2 [ 95% ci 4.511.4 ] for those with hvc-2 ) compared with those with either isolated avs ( 3.2 [ 2.24.8 ] ) or isolated mac ( 2.4 [ 1.34.1 ] ) , respectively . in addition , a stronger association with the risk of mortality was also observed with the amount of calcium at the level of aortic valve ; the hr for all - cause mortality increased from 3.2 ( 95% ci 2.24.8 ) in those with isolated avs to 4.4 ( 2.27.7 ) in those with aortic stenosis at baseline . notably , after exclusion of patients who had aortic stenosis at baseline ( n = 56 ) or developed aortic stenosis during the follow - up ( n = 13 ) , the value of hvc score in predicting all - cause mortality remained essentially unchanged ( adjusted hr 2.3 [ 95% ci 1.53.5 ] for hvc-1 and hr 5.1 [ 3.18.5 ] for hvc-2 , respectively ) . of the initial sample of 937 type 2 diabetic patients , 35 who had either aortic valve prosthesis or rheumatic or congenital aortic valve disease were excluded from analysis . thus , 902 ( 362 women ) patients with type 2 diabetes represent the final analytical sample . overall , 477 ( 52.9% ) patients were free of any thickening or calcification at the valvular level ( i.e. , hvc-0 ) , and 304 ( 33.7% ) patients had either isolated mac or isolated avs ( hvc-1 ) ; i.e. , 43 ( 4.8% ) patients had isolated mac and 261 ( 28.9% ) patients had avs , and 17 of them had aortic stenosis . the remaining 121 ( 13.4% ) patients had combined avs and mac ( hvc-2 ) , and 39 of them had aortic stenosis . as shown in table 1 , age , systolic blood pressure , ldl cholesterol , albuminuria , and the prevalence of hypertension significantly increased , whereas bmi , triglycerides , and egfr decreased across the hvc score . sex , smoking history , duration of diabetes , frequency of diabetic retinopathy , fasting plasma glucose , a1c , and hdl cholesterol did not significantly change across the hvc score . among the echocardiographic parameters , only la diameter increased across the hvc score , whereas lv mass , lv - ef , and wmsi were not significantly different among the groups . baseline clinical , biochemical , and echocardiographic characteristics of type 2 diabetic patients , stratified by hvc score during a mean follow - up period of 9 years ( median 7.6 years ) , 137 ( 15.2% of total ) patients died ( 78 of these from cvd causes ) . 1a , the hvc score was strongly associated with an increased risk of death from all causes and from cvd causes . during the follow - up period , patients with hvc-2 had an 20% overall survival rate compared with 80% of those with hvc-0 and with an intermediate survival rate ( 40% ) in those with hvc-1 . the differences among these curves were statistically significant ( p < 0.0001 by the log - rank test ) . kaplan - meier survival analysis for all - cause ( a ) and cardiovascular ( b ) mortality in 902 type 2 diabetic patients , stratified by the hvc score . in cox univariate survival analysis ( see unadjusted models in tables 2 and 3 ) , both hvc-1 and hvc-2 were associated with an increased risk of all - cause and cvd mortality . as also shown in tables 2 and 3 , the results remained essentially unchanged after adjustment for age , sex , bmi , smoking status , systolic blood pressure , duration of diabetes , a1c , ldl cholesterol , egfr , history of myocardial infarction , and current use of any antihypertensive and lipid - lowering medications ( regression models 1 and 2 ) or after adjustment for age , sex , and other baseline echocardiographic parameters , such as lv mass , lv - ef < 50% , wmsi , and la diameter ( model 3 ) . association between hvc score and the risk of all - cause mortality in patients with type 2 diabetes association between hvc score and the risk of cardiovascular mortality in patients with type 2 diabetes notably , other independent predictors of cvd and all - cause mortality ( p < 0.010.001 ) were older age , longer duration of diabetes , smoking status , lower egfr , and greater lv mass ( data not shown ) . the significant , graded association between the hvc score and the risk of both all - cause and cvd mortality was consistent in men versus women ( as shown in supplementary tables 14 ) and in younger versus older individuals ( data not shown ) . almost identical results were also found when separate analyses were performed in those with ( n = 210 ) and those without ( n = 692 ) a previous history of myocardial infarction . in both of these subgroups , increasing hvc score predicted all - cause and cvd mortality , independently of potential confounders . in particular , among those with a history of myocardial infarction , the hvc score was associated with both all - cause mortality ( hr 4.9 [ 95% ci 2.410.4 ] , p < 0.001 for hvc-1 , and 7.5 [ 2.720.6 ] , p < 0.001 for hvc-2 ) and cvd mortality ( 4.2 [ 1.710.9 ] , p < 0.005 , and 9.0 [ 2.631.1 ] , p < 0.001 ) even after adjustment for age , sex , lv mass , lv - ef < 50% , wmsi , and la diameter . the strong association between the hvc score and mortality was further reinforced by the finding of a positive , graded relationship between the extent of mitral and aortic annular calcification and the clinical outcomes . indeed , the hr for all - cause mortality was more than doubled in those with both valves affected ( unadjusted hr 7.2 [ 95% ci 4.511.4 ] for those with hvc-2 ) compared with those with either isolated avs ( 3.2 [ 2.24.8 ] ) or isolated mac ( 2.4 [ 1.34.1 ] ) , respectively . in addition , a stronger association with the risk of mortality was also observed with the amount of calcium at the level of aortic valve ; the hr for all - cause mortality increased from 3.2 ( 95% ci 2.24.8 ) in those with isolated avs to 4.4 ( 2.27.7 ) in those with aortic stenosis at baseline . notably , after exclusion of patients who had aortic stenosis at baseline ( n = 56 ) or developed aortic stenosis during the follow - up ( n = 13 ) , the value of hvc score in predicting all - cause mortality remained essentially unchanged ( adjusted hr 2.3 [ 95% ci 1.53.5 ] for hvc-1 and hr 5.1 [ 3.18.5 ] for hvc-2 , respectively ) . to our knowledge , this study is the first to specifically address the value of avs and mac in predicting all - cause and cvd mortality in a large sample of type 2 diabetic patients referred for clinically indicated echocardiograms . study is that avs and mac , singly or in combination , predicted an increased risk of cvd and all - cause mortality , independently of traditional risk factors , diabetes - related variables , kidney function parameters ( egfr ) , and other baseline echocardiographic variables ( lv mass , lv - ef , wmsi , and la diameter ) . notably , the combined presence of avs and mac was more strongly associated with an increased risk of all - cause and cvd mortality than the presence of either item alone . collectively , the results of this study complement and expand previous observations on the value of avs and mac for predicting mortality both in the general population and in other nondiabetic high - risk patient populations ( 1,49,18 ) . the biological mechanisms accounting for the association of avs and mac with an increased risk of all - cause and cvd mortality have still not been fully elucidated . patients with mac or avs , especially those with avs , may progress to aortic stenosis , which is a pathologic condition that is associated with an increased risk of mortality ( 19 ) . however , it should also be noted that in our study the strength of the association between the hvc score and the clinical outcomes remained essentially unaltered even after exclusion of patients who either had aortic stenosis at baseline or developed aortic stenosis during the follow - up . thus , in accord with the results of previous studies ( 13,20,21 ) , we believe that a plausible explanation for our findings is that avs and mac are reliable markers of the severity of systemic atherosclerosis . it is known that there are similarities in the histopathologic lesions of avs , mac , and coronary atherosclerosis and that these three diseases share multiple cvd risk factors ( e.g. , hypertension , smoking , dyslipidemia , and diabetes ) and common pathophysiologic mechanisms ( 13,18,21 ) . more recent studies suggested that avs and mac are not only associated with coronary atherosclerosis but also with atherosclerosis in other vascular beds ( 2125 ) . however , since in our study the hvc score strongly predicted cvd mortality independently of traditional cvd risk factors and other potential confounders , it is also possible to speculate that avs and mac , singly or in combination , reflect not only the burden of systemic atherosclerosis ( 13,21,26,27 ) but also the presence of other underlying conditions , which may predispose patients to active and accelerated atherosclerosis , such as systemic chronic inflammation ( 21,28 ) , platelet aggregation abnormalities ( 21,29 ) , and genetic predisposition ( 30 ) . the use of risk estimation systems such as the framingham risk score has been recommended as the cornerstone for the estimation of the cvd risk in persons without previous cvd events ( 31,32 ) , but their ability to identify patients who will develop future cvd events and death remains limited , particularly in those with type 2 diabetes ( 33,34 ) . great attention has recently been paid to coronary artery calcium ( cac ) score , as measured by computed tomography ( 35,36 ) . however , the cac evaluation has some important disadvantages such as its high costs and radiation exposure . moreover , the incremental value of cac score to the prediction of major cvd events and death is marginal ( 37 ) . conversely , the echocardiographic evaluation of avs and mac is easy , reproducible , safe , and cheap ( 15 ) . overall , our findings provide strong evidence that avs and mac , singly or in combination , are powerful independent predictors of all - cause and cvd mortality in patients with type 2 diabetes . asymptomatic mac and avs can identify a subgroup of patients with higher likelihood of diffuse vascular atherosclerotic processes and should prompt a careful evaluation of conventional cvd risk factors and the institution of proper prevention or treatment strategies . the major limitations of this study are its retrospective , longitudinal design ( which does not allow us to draw any firm conclusion about causality ) and a possible selection bias of excluding the patients who had missing echocardiographic data at baseline . there was also a relatively small number of clinical events during the follow - up ( i.e. , the cumulative incidence of all - cause mortality in the whole sample was 15% [ 137 deaths ] ) , and , therefore , the results should be interpreted with some caution . in addition , although our statistical models were extensive , unmeasured confounders ( e.g. , plasma inflammatory biomarkers ) could potentially explain the observed associations . finally , because our sample comprised white type 2 diabetic individuals who were followed at an outpatient diabetes clinic and who were referred for echocardiography for clinical reasons , our results may not necessarily be generalizable to other diabetic populations . the strengths of our study include the relatively large number of participants of both sexes , the long duration of the follow - up , the complete nature of the dataset , and the ability to adjust for several established risk factors and potential confounders . in conclusion , this study is the first to demonstrate a graded and independent association of avs and mac with the risk of all - cause and cvd mortality in a sample of type 2 diabetic patients referred for clinically indicated echocardiograms . further follow - up studies on larger cohorts of patients are needed to confirm the reproducibility of our results and to determine whether intervening in the progression of calcific annular or valvular disease can reduce the incidence of cvd events and death in patients with type 2 diabetes .

objectiveto examine the association of aortic valve sclerosis ( avs ) and mitral annulus calcification ( mac ) with all - cause and cardiovascular mortality in type 2 diabetic individuals.research design and methodswe retrospectively analyzed the data from 902 type 2 diabetic outpatients , who had undergone a transthoracic echocardiography for clinical reasons during the years 19922007 . avs and mac were diagnosed by echocardiography , and a heart valve calcium ( hvc ) score was calculated by summing up the avs and mac variables . the study outcomes were all - cause and cardiovascular mortality.resultsat baseline , 477 ( 52.9% ) patients had no heart valves affected ( hvc-0 ) , 304 ( 33.7% ) had one valve affected ( hvc-1 ) , and 121 ( 13.4% ) had both valves affected ( hvc-2 ) . during a mean follow - up of 9 years , 137 ( 15.2% ) patients died , 78 of them from cardiovascular causes . compared with patients with hvc-0 , those with hvc-2 had the highest risk of all - cause and cardiovascular mortality , whereas those with hvc-1 had an intermediate risk ( p < 0.0001 by the log - rank test ) . after adjustment for sex , age , bmi , systolic blood pressure , diabetes duration , a1c , ldl cholesterol , estimated glomerular filtration rate , smoking , history of myocardial infarction , and use of antihypertensive and lipid - lowering drugs , the hazard ratio of all - cause mortality was 2.3 ( 95% ci 1.14.9 ; p < 0.01 ) for patients with hvc-1 and 9.3 ( 3.917.4 ; p < 0.001 ) for those with hvc-2 . similar results were found for cardiovascular mortality.conclusionsour findings indicate that avs and mac , singly or in combination , are independently associated with all - cause and cardiovascular mortality in type 2 diabetic patients .

RESEARCH DESIGN AND METHODS Clinical and laboratory data Conventional echocardiography Mortality follow-up Statistical analysis RESULTS Baseline characteristics Survival analysis CONCLUSIONS Supplementary Material

the study was performed within the frame of the verona diabetes study , an observational longitudinal study on chronic complications in type 2 diabetic outpatients attending the diabetes clinic at the university hospital of verona ( 13 ) . briefly , for this analysis we retrospectively analyzed the electronic records of all caucasian type 2 diabetic outpatients , who regularly attended our diabetes clinic during the years 19922007 . of these , we selected all patients with type 2 diabetes who had undergone a first transthoracic echocardiography for clinical reasons ( e.g. , dyspnea , palpitations , chest pain , heart murmur , history of myocardial infarction , electrocardiographic abnormalities , assessment of left ventricular wall thickness , presence of multiple cvd risk factors ) at our institution during the same period of time . left ventricular ( lv ) chamber dimensions and wall thickness were measured from m - mode recordings as suggested by the american society of echocardiography , and lv mass was calculated using a necropsy validated equation ( 15 ) . in accord with a previous study ( 5 ) , we calculated a heart valve calcium ( hvc ) score combining the presence of calcium at the level of aortic and mitral valve as follows : hvc-0 , absence of any thickening or calcification ; hvc-1 , presence of either isolated avs or isolated mac ; and hvc-2 , coexistence of avs and mac . univariate survival analysis was performed by the kaplan - meier analysis , and the overall significance was calculated by the log - rank test . cox regression analysis was used to study the association between the hvc score and the risk of all - cause and cvd mortality after adjustment for potential confounders . four forced - entry cox regression models were performed : an unadjusted model ; a model adjusted for age and sex ( model 1 ) ; a model adjusted for age , sex , bmi , duration of diabetes , smoking status , systolic blood pressure , a1c , ldl cholesterol , estimated glomerular filtration rate ( egfr ) , history of myocardial infarction , and use of any antihypertensive and lipid - lowering medications ( model 2 , where the number of subjects with available data for analysis was 792 ) ; and a model adjusted for age , sex , lv mass , lv - ef < 50% , wmsi , and la diameter ( model 3 , where all subjects had available data for analysis ) . left ventricular ( lv ) chamber dimensions and wall thickness were measured from m - mode recordings as suggested by the american society of echocardiography , and lv mass was calculated using a necropsy validated equation ( 15 ) . in accord with a previous study ( 5 ) , we calculated a heart valve calcium ( hvc ) score combining the presence of calcium at the level of aortic and mitral valve as follows : hvc-0 , absence of any thickening or calcification ; hvc-1 , presence of either isolated avs or isolated mac ; and hvc-2 , coexistence of avs and mac . univariate survival analysis was performed by the kaplan - meier analysis , and the overall significance was calculated by the log - rank test . cox regression analysis was used to study the association between the hvc score and the risk of all - cause and cvd mortality after adjustment for potential confounders . four forced - entry cox regression models were performed : an unadjusted model ; a model adjusted for age and sex ( model 1 ) ; a model adjusted for age , sex , bmi , duration of diabetes , smoking status , systolic blood pressure , a1c , ldl cholesterol , estimated glomerular filtration rate ( egfr ) , history of myocardial infarction , and use of any antihypertensive and lipid - lowering medications ( model 2 , where the number of subjects with available data for analysis was 792 ) ; and a model adjusted for age , sex , lv mass , lv - ef < 50% , wmsi , and la diameter ( model 3 , where all subjects had available data for analysis ) . of the initial sample of 937 type 2 diabetic patients , 35 who had either aortic valve prosthesis or rheumatic or congenital aortic valve disease were excluded from analysis . overall , 477 ( 52.9% ) patients were free of any thickening or calcification at the valvular level ( i.e. , hvc-0 ) , and 304 ( 33.7% ) patients had either isolated mac or isolated avs ( hvc-1 ) ; i.e. , 43 ( 4.8% ) patients had isolated mac and 261 ( 28.9% ) patients had avs , and 17 of them had aortic stenosis . the remaining 121 ( 13.4% ) patients had combined avs and mac ( hvc-2 ) , and 39 of them had aortic stenosis . as shown in table 1 , age , systolic blood pressure , ldl cholesterol , albuminuria , and the prevalence of hypertension significantly increased , whereas bmi , triglycerides , and egfr decreased across the hvc score . sex , smoking history , duration of diabetes , frequency of diabetic retinopathy , fasting plasma glucose , a1c , and hdl cholesterol did not significantly change across the hvc score . baseline clinical , biochemical , and echocardiographic characteristics of type 2 diabetic patients , stratified by hvc score during a mean follow - up period of 9 years ( median 7.6 years ) , 137 ( 15.2% of total ) patients died ( 78 of these from cvd causes ) . 1a , the hvc score was strongly associated with an increased risk of death from all causes and from cvd causes . during the follow - up period , patients with hvc-2 had an 20% overall survival rate compared with 80% of those with hvc-0 and with an intermediate survival rate ( 40% ) in those with hvc-1 . the differences among these curves were statistically significant ( p < 0.0001 by the log - rank test ) . kaplan - meier survival analysis for all - cause ( a ) and cardiovascular ( b ) mortality in 902 type 2 diabetic patients , stratified by the hvc score . in cox univariate survival analysis ( see unadjusted models in tables 2 and 3 ) , both hvc-1 and hvc-2 were associated with an increased risk of all - cause and cvd mortality . as also shown in tables 2 and 3 , the results remained essentially unchanged after adjustment for age , sex , bmi , smoking status , systolic blood pressure , duration of diabetes , a1c , ldl cholesterol , egfr , history of myocardial infarction , and current use of any antihypertensive and lipid - lowering medications ( regression models 1 and 2 ) or after adjustment for age , sex , and other baseline echocardiographic parameters , such as lv mass , lv - ef < 50% , wmsi , and la diameter ( model 3 ) . association between hvc score and the risk of all - cause mortality in patients with type 2 diabetes association between hvc score and the risk of cardiovascular mortality in patients with type 2 diabetes notably , other independent predictors of cvd and all - cause mortality ( p < 0.010.001 ) were older age , longer duration of diabetes , smoking status , lower egfr , and greater lv mass ( data not shown ) . the significant , graded association between the hvc score and the risk of both all - cause and cvd mortality was consistent in men versus women ( as shown in supplementary tables 14 ) and in younger versus older individuals ( data not shown ) . almost identical results were also found when separate analyses were performed in those with ( n = 210 ) and those without ( n = 692 ) a previous history of myocardial infarction . in both of these subgroups , increasing hvc score predicted all - cause and cvd mortality , independently of potential confounders . in particular , among those with a history of myocardial infarction , the hvc score was associated with both all - cause mortality ( hr 4.9 [ 95% ci 2.410.4 ] , p < 0.001 for hvc-1 , and 7.5 [ 2.720.6 ] , p < 0.001 for hvc-2 ) and cvd mortality ( 4.2 [ 1.710.9 ] , p < 0.005 , and 9.0 [ 2.631.1 ] , p < 0.001 ) even after adjustment for age , sex , lv mass , lv - ef < 50% , wmsi , and la diameter . indeed , the hr for all - cause mortality was more than doubled in those with both valves affected ( unadjusted hr 7.2 [ 95% ci 4.511.4 ] for those with hvc-2 ) compared with those with either isolated avs ( 3.2 [ 2.24.8 ] ) or isolated mac ( 2.4 [ 1.34.1 ] ) , respectively . in addition , a stronger association with the risk of mortality was also observed with the amount of calcium at the level of aortic valve ; the hr for all - cause mortality increased from 3.2 ( 95% ci 2.24.8 ) in those with isolated avs to 4.4 ( 2.27.7 ) in those with aortic stenosis at baseline . notably , after exclusion of patients who had aortic stenosis at baseline ( n = 56 ) or developed aortic stenosis during the follow - up ( n = 13 ) , the value of hvc score in predicting all - cause mortality remained essentially unchanged ( adjusted hr 2.3 [ 95% ci 1.53.5 ] for hvc-1 and hr 5.1 [ 3.18.5 ] for hvc-2 , respectively ) . of the initial sample of 937 type 2 diabetic patients , 35 who had either aortic valve prosthesis or rheumatic or congenital aortic valve disease were excluded from analysis . overall , 477 ( 52.9% ) patients were free of any thickening or calcification at the valvular level ( i.e. , hvc-0 ) , and 304 ( 33.7% ) patients had either isolated mac or isolated avs ( hvc-1 ) ; i.e. , 43 ( 4.8% ) patients had isolated mac and 261 ( 28.9% ) patients had avs , and 17 of them had aortic stenosis . the remaining 121 ( 13.4% ) patients had combined avs and mac ( hvc-2 ) , and 39 of them had aortic stenosis . as shown in table 1 , age , systolic blood pressure , ldl cholesterol , albuminuria , and the prevalence of hypertension significantly increased , whereas bmi , triglycerides , and egfr decreased across the hvc score . sex , smoking history , duration of diabetes , frequency of diabetic retinopathy , fasting plasma glucose , a1c , and hdl cholesterol did not significantly change across the hvc score . baseline clinical , biochemical , and echocardiographic characteristics of type 2 diabetic patients , stratified by hvc score during a mean follow - up period of 9 years ( median 7.6 years ) , 137 ( 15.2% of total ) patients died ( 78 of these from cvd causes ) . during the follow - up period , patients with hvc-2 had an 20% overall survival rate compared with 80% of those with hvc-0 and with an intermediate survival rate ( 40% ) in those with hvc-1 . the differences among these curves were statistically significant ( p < 0.0001 by the log - rank test ) . kaplan - meier survival analysis for all - cause ( a ) and cardiovascular ( b ) mortality in 902 type 2 diabetic patients , stratified by the hvc score . in cox univariate survival analysis ( see unadjusted models in tables 2 and 3 ) , both hvc-1 and hvc-2 were associated with an increased risk of all - cause and cvd mortality . as also shown in tables 2 and 3 , the results remained essentially unchanged after adjustment for age , sex , bmi , smoking status , systolic blood pressure , duration of diabetes , a1c , ldl cholesterol , egfr , history of myocardial infarction , and current use of any antihypertensive and lipid - lowering medications ( regression models 1 and 2 ) or after adjustment for age , sex , and other baseline echocardiographic parameters , such as lv mass , lv - ef < 50% , wmsi , and la diameter ( model 3 ) . association between hvc score and the risk of all - cause mortality in patients with type 2 diabetes association between hvc score and the risk of cardiovascular mortality in patients with type 2 diabetes notably , other independent predictors of cvd and all - cause mortality ( p < 0.010.001 ) were older age , longer duration of diabetes , smoking status , lower egfr , and greater lv mass ( data not shown ) . the significant , graded association between the hvc score and the risk of both all - cause and cvd mortality was consistent in men versus women ( as shown in supplementary tables 14 ) and in younger versus older individuals ( data not shown ) . almost identical results were also found when separate analyses were performed in those with ( n = 210 ) and those without ( n = 692 ) a previous history of myocardial infarction . in both of these subgroups , increasing hvc score predicted all - cause and cvd mortality , independently of potential confounders . in particular , among those with a history of myocardial infarction , the hvc score was associated with both all - cause mortality ( hr 4.9 [ 95% ci 2.410.4 ] , p < 0.001 for hvc-1 , and 7.5 [ 2.720.6 ] , p < 0.001 for hvc-2 ) and cvd mortality ( 4.2 [ 1.710.9 ] , p < 0.005 , and 9.0 [ 2.631.1 ] , p < 0.001 ) even after adjustment for age , sex , lv mass , lv - ef < 50% , wmsi , and la diameter . indeed , the hr for all - cause mortality was more than doubled in those with both valves affected ( unadjusted hr 7.2 [ 95% ci 4.511.4 ] for those with hvc-2 ) compared with those with either isolated avs ( 3.2 [ 2.24.8 ] ) or isolated mac ( 2.4 [ 1.34.1 ] ) , respectively . in addition , a stronger association with the risk of mortality was also observed with the amount of calcium at the level of aortic valve ; the hr for all - cause mortality increased from 3.2 ( 95% ci 2.24.8 ) in those with isolated avs to 4.4 ( 2.27.7 ) in those with aortic stenosis at baseline . notably , after exclusion of patients who had aortic stenosis at baseline ( n = 56 ) or developed aortic stenosis during the follow - up ( n = 13 ) , the value of hvc score in predicting all - cause mortality remained essentially unchanged ( adjusted hr 2.3 [ 95% ci 1.53.5 ] for hvc-1 and hr 5.1 [ 3.18.5 ] for hvc-2 , respectively ) . to our knowledge , this study is the first to specifically address the value of avs and mac in predicting all - cause and cvd mortality in a large sample of type 2 diabetic patients referred for clinically indicated echocardiograms . study is that avs and mac , singly or in combination , predicted an increased risk of cvd and all - cause mortality , independently of traditional risk factors , diabetes - related variables , kidney function parameters ( egfr ) , and other baseline echocardiographic variables ( lv mass , lv - ef , wmsi , and la diameter ) . notably , the combined presence of avs and mac was more strongly associated with an increased risk of all - cause and cvd mortality than the presence of either item alone . the biological mechanisms accounting for the association of avs and mac with an increased risk of all - cause and cvd mortality have still not been fully elucidated . patients with mac or avs , especially those with avs , may progress to aortic stenosis , which is a pathologic condition that is associated with an increased risk of mortality ( 19 ) . however , it should also be noted that in our study the strength of the association between the hvc score and the clinical outcomes remained essentially unaltered even after exclusion of patients who either had aortic stenosis at baseline or developed aortic stenosis during the follow - up . thus , in accord with the results of previous studies ( 13,20,21 ) , we believe that a plausible explanation for our findings is that avs and mac are reliable markers of the severity of systemic atherosclerosis . more recent studies suggested that avs and mac are not only associated with coronary atherosclerosis but also with atherosclerosis in other vascular beds ( 2125 ) . however , since in our study the hvc score strongly predicted cvd mortality independently of traditional cvd risk factors and other potential confounders , it is also possible to speculate that avs and mac , singly or in combination , reflect not only the burden of systemic atherosclerosis ( 13,21,26,27 ) but also the presence of other underlying conditions , which may predispose patients to active and accelerated atherosclerosis , such as systemic chronic inflammation ( 21,28 ) , platelet aggregation abnormalities ( 21,29 ) , and genetic predisposition ( 30 ) . conversely , the echocardiographic evaluation of avs and mac is easy , reproducible , safe , and cheap ( 15 ) . overall , our findings provide strong evidence that avs and mac , singly or in combination , are powerful independent predictors of all - cause and cvd mortality in patients with type 2 diabetes . , the cumulative incidence of all - cause mortality in the whole sample was 15% [ 137 deaths ] ) , and , therefore , the results should be interpreted with some caution . finally , because our sample comprised white type 2 diabetic individuals who were followed at an outpatient diabetes clinic and who were referred for echocardiography for clinical reasons , our results may not necessarily be generalizable to other diabetic populations . the strengths of our study include the relatively large number of participants of both sexes , the long duration of the follow - up , the complete nature of the dataset , and the ability to adjust for several established risk factors and potential confounders . in conclusion , this study is the first to demonstrate a graded and independent association of avs and mac with the risk of all - cause and cvd mortality in a sample of type 2 diabetic patients referred for clinically indicated echocardiograms . further follow - up studies on larger cohorts of patients are needed to confirm the reproducibility of our results and to determine whether intervening in the progression of calcific annular or valvular disease can reduce the incidence of cvd events and death in patients with type 2 diabetes .

puromycin aminonucleoside ( pan)-induced nephrotic syndrome is one of the most extensively studied models of glomerulonephritis in rats . this experimental model mimics the minimal change disease glomerulopathy found in human pathology . in vivo micropuncture studies in the unilateral model of pan - induced nephrotic syndrome have shown that sodium reabsorption is specifically increased in the collecting duct and not in the proximal tubule and distal nephron11 ) . micropuncture of the accessible distal convoluted tubule revealed that the tubular sodium load was similar in pan - treated kidney and control kidney of the same rat , and the final urine sodium excretion was threefold lower in urine collected from the pan - treated kidney compared to that in the untreated control kidney in the same animal11 ) . thus , this study directly point to a role of increased sodium reabsorption in the collecting duct and , hence , it may be hypothesized that dysregulation of the key sodium channels and transporters in the collecting duct may be responsible for this . a few studies have subsequently demonstrated that sodium retention in pan nephrotic rats is correlated with increased na , k - atpase activity and expression in the cortical collecting duct12 , 13 ) . we demonstrated that pan - induced nephrotic syndrome is associated with 1 ) sodium retention , decreased urinary excretion of sodium , a marked ascites , and increased plasma aldosterone level , 2 ) upregulation of protein abundance of specific enac subunits in cortex , outer medulla and inner medulla , and 3 ) increased apical targeting of enac subunits in connecting tubule and collecting duct segments observed as increased immunolabeling in apical plasma membrane domains14 ) . in contrast , the protein levels of other major sodium transporters expressed in nephron segments at a site proximal to the connecting tubule ( i.e. , nhe3 , nkcc2 , na , k - atpase and ncc ) were significantly reduced in these segments14 ) . indeed , there was no downregulation of the na , k - atpase expression in the collecting duct . taken together , these observations therefore strongly support the view that the renal sodium retention associated with pan - induced nephrotic syndrome is caused by increased sodium reabsorption in connecting tubule and collecting duct14 ) . thus , this is likely to represent a key molecular basis for the sodium retention associated with pan - induced nephrotic syndrome combined with the previously demonstrated increase in collecting duct na , katpase activity and protein abundance12 , 13 ) . moreover , our results extend this study by demonstrating the segmental specific upregulation of all three enac subunits , and most importantly provide evidence for a significantly increased apical targeting by immunoelectron microscopy . the latter is essential since it is important to document that the increased expression is at the site of function i.e. in the plasma membrane . finally , we also confirm that there is a uniform downregulation of all investigated sodium transporters in the thick ascending limb of henle ( tal ) and proximal tubule . this together with observed increased expression and increased targeting of enac subunits in combination with the previous functional micropuncture studies strongly support the view that the sodium retention in the nephrotic syndrome may occur in the connecting tubule and collecting duct15 ) . nephrotic syndrome may develop as a result of primary diseases such as minimal change disease or immune glomerulonephritis . membranous glomerulonephritis ( mgn ) remains the most common cause of primary nephrotic syndrome in adult . a further reason for its importance is that approximately 25 to 50% of patients progress to end - stage kidney disease over 10 years . thus , mgn has a different and more progressive clinical course as compared to the non - progressive benign character of minimal change nephrotic syndrome16 ) . mercury chloride has been known to induce a systemic autoimmune disease including membranous nephropathy with igg deposits . this nephropathy is responsible for the development of high - range proteinuria and a full - blown nephrotic syndrome associated with generalized edema and ascites17 ) . to elucidate whether the changes in expression and plasma membrane targeting of enac subunits in the pan - induced minimal change nephrotic syndrome ( caused by podocyte injury , not immune complex mediated ) is unique to the pan model or may be a more general characteristic of nephrotic syndrome including immune complex mediated glomerulonephritis , and to investigate the physiologic role of changes in subcellular distribution of enac subunits in the abnormal sodium retention and ascites formation during disease states , we examined the changes of enac abundance and/or trafficking and 11hsd2 in hgcl2 induced nephritic syndrome in brown norway rats . the results demonstrated that hgcl2-induced immune complex glomerulonephritis was associated with 1 ) sodium retention , decreased urinary sodium excretion , development of ascites , and increased plasma aldosterone level ; 2 ) increased abundance of enac ( fig . 1 ) 3 ) increased apical targeting of enac subunits in connecting tubule and collecting duct segments ; and 4 ) decreased protein abundance of 11hsd2 ( fig . these observations therefore strongly support the view that the renal sodium retention associated with hgcl2 induced nephrotic syndrome is caused by increased sodium reabsorption in the distal nephron including the connecting tubule and collecting duct . these results are consistent with the results of our previous studies of nephrotic syndrome induced by pan14 ) . thus , a straightforward interpretation of these observations would be that increased apical targeting of enac subunits plays a role in the development of sodium retention in hgcl2 nephrotic syndrome as well , and it could be argued that this mechanism in fact could be a general characteristic of the nephrotic syndrome including both minimal change disease and immune complex mediated glomerulonephritis . apical targeting of enac subunits and protein abundance of enac can be regulated by aldosterone7,8 ) . indeed , plasma aldosterone levels were significantly increased in hgcl2 nephropathy , and thus , it can be speculated that aldosterone stimulate the sodium reabsorption in hgcl2 nephropathy . however , substantial evidence argue against a major role of aldosterone in nephrotic syndrome : 1 ) ascites and edema occur in rats with nephrosis attributable to rabbit anti - rat kidney serum injection , without elevated aldosterone levels19 ) . 2 ) inhibition of angiotensin converting enzyme by captopril failed to induce natriuresis in rats with pan nephrosis despite decreased aldosterone levels20 ) . and 3 ) sodium retention was observed only in the affected proteinuria kidney in rats unilateral models of pan nephrosis11 ) . these observations suggest that aldosterone may not alone be involved in the sodium retention and presumably in the enac regulation in the nephrotic syndrome . it has been suggested that the activity of type 2 11hydroxysteroid dehydrogenase ( 11hsd2 ) can regulate the sodium reabsorption in the aldosterone responsive renal tubules by glucocorticoid induced activation of mineralocorticoid receptor ( mr)21 ) . we also demonstrate that there is a downregulation of 11hsd2 protein expression in the kidney in hgcl2 nephrotic syndrome as well as in pan nephrosis . this finding is in line with recent studies in nephrotic patients22 ) and in experimental nephrotic syndrome induced by pan or adriamycin23 ) . the investigators observed a rise in the plasma ratio of corticosterone/11-dehydrocorticosterone as well as in the urinary ratio of ( thb+5-thb)/tha in nephritic rats23 ) , and interpreted this change as a result of a decreased activity of 11-hydroxysteroid dehydrogenase . we here propose that this may occur via a downregulated protein expression of 11hsd2 in nephrotic syndrome . these findings suggest that downregulation of 11hsd2 provides access of glucocorticoids to the mr , resulting in increased activity of mr and collecting duct sodium retention in rats with nephrotic syndrome . systemic and local factors are known to regulate enac abundance , targeting and activity as well as na , k - atpase activity . these include aldosterone7 , 8) , vasopressin6 ) and angiotensin ii ( ang ii)24 ) . aldosterone is known to increase na , k - atpase activity in the cortical collecting duct and to increase enac redistribution to the apical membrane of connecting tubule and collecting duct principal cells and to promote the expression of a 70 kda form of the gamma enac subunit , thought to be linked to activation of the channel by proteolytic cleavage7 , 8) . it is therefore tempting to link this hormone with the findings observed in pan nephrosis . moreover levels of vasopressin , ang ii and aldosterone are increased in the initial phase of nephrosis14 , 25 , 26 ) , leading to the hypothesis that they may play significant roles in the development of sodium retention . as the time course and magnitude of sodium retention is indistinguishable in nephrotic brattleboro rats ( vasopressin deficient ) compared to normal nephrotic rats12 ) , vasopressin is not considered to be a major factor for the development of sodium retention in nephrotic syndrome . we have also demonstrated that ang ii at1 receptor blockade , candesartan , did not improve the sodium retention associated with pan - induced nephrotic syndrome nor the time course of its development27 ) . there have been contradictory studies on the impact of aldosterone absence on the development of edema in nephrotic syndrome28 , 29 ) . furthermore , even though pharmacologic blockade of aldosterone has been proven to be unsuccessful regarding sodium retention in experimental nephrosis15 ) , this can not rule out aldosterone effect as pharmacologic mineralocorticoid receptor blockade has been demonstrated to be useless against enac trafficking30 ) and would not avoid aldosterone action through the glucocorticoid receptor31 ) . finally , na , k - atpase in the collecting duct of nephrotic rats is still activated in the absence of aldosterone29 ) . it is therefore not known whether the enhanced enac targeting observed in pan nephrosis is a primary dysregulation in nephrotic syndrome or is secondary to the high aldosterone levels observed in this condition . we therefore studied the development of nephrotic syndrome in adrenalectomized rats , aiming at defining the role of aldosterone on sodium retention and enac targeting observed in this pathologic condition . puromycin treatment of adrenalectomized rats supplemented with dexamethasone induced sodium retention in spite of the absence of aldosterone . immunocytochemical analyses revealed absence of enhanced apical targeting of enac subunits in pan treated adrenalectomized ( adx - pan ) rats with distribution of labelling similar to adrenalectomized dexamethasone - treated control rats ( adx ) . key sodium transporters were downregulated as previously observed in non - adrenalectomized puromycin - treated rats , whereas the global expression of the alpha-1 subunit of the na , k - atpase was unchanged . taken together , pan treatment in the absence of aldosterone induced sodium retention , increased enac expression but did not change the subcellular distribution of enac . this indicates that the previously observed enhanced apical targeting of enac in pan induced neprotic syndrome is caused by aldosterone and that development of sodium retention can occur in the absence of aldosterone in nephrotic syndrome . however , it is not entirely clear whether whether puromycin - treatment results in an increased sensitivity of kidneys to systemic hormonal changes and which local factors might influence the sodium retention and enac activity . renal sodium and water retention has been shown to be responsible for the development of ascites not only in patients with liver cirrhosis but also in experimental cirrhotic rats . atrial natriuretic peptide resistance , arginine vasopressin ( avp ) , renin - angiotensin - aldosterone , and sympathetic nerve overactivity have been shown to modulate renal tubular functions and have been considered to be mainly involved in sodium and water retention in liver cirrhosis . in kidneys of cirrhotic rat , increased reabsorption of sodium and water in the distal nephron and collecting duct has been suggested to be one of the most important renal tubular dysfuctions involved in the pathogenesis of ascites . however , the underlying molecular and cellular mechanisms for the sodium retention are still incompletely understood . in particular , the role of aldosterone in sodium retention and ascites formation in liver cirrhosis is still unclear . previous studies demonstrated that plasma aldosterone levels were usually elevated in liver cirrhosis with ascites33 ) and that spironolactone , a mineralocorticoid receptor antagonist , increased sodium excretion in these patients34 ) . there has been some controversy as to the role of regulation of the renal sodium transporters and channels in the development of edema in animal models of cirrhosis . rats are treated with carbon tetrachloride ( ccl4 ) in combination with phenobarbital supplied in the drinking water , which results in liver toxicity . in the second , rats have their common bile duct ligated , and increased circulating levels of bile acids ( cholestasis ) results in hepatotoxicity and eventually cirrhosis . differences in the severity , reproducibility , and rapidity of the manifestation of ascites in the two models probably play a role in some discrepancies35 ) . the renal responses for sodium retention displayed wide variations among the rats with ccl4-induced liver cirrhosis . some rats showed markedly decreased urinary sodium excretion ( sodium retaining stage , group a ) whereas the others exhibited unchanged urinary sodium excretion ( maintenance stage , group b ) compared with controls , even though all ccl4-treated rats had significant amount of ascites ( fig . the results demonstrated that ccl4-induced sodium retaining stage ( group a ) of liver cirrhosis was associated with 1 ) decreased urinary sodium excretion , and increased or maintained plasma aldosterone levels ; 2 ) increased apical targeting of enac subunits in dct2 , cnt and collecting duct segments ; and 3 ) decreased protein abundance of 11hsd2 . in contrast , maintenance stage ( group b ) of liver cirrhosis was associated with no changes in the urinary sodium excretion , trafficking and abundance of enac subunits and the abundance of 11hsd236 ) . the most important finding is the striking increase in targeting of all enac subunits to the apical plasma membrane domain in dct2 , cnt and collecting duct in the sodium retaining stage ( group a ) , but not in the maintenance stage ( group b ) of liver cirrhosis . since an increased targeting of all enac subunits to the apical plasma membrane is associated with increased sodium reabsorption , the present finding of an increased enac targeting in the sodium - retaining stage of liver cirrhosis could contribute significantly to the increased renal tubular sodium reabsorption . these observations therefore strongly support the view that the renal sodium retention in the decompensated liver cirrhosis is mainly caused by an increased sodium reabsorption in distal nephron including the collecting duct37 ) . we demonstrated that the abundance of enac was decreased or unchanged , and the abundance of the 70-kda form of enac was increased while the 85-kda band was markedly decreased in the sodium - retaining stage ( group a ) of cirrhotic rats ( fig . previous studies demonstrated that aldosterone causes a mobility shift of enac from an 85 kda band to 70 kda band without a change in total enac protein abundance7 ) . the same changes are observed in chronically sodium - restricted rats in addition to a significant downregulation of the enac subunit7 ) . thus , the observed increased apical targeting and altered expression of - and enac subunits in group a could be caused by the stimulation of mineralocorticoid receptor ( mr ) in the aldosterone - responsive epithelium . long - term bile duct ligation in rats is a well known experimental model of liver cirrhosis and portal hypertension , and has been widely used to study the renal mechanisms of sodium retention , as well as systemic and splanchnic hemodynamics38 ) . cbdl rats showed decreased fractional excretion of sodium and hence , positive sodium balance in the sodium retaining stage of liver cirrhosis ( at 6 wks ) . it is suggested that increased renal tubular absorption of sodium may account for the sodium retention and pathogenesis of ascites formation . it is noteworthy that at 8 weeks of liver cirrhosis we did not see any evidence of positive sodium balance even though all rats showed marked ascites39 ) . these findings may suggest that there are dynamic changes in sodium retention at different stages ( i.e. , a sodium retaining stage and a compensatory stage ) of liver cirrhosis . it should be emphasized that the two time periods are two in a continuum of changes during the development of sodium retention and the stages with partial of full compensation . recognition of this apparent biphasic renal response to cirrhosis , with progression from the early to the late phase of decompensation , may help to clarify apparent discrepancies in a variety of earlier studies . the most important finding is the increased targeting of all enac subunits to the apical plasma membrane domain in dct2 , cnt and collecting duct in the sodium retaining cirrhotic rats ( fig . 5 ) , but not in 8 week liver cirrhotic rats without positive sodium balance39 ) . since increased enac plasma membrane targeting is strongly associated with increased sodium reabsorption , this finding very strongly suggests that increased enac targetings contribute significantly to the increased renal tubular sodium reabsorption . the increased apical targeting of enac subunits in cbdl - induced liver cirrhosis may be caused by increased aldosterone activity via stimulation of mineralocorticoid receptor ( mr ) in the aldosterone responsive epithelium . since there was a markedly enhanced apical plasma membrane expression of all enac subunits at 6 wk liver cirrhosis associated with maintained plasma aldosterone levels but not at 8 wk liver cirrhosis where aldosterone levels were markedly reduced , it is likely that aldosterone plays an essential role . have demonstrated that the mr has an equal affinity for aldosterone and glucocorticoids , yet in vivo it displays specificity for aldosterone in the face of much higher circulating concentrations of glucocorticoids . this specificity is conferred by 11hsd2 , an enzyme that converts glucocorticoids to inactive 11-ketosteroid derivatives that have weak or no affinity for the mr in mineralocorticoid sensitive tissues including the distal nephron21 ) . loss of function mutation of 11hsd2 or inhibition of 11hsd2 activity allows glucocortiocids to promote renal sodium retention and potassium excretion in the cortical collecting tubule40 , 41 ) . these findings suggest that the 11hsd2 regulates the sodium reabsorption in the aldosterone responsive renal tubules by glucocorticoid - induced activation of the mr . we demonstrate in the present studies that there is downregulation of 11hsd2 abundance in the kidney in decompensated liver cirrhosis rats . this finding is in line with recent studies in compensated liver cirrhosis reported by quattropani et al.42 ) and escher et al.43 ) . they demonstrated that 11 hsd2 activity in the kidney was reduced in liver cirrhosis following bile duct ligation42 , 43 ) . these findings together suggest that reduced expression and activity provides basis for access of glucocorticoids to the mr , thereby promoting increased collecting duct sodium reabsorption despite normal plasma levels of aldosterone in rats with experimental liver cirrhosis . this notion was strongly supported by the increased apical targeting of enac subunits in rats with glycyrrhizic acid - induced inhibition of 11hsd2 activity44 ) . plasma aldosterone levels , at least in humans and rats without liver cirrhosis , have been shown to be reduced in conditions with low expression of 11hsd240 , 41 ) . it is noteworthy that plasma aldosterone levels were not reduced in 6 wk liver cirrhosis rats , despite that the protein abundance of 11hsd2 was significantly decreased . thus , coordinated activation of mr by both glucocorticoid as a consequence of reduced activity of 11hsd2 and by relatively high plasma aldosterone may enhance distal renal tubular sodium reabsorption and potassium loss , and hence play important roles for the pathogenesis of sodium retention and possible ascites formation in cbdl - induced liver cirrhosis . in contrast , in response to liver cirrhosis for 8 weeks enac abundance was decreased ( fig . in addition , the increased apical targeting of enac subunits was retrieved in spite of decreased abundance of 11hsd2 . it can be speculated that 11hsd2 is still functioning but not at full power ( thus aldosterone may play a major role ) , or it may be speculated that differences in enac trafficking between 6 and 8 weeks are independent of aldosterone and/or 11hsd2 . in normal rats both the apical targeting and protein abundance of enac however , in the present study we did not observe an increase in enac protein expression in the cortex in kidneys from rats with cbdl - induced liver cirrhosis for 6 weeks although increased abundance of enac was seen in inner stripe of the outer medulla . it may be speculated that glucocorticoids may be involved in this regulation , and if this is the case the observations suggest that regulation of enac abundance is not as sensitive , as is the regulation of changes in the subcellular localization of enac , to mr activation by glucocorticoids . whether the differential regulation of enac subunit targeting and expression may be related to differences in the kinetics of mr - ligand interaction remains unknown ; aldosterone dissociates more slowly from mr than cortisol does , indicating that the stability of the aldosterone - mr complex is higher than that of the cortisol - mr complex45 , 46 ) . in addition , the mr activity can be regulated by at the level of transcription activation or repression by cell specific co - factors47 ) . it is also interesting to note that the rats with liver cirrhosis apparently exhibited dynamic changes in sodium retention and enac trafficking . after 6 weeks , liver cirrhosis animals exhibited sodium retention , potassium loss and decreased urine na / k ratio indicating increased aldosterone - like activity . these findings can be explained in part by decreased abundance of 11hsd2 , which permits the stimulation of mr by glucocorticoid resulting in increased apical targeting of enac subunits . on the other hand , at 8 weeks the rats with liver cirrhosis did not exhibit any changes in urinary sodium and potassium excretion even though they all had marked ascites . the underlying mechanism for the time course changes in circulating aldosterone levels and changes in enac subunits trafficking remains undefined . however , it can be speculated that at later stages of liver cirrhosis the rise in extracellular fluid volume and enhanced secretion of atrial natriuretic peptaide ( anp ) contribute to the decrease in plasma aldosterone levels and , hence , limit enac abundance / trafficking since anp has been known to directly inhibit aldosterone secretion in the adrenal glands through specific anp receptors in the zona glomerulosa48 ) . the results strongly suggests that the marked increase in apical enac subunit targeting combined with diminished abundance of 11hsd2 in the dct2 , connecting tubule and collecting duct is likely to play key roles for the sodium retention associated with cbdl - induced liver cirrhosis in rats in the sodium retaining stage ( i.e. in this model after 6 weeks of cbdl ) . the subsequent decrease in the expression of enac subunits and the reduced targeting after 8 weeks of cbdl - induced liver cirrhosis may contribute to promote sodium excretion at later stages of liver cirrhosis . this pahse may represent active and successful escape from the sodium retention by means of down - regulation of the activity of the enac . the renal responses for sodium retention displayed wide variations among the rats with ccl4-induced liver cirrhosis . some rats showed markedly decreased urinary sodium excretion ( sodium retaining stage , group a ) whereas the others exhibited unchanged urinary sodium excretion ( maintenance stage , group b ) compared with controls , even though all ccl4-treated rats had significant amount of ascites ( fig . the results demonstrated that ccl4-induced sodium retaining stage ( group a ) of liver cirrhosis was associated with 1 ) decreased urinary sodium excretion , and increased or maintained plasma aldosterone levels ; 2 ) increased apical targeting of enac subunits in dct2 , cnt and collecting duct segments ; and 3 ) decreased protein abundance of 11hsd2 . in contrast , maintenance stage ( group b ) of liver cirrhosis was associated with no changes in the urinary sodium excretion , trafficking and abundance of enac subunits and the abundance of 11hsd236 ) . the most important finding is the striking increase in targeting of all enac subunits to the apical plasma membrane domain in dct2 , cnt and collecting duct in the sodium retaining stage ( group a ) , but not in the maintenance stage ( group b ) of liver cirrhosis . since an increased targeting of all enac subunits to the apical plasma membrane is associated with increased sodium reabsorption , the present finding of an increased enac targeting in the sodium - retaining stage of liver cirrhosis could contribute significantly to the increased renal tubular sodium reabsorption . these observations therefore strongly support the view that the renal sodium retention in the decompensated liver cirrhosis is mainly caused by an increased sodium reabsorption in distal nephron including the collecting duct37 ) . we demonstrated that the abundance of enac was decreased or unchanged , and the abundance of the 70-kda form of enac was increased while the 85-kda band was markedly decreased in the sodium - retaining stage ( group a ) of cirrhotic rats ( fig . previous studies demonstrated that aldosterone causes a mobility shift of enac from an 85 kda band to 70 kda band without a change in total enac protein abundance7 ) . the same changes are observed in chronically sodium - restricted rats in addition to a significant downregulation of the enac subunit7 ) . thus , the observed increased apical targeting and altered expression of - and enac subunits in group a could be caused by the stimulation of mineralocorticoid receptor ( mr ) in the aldosterone - responsive epithelium . long - term bile duct ligation in rats is a well known experimental model of liver cirrhosis and portal hypertension , and has been widely used to study the renal mechanisms of sodium retention , as well as systemic and splanchnic hemodynamics38 ) . cbdl rats showed decreased fractional excretion of sodium and hence , positive sodium balance in the sodium retaining stage of liver cirrhosis ( at 6 wks ) . it is suggested that increased renal tubular absorption of sodium may account for the sodium retention and pathogenesis of ascites formation . it is noteworthy that at 8 weeks of liver cirrhosis we did not see any evidence of positive sodium balance even though all rats showed marked ascites39 ) . these findings may suggest that there are dynamic changes in sodium retention at different stages ( i.e. , a sodium retaining stage and a compensatory stage ) of liver cirrhosis . it should be emphasized that the two time periods are two in a continuum of changes during the development of sodium retention and the stages with partial of full compensation . recognition of this apparent biphasic renal response to cirrhosis , with progression from the early to the late phase of decompensation , may help to clarify apparent discrepancies in a variety of earlier studies . the most important finding is the increased targeting of all enac subunits to the apical plasma membrane domain in dct2 , cnt and collecting duct in the sodium retaining cirrhotic rats ( fig . 5 ) , but not in 8 week liver cirrhotic rats without positive sodium balance39 ) . since increased enac plasma membrane targeting is strongly associated with increased sodium reabsorption , this finding very strongly suggests that increased enac targetings contribute significantly to the increased renal tubular sodium reabsorption . the increased apical targeting of enac subunits in cbdl - induced liver cirrhosis may be caused by increased aldosterone activity via stimulation of mineralocorticoid receptor ( mr ) in the aldosterone responsive epithelium . since there was a markedly enhanced apical plasma membrane expression of all enac subunits at 6 wk liver cirrhosis associated with maintained plasma aldosterone levels but not at 8 wk liver cirrhosis where aldosterone levels were markedly reduced , it is likely that aldosterone plays an essential role . have demonstrated that the mr has an equal affinity for aldosterone and glucocorticoids , yet in vivo it displays specificity for aldosterone in the face of much higher circulating concentrations of glucocorticoids . this specificity is conferred by 11hsd2 , an enzyme that converts glucocorticoids to inactive 11-ketosteroid derivatives that have weak or no affinity for the mr in mineralocorticoid sensitive tissues including the distal nephron21 ) . loss of function mutation of 11hsd2 or inhibition of 11hsd2 activity allows glucocortiocids to promote renal sodium retention and potassium excretion in the cortical collecting tubule40 , 41 ) . these findings suggest that the 11hsd2 regulates the sodium reabsorption in the aldosterone responsive renal tubules by glucocorticoid - induced activation of the mr . we demonstrate in the present studies that there is downregulation of 11hsd2 abundance in the kidney in decompensated liver cirrhosis rats . this finding is in line with recent studies in compensated liver cirrhosis reported by quattropani et al.42 ) and escher et al.43 ) . they demonstrated that 11 hsd2 activity in the kidney was reduced in liver cirrhosis following bile duct ligation42 , 43 ) . these findings together suggest that reduced expression and activity provides basis for access of glucocorticoids to the mr , thereby promoting increased collecting duct sodium reabsorption despite normal plasma levels of aldosterone in rats with experimental liver cirrhosis . this notion was strongly supported by the increased apical targeting of enac subunits in rats with glycyrrhizic acid - induced inhibition of 11hsd2 activity44 ) . plasma aldosterone levels , at least in humans and rats without liver cirrhosis , have been shown to be reduced in conditions with low expression of 11hsd240 , 41 ) . it is noteworthy that plasma aldosterone levels were not reduced in 6 wk liver cirrhosis rats , despite that the protein abundance of 11hsd2 was significantly decreased . thus , coordinated activation of mr by both glucocorticoid as a consequence of reduced activity of 11hsd2 and by relatively high plasma aldosterone may enhance distal renal tubular sodium reabsorption and potassium loss , and hence play important roles for the pathogenesis of sodium retention and possible ascites formation in cbdl - induced liver cirrhosis . in contrast , in response to liver cirrhosis for 8 weeks enac abundance was decreased ( fig . in addition , the increased apical targeting of enac subunits was retrieved in spite of decreased abundance of 11hsd2 . it can be speculated that 11hsd2 is still functioning but not at full power ( thus aldosterone may play a major role ) , or it may be speculated that differences in enac trafficking between 6 and 8 weeks are independent of aldosterone and/or 11hsd2 . in normal rats both the apical targeting and protein abundance of enac are increased by high dose aldosterone treatment7 ) . however , in the present study we did not observe an increase in enac protein expression in the cortex in kidneys from rats with cbdl - induced liver cirrhosis for 6 weeks although increased abundance of enac was seen in inner stripe of the outer medulla . it may be speculated that glucocorticoids may be involved in this regulation , and if this is the case the observations suggest that regulation of enac abundance is not as sensitive , as is the regulation of changes in the subcellular localization of enac , to mr activation by glucocorticoids . whether the differential regulation of enac subunit targeting and expression may be related to differences in the kinetics of mr - ligand interaction remains unknown ; aldosterone dissociates more slowly from mr than cortisol does , indicating that the stability of the aldosterone - mr complex is higher than that of the cortisol - mr complex45 , 46 ) . in addition , the mr activity can be regulated by at the level of transcription activation or repression by cell specific co - factors47 ) . it is also interesting to note that the rats with liver cirrhosis apparently exhibited dynamic changes in sodium retention and enac trafficking . after 6 weeks , liver cirrhosis animals exhibited sodium retention , potassium loss and decreased urine na / k ratio indicating increased aldosterone - like activity . these findings can be explained in part by decreased abundance of 11hsd2 , which permits the stimulation of mr by glucocorticoid resulting in increased apical targeting of enac subunits . on the other hand , at 8 weeks the rats with liver cirrhosis did not exhibit any changes in urinary sodium and potassium excretion even though they all had marked ascites . the underlying mechanism for the time course changes in circulating aldosterone levels and changes in enac subunits trafficking remains undefined . however , it can be speculated that at later stages of liver cirrhosis the rise in extracellular fluid volume and enhanced secretion of atrial natriuretic peptaide ( anp ) contribute to the decrease in plasma aldosterone levels and , hence , limit enac abundance / trafficking since anp has been known to directly inhibit aldosterone secretion in the adrenal glands through specific anp receptors in the zona glomerulosa48 ) . the results strongly suggests that the marked increase in apical enac subunit targeting combined with diminished abundance of 11hsd2 in the dct2 , connecting tubule and collecting duct is likely to play key roles for the sodium retention associated with cbdl - induced liver cirrhosis in rats in the sodium retaining stage ( i.e. in this model after 6 weeks of cbdl ) . the subsequent decrease in the expression of enac subunits and the reduced targeting after 8 weeks of cbdl - induced liver cirrhosis may contribute to promote sodium excretion at later stages of liver cirrhosis . this pahse may represent active and successful escape from the sodium retention by means of down - regulation of the activity of the enac . increased abundance and/or apical targeting of enac subunits in the connecting tubule and collecting duct play an important role in the pathogenesis of sodium retention in nephrotic syndrome and liver cirrhosis .

nephrotic syndrome and liver cirrhosis are common clinical manifestations , and are associated with avid sodium retention leading to the development of edema and ascites . however , the mechanism for the sodium retention is still incompletely understood and the molecular basis remains undefined . we examined the changes of sodium ( co)transporters and epithelial sodium channels ( enacs ) in the kidneys of experimental nephrotic syndrome and liver cirrhosis . the results demonstrated that puromycin- or hgcl2-induced nephrotic syndrome was associated with 1 ) sodium retention , decreased urinary sodium excretion , development of ascites , and increased plasma aldosterone level ; 2 ) increased apical targeting of enac subunits in connecting tubule and collecting duct segments ; and 3 ) decreased protein abundance of type 2 11-hydroxysteroid dehydrogenase ( 11hsd2 ) . experimental liver cirrhosis was induced in rats by ccl4 treatment or common bile duct ligation . an increased apical targeting of alpha- , beta- , and gamma - enac subunits in connecting tubule , and cortical and medullary collecting duct segments in sodium retaining phase of liver cirhosis but not in escape phase of sodium retention . immunolabeling intensity of 11hsd2 in the connecting tubule and cortical collecting duct was significantly reduced in sodium retaining phase of liver cirrhosis , and this was confirmed by immunoblotting . these observations therefore strongly support the view that the renal sodium retention associated with nephrotic syndrome and liver cirrhosis is caused by increased sodium reabsorption in the aldosterone sensitive distal nephron including the connecting tubule and collecting duct , and increased apical targeting of enac subunits plays a role in the development of sodium retention in nephrotic syndrome and liver cirrhosis .

Nephrotic syndrome Liver cirrhosis 1. CCl 2. Common bile duct ligation (CBDL) induced-biliary liver cirrhosis Conclusions

in vivo micropuncture studies in the unilateral model of pan - induced nephrotic syndrome have shown that sodium reabsorption is specifically increased in the collecting duct and not in the proximal tubule and distal nephron11 ) . thus , this study directly point to a role of increased sodium reabsorption in the collecting duct and , hence , it may be hypothesized that dysregulation of the key sodium channels and transporters in the collecting duct may be responsible for this . a few studies have subsequently demonstrated that sodium retention in pan nephrotic rats is correlated with increased na , k - atpase activity and expression in the cortical collecting duct12 , 13 ) . we demonstrated that pan - induced nephrotic syndrome is associated with 1 ) sodium retention , decreased urinary excretion of sodium , a marked ascites , and increased plasma aldosterone level , 2 ) upregulation of protein abundance of specific enac subunits in cortex , outer medulla and inner medulla , and 3 ) increased apical targeting of enac subunits in connecting tubule and collecting duct segments observed as increased immunolabeling in apical plasma membrane domains14 ) . taken together , these observations therefore strongly support the view that the renal sodium retention associated with pan - induced nephrotic syndrome is caused by increased sodium reabsorption in connecting tubule and collecting duct14 ) . thus , this is likely to represent a key molecular basis for the sodium retention associated with pan - induced nephrotic syndrome combined with the previously demonstrated increase in collecting duct na , katpase activity and protein abundance12 , 13 ) . this together with observed increased expression and increased targeting of enac subunits in combination with the previous functional micropuncture studies strongly support the view that the sodium retention in the nephrotic syndrome may occur in the connecting tubule and collecting duct15 ) . this nephropathy is responsible for the development of high - range proteinuria and a full - blown nephrotic syndrome associated with generalized edema and ascites17 ) . to elucidate whether the changes in expression and plasma membrane targeting of enac subunits in the pan - induced minimal change nephrotic syndrome ( caused by podocyte injury , not immune complex mediated ) is unique to the pan model or may be a more general characteristic of nephrotic syndrome including immune complex mediated glomerulonephritis , and to investigate the physiologic role of changes in subcellular distribution of enac subunits in the abnormal sodium retention and ascites formation during disease states , we examined the changes of enac abundance and/or trafficking and 11hsd2 in hgcl2 induced nephritic syndrome in brown norway rats . the results demonstrated that hgcl2-induced immune complex glomerulonephritis was associated with 1 ) sodium retention , decreased urinary sodium excretion , development of ascites , and increased plasma aldosterone level ; 2 ) increased abundance of enac ( fig . 1 ) 3 ) increased apical targeting of enac subunits in connecting tubule and collecting duct segments ; and 4 ) decreased protein abundance of 11hsd2 ( fig . these observations therefore strongly support the view that the renal sodium retention associated with hgcl2 induced nephrotic syndrome is caused by increased sodium reabsorption in the distal nephron including the connecting tubule and collecting duct . thus , a straightforward interpretation of these observations would be that increased apical targeting of enac subunits plays a role in the development of sodium retention in hgcl2 nephrotic syndrome as well , and it could be argued that this mechanism in fact could be a general characteristic of the nephrotic syndrome including both minimal change disease and immune complex mediated glomerulonephritis . apical targeting of enac subunits and protein abundance of enac can be regulated by aldosterone7,8 ) . however , substantial evidence argue against a major role of aldosterone in nephrotic syndrome : 1 ) ascites and edema occur in rats with nephrosis attributable to rabbit anti - rat kidney serum injection , without elevated aldosterone levels19 ) . and 3 ) sodium retention was observed only in the affected proteinuria kidney in rats unilateral models of pan nephrosis11 ) . it has been suggested that the activity of type 2 11hydroxysteroid dehydrogenase ( 11hsd2 ) can regulate the sodium reabsorption in the aldosterone responsive renal tubules by glucocorticoid induced activation of mineralocorticoid receptor ( mr)21 ) . these findings suggest that downregulation of 11hsd2 provides access of glucocorticoids to the mr , resulting in increased activity of mr and collecting duct sodium retention in rats with nephrotic syndrome . aldosterone is known to increase na , k - atpase activity in the cortical collecting duct and to increase enac redistribution to the apical membrane of connecting tubule and collecting duct principal cells and to promote the expression of a 70 kda form of the gamma enac subunit , thought to be linked to activation of the channel by proteolytic cleavage7 , 8) . moreover levels of vasopressin , ang ii and aldosterone are increased in the initial phase of nephrosis14 , 25 , 26 ) , leading to the hypothesis that they may play significant roles in the development of sodium retention . as the time course and magnitude of sodium retention is indistinguishable in nephrotic brattleboro rats ( vasopressin deficient ) compared to normal nephrotic rats12 ) , vasopressin is not considered to be a major factor for the development of sodium retention in nephrotic syndrome . we have also demonstrated that ang ii at1 receptor blockade , candesartan , did not improve the sodium retention associated with pan - induced nephrotic syndrome nor the time course of its development27 ) . immunocytochemical analyses revealed absence of enhanced apical targeting of enac subunits in pan treated adrenalectomized ( adx - pan ) rats with distribution of labelling similar to adrenalectomized dexamethasone - treated control rats ( adx ) . this indicates that the previously observed enhanced apical targeting of enac in pan induced neprotic syndrome is caused by aldosterone and that development of sodium retention can occur in the absence of aldosterone in nephrotic syndrome . renal sodium and water retention has been shown to be responsible for the development of ascites not only in patients with liver cirrhosis but also in experimental cirrhotic rats . in kidneys of cirrhotic rat , increased reabsorption of sodium and water in the distal nephron and collecting duct has been suggested to be one of the most important renal tubular dysfuctions involved in the pathogenesis of ascites . however , the underlying molecular and cellular mechanisms for the sodium retention are still incompletely understood . in particular , the role of aldosterone in sodium retention and ascites formation in liver cirrhosis is still unclear . previous studies demonstrated that plasma aldosterone levels were usually elevated in liver cirrhosis with ascites33 ) and that spironolactone , a mineralocorticoid receptor antagonist , increased sodium excretion in these patients34 ) . there has been some controversy as to the role of regulation of the renal sodium transporters and channels in the development of edema in animal models of cirrhosis . the results demonstrated that ccl4-induced sodium retaining stage ( group a ) of liver cirrhosis was associated with 1 ) decreased urinary sodium excretion , and increased or maintained plasma aldosterone levels ; 2 ) increased apical targeting of enac subunits in dct2 , cnt and collecting duct segments ; and 3 ) decreased protein abundance of 11hsd2 . in contrast , maintenance stage ( group b ) of liver cirrhosis was associated with no changes in the urinary sodium excretion , trafficking and abundance of enac subunits and the abundance of 11hsd236 ) . the most important finding is the striking increase in targeting of all enac subunits to the apical plasma membrane domain in dct2 , cnt and collecting duct in the sodium retaining stage ( group a ) , but not in the maintenance stage ( group b ) of liver cirrhosis . since an increased targeting of all enac subunits to the apical plasma membrane is associated with increased sodium reabsorption , the present finding of an increased enac targeting in the sodium - retaining stage of liver cirrhosis could contribute significantly to the increased renal tubular sodium reabsorption . these observations therefore strongly support the view that the renal sodium retention in the decompensated liver cirrhosis is mainly caused by an increased sodium reabsorption in distal nephron including the collecting duct37 ) . we demonstrated that the abundance of enac was decreased or unchanged , and the abundance of the 70-kda form of enac was increased while the 85-kda band was markedly decreased in the sodium - retaining stage ( group a ) of cirrhotic rats ( fig . thus , the observed increased apical targeting and altered expression of - and enac subunits in group a could be caused by the stimulation of mineralocorticoid receptor ( mr ) in the aldosterone - responsive epithelium . long - term bile duct ligation in rats is a well known experimental model of liver cirrhosis and portal hypertension , and has been widely used to study the renal mechanisms of sodium retention , as well as systemic and splanchnic hemodynamics38 ) . cbdl rats showed decreased fractional excretion of sodium and hence , positive sodium balance in the sodium retaining stage of liver cirrhosis ( at 6 wks ) . it is suggested that increased renal tubular absorption of sodium may account for the sodium retention and pathogenesis of ascites formation . it should be emphasized that the two time periods are two in a continuum of changes during the development of sodium retention and the stages with partial of full compensation . the most important finding is the increased targeting of all enac subunits to the apical plasma membrane domain in dct2 , cnt and collecting duct in the sodium retaining cirrhotic rats ( fig . the increased apical targeting of enac subunits in cbdl - induced liver cirrhosis may be caused by increased aldosterone activity via stimulation of mineralocorticoid receptor ( mr ) in the aldosterone responsive epithelium . since there was a markedly enhanced apical plasma membrane expression of all enac subunits at 6 wk liver cirrhosis associated with maintained plasma aldosterone levels but not at 8 wk liver cirrhosis where aldosterone levels were markedly reduced , it is likely that aldosterone plays an essential role . these findings suggest that the 11hsd2 regulates the sodium reabsorption in the aldosterone responsive renal tubules by glucocorticoid - induced activation of the mr . they demonstrated that 11 hsd2 activity in the kidney was reduced in liver cirrhosis following bile duct ligation42 , 43 ) . these findings together suggest that reduced expression and activity provides basis for access of glucocorticoids to the mr , thereby promoting increased collecting duct sodium reabsorption despite normal plasma levels of aldosterone in rats with experimental liver cirrhosis . this notion was strongly supported by the increased apical targeting of enac subunits in rats with glycyrrhizic acid - induced inhibition of 11hsd2 activity44 ) . it is noteworthy that plasma aldosterone levels were not reduced in 6 wk liver cirrhosis rats , despite that the protein abundance of 11hsd2 was significantly decreased . thus , coordinated activation of mr by both glucocorticoid as a consequence of reduced activity of 11hsd2 and by relatively high plasma aldosterone may enhance distal renal tubular sodium reabsorption and potassium loss , and hence play important roles for the pathogenesis of sodium retention and possible ascites formation in cbdl - induced liver cirrhosis . in addition , the increased apical targeting of enac subunits was retrieved in spite of decreased abundance of 11hsd2 . in normal rats both the apical targeting and protein abundance of enac however , in the present study we did not observe an increase in enac protein expression in the cortex in kidneys from rats with cbdl - induced liver cirrhosis for 6 weeks although increased abundance of enac was seen in inner stripe of the outer medulla . these findings can be explained in part by decreased abundance of 11hsd2 , which permits the stimulation of mr by glucocorticoid resulting in increased apical targeting of enac subunits . however , it can be speculated that at later stages of liver cirrhosis the rise in extracellular fluid volume and enhanced secretion of atrial natriuretic peptaide ( anp ) contribute to the decrease in plasma aldosterone levels and , hence , limit enac abundance / trafficking since anp has been known to directly inhibit aldosterone secretion in the adrenal glands through specific anp receptors in the zona glomerulosa48 ) . the results strongly suggests that the marked increase in apical enac subunit targeting combined with diminished abundance of 11hsd2 in the dct2 , connecting tubule and collecting duct is likely to play key roles for the sodium retention associated with cbdl - induced liver cirrhosis in rats in the sodium retaining stage ( i.e. the subsequent decrease in the expression of enac subunits and the reduced targeting after 8 weeks of cbdl - induced liver cirrhosis may contribute to promote sodium excretion at later stages of liver cirrhosis . the results demonstrated that ccl4-induced sodium retaining stage ( group a ) of liver cirrhosis was associated with 1 ) decreased urinary sodium excretion , and increased or maintained plasma aldosterone levels ; 2 ) increased apical targeting of enac subunits in dct2 , cnt and collecting duct segments ; and 3 ) decreased protein abundance of 11hsd2 . in contrast , maintenance stage ( group b ) of liver cirrhosis was associated with no changes in the urinary sodium excretion , trafficking and abundance of enac subunits and the abundance of 11hsd236 ) . the most important finding is the striking increase in targeting of all enac subunits to the apical plasma membrane domain in dct2 , cnt and collecting duct in the sodium retaining stage ( group a ) , but not in the maintenance stage ( group b ) of liver cirrhosis . since an increased targeting of all enac subunits to the apical plasma membrane is associated with increased sodium reabsorption , the present finding of an increased enac targeting in the sodium - retaining stage of liver cirrhosis could contribute significantly to the increased renal tubular sodium reabsorption . these observations therefore strongly support the view that the renal sodium retention in the decompensated liver cirrhosis is mainly caused by an increased sodium reabsorption in distal nephron including the collecting duct37 ) . we demonstrated that the abundance of enac was decreased or unchanged , and the abundance of the 70-kda form of enac was increased while the 85-kda band was markedly decreased in the sodium - retaining stage ( group a ) of cirrhotic rats ( fig . thus , the observed increased apical targeting and altered expression of - and enac subunits in group a could be caused by the stimulation of mineralocorticoid receptor ( mr ) in the aldosterone - responsive epithelium . long - term bile duct ligation in rats is a well known experimental model of liver cirrhosis and portal hypertension , and has been widely used to study the renal mechanisms of sodium retention , as well as systemic and splanchnic hemodynamics38 ) . cbdl rats showed decreased fractional excretion of sodium and hence , positive sodium balance in the sodium retaining stage of liver cirrhosis ( at 6 wks ) . it should be emphasized that the two time periods are two in a continuum of changes during the development of sodium retention and the stages with partial of full compensation . the most important finding is the increased targeting of all enac subunits to the apical plasma membrane domain in dct2 , cnt and collecting duct in the sodium retaining cirrhotic rats ( fig . the increased apical targeting of enac subunits in cbdl - induced liver cirrhosis may be caused by increased aldosterone activity via stimulation of mineralocorticoid receptor ( mr ) in the aldosterone responsive epithelium . these findings suggest that the 11hsd2 regulates the sodium reabsorption in the aldosterone responsive renal tubules by glucocorticoid - induced activation of the mr . they demonstrated that 11 hsd2 activity in the kidney was reduced in liver cirrhosis following bile duct ligation42 , 43 ) . these findings together suggest that reduced expression and activity provides basis for access of glucocorticoids to the mr , thereby promoting increased collecting duct sodium reabsorption despite normal plasma levels of aldosterone in rats with experimental liver cirrhosis . this notion was strongly supported by the increased apical targeting of enac subunits in rats with glycyrrhizic acid - induced inhibition of 11hsd2 activity44 ) . it is noteworthy that plasma aldosterone levels were not reduced in 6 wk liver cirrhosis rats , despite that the protein abundance of 11hsd2 was significantly decreased . thus , coordinated activation of mr by both glucocorticoid as a consequence of reduced activity of 11hsd2 and by relatively high plasma aldosterone may enhance distal renal tubular sodium reabsorption and potassium loss , and hence play important roles for the pathogenesis of sodium retention and possible ascites formation in cbdl - induced liver cirrhosis . in addition , the increased apical targeting of enac subunits was retrieved in spite of decreased abundance of 11hsd2 . however , in the present study we did not observe an increase in enac protein expression in the cortex in kidneys from rats with cbdl - induced liver cirrhosis for 6 weeks although increased abundance of enac was seen in inner stripe of the outer medulla . these findings can be explained in part by decreased abundance of 11hsd2 , which permits the stimulation of mr by glucocorticoid resulting in increased apical targeting of enac subunits . however , it can be speculated that at later stages of liver cirrhosis the rise in extracellular fluid volume and enhanced secretion of atrial natriuretic peptaide ( anp ) contribute to the decrease in plasma aldosterone levels and , hence , limit enac abundance / trafficking since anp has been known to directly inhibit aldosterone secretion in the adrenal glands through specific anp receptors in the zona glomerulosa48 ) . the results strongly suggests that the marked increase in apical enac subunit targeting combined with diminished abundance of 11hsd2 in the dct2 , connecting tubule and collecting duct is likely to play key roles for the sodium retention associated with cbdl - induced liver cirrhosis in rats in the sodium retaining stage ( i.e. the subsequent decrease in the expression of enac subunits and the reduced targeting after 8 weeks of cbdl - induced liver cirrhosis may contribute to promote sodium excretion at later stages of liver cirrhosis . increased abundance and/or apical targeting of enac subunits in the connecting tubule and collecting duct play an important role in the pathogenesis of sodium retention in nephrotic syndrome and liver cirrhosis .

produced by various processes ( i.e. , cold vapor deposition , laser ablation , and arc discharge surface mediated growth ) , carbon nanotubes ( cnts ) contain metal impurities ( e.g. , fe , co , mo , ni , and y ) [ 15 ] which are introduced in the manufacturing process as catalysts for carbon structure catalytic growth or as contaminants [ 2 , 46 ] . it has been observed that even after rigorous purification those embedded impurities still persist in the cnt graphitic structure [ 2 , 3 , 69 ] . for example , an icp - ms study of commercially important cnts showed that impurity concentrations in three multiwall cnts ( mwcnts ) were between 4738 and 51867 g / g for ni and in two commercially relevant single - wall cnts ( swcnts ) between 1798 and 4217 g / g for co and 14721672 g / g mo . the currently accepted method for determination of cnts in workplace air is thermooptical analysis ( niosh method # 5040 ) for determination of elemental carbon . a drawback of this approach is that it does not distinguish between cnt and non - cnt sources of elemental carbon such as diesel exhaust [ 11 , 12 ] . the detection of cnt - bound metal impurities in environmental samples provides a means to distinguish cnt emissions from non - cnt background sources of elemental carbon [ 1215 ] . worker exposure to cnts can occur at any stage of the manufacturing process and during transport , storage , and handling of cnts , with inhalation being the major route of exposure [ 11 , 16 ] . different sampling methods have been suggested as possible strategies to collect samples for workplace monitoring of metal impurities in cnts such as filter cassettes , electrostatic precipitation , and surface wipes [ 13 , 15 ] . wipe sampling has emerged as a useful and cost - effective qualitative technique suitable for representative mapping of spatial variations of surface metal contamination within a workplace . the method provides an additional tool to detect workplace releases of cnts ( and potential exposures ) , evaluate housekeeping or clean - up procedures , and monitor the effectiveness of engineering controls . niosh and oecd provided guidance on collecting surface wipe samples in nanotechnology applications using premoistened ghostwipes towelettes , but neither included an analytical methodology for determining cnts in wipe samples . wipe sampling for cnt metal tracers is considered a qualitative indicator of the presence of cnts , not a quantitative exposure metric . as described by the oecd tiered approach , such qualitative assessments are useful for comparing particle concentrations at the emission source with background particle concentrations . even though wipe sampling may be considered qualitative , an analytical method is needed that includes an efficient digestion step and adequate detection limits to ensure reliable and reproducible metal determinations . the most commonly used analytical methods for determining metal impurities in cnts are neutron activation analysis ( naa ) [ 1 , 3 , 2022 ] , direct solid sampling electrothermal atomic absorption spectrometry ( dss - et aas ) , high resolution continuum source graphite furnace atomic absorption spectrometry ( hr cs gfaas ) , inductively coupled plasma optical emission spectrometry ( icp - oes ) [ 1 , 4 , 20 , 21 , 23 , 24 ] , and inductively coupled plasma mass spectrometry ( icp - ms ) [ 1 , 3 , 4 , 7 , 20 , 21 ] . out of all of these detection methods , icp - ms is a suitably sensitive approach for determination of particle - bound metals on wipe substrates , but an appropriate extraction method is needed for cnt - bound metals . the key challenge is the difficulty in quantitatively extracting cnt metal impurities during the digestion step [ 3 , 4 , 7 , 22 , 26 ] . a variety of sample pretreatments have been employed for determination of metals in cnt samples including wet digestion [ 1 , 3 , 23 ] , dry ashing combined with acid digestion [ 1 , 3 , 4 ] , carius tube digestion , microwave - assisted acid digestion [ 1 , 3 , 4 , 20 , 26 ] , and microwave - induced combustion [ 4 , 21 , 24 ] . grinberg and coworkers reported incomplete recovery of metals from cnts with the single - step microwave digestion ( hno3 and h2o2 ) recommended by decker et al . and proposed a multistep microwave - assisted procedure that involved the repeated use of strong acids as follows : hno3 combined with h2o2 ( 1x ) , hno3 alone ( 3x ) , evaporation near dryness , hno3 and hclo4 followed by hno3 addition and evaporation to dryness ( 4x ) , and finally dissolution and dilution in 1% hno3 . grinberg et al . found that this lengthy and aggressive procedure was necessary to ensure complete sample digestion and efficient recovery of residual catalysts ( co and ni ) and trace metal impurity content ( fe , mo , pb , and hg ) from the nrc swcnt-1 reference material investigated . iso / iec 13278 also recommended repeating the heating cycles with new reagent addition several times ( i.e. , three to six heating cycles depending on cnt structure ) when using microwave digestion to measure cnt metal impurities . yang et al . emphasized the importance of the digestion step as they observed positive nonspectral interferences from undigested carbon residues remaining in the sample solution which they identified by comparing icp - ms and icp - oes results for the same swcnt digest . the purpose of this study is to identify an appropriate digestion approach for icp - ms determination of metal impurities in cnts collected using wipe samples . two approaches are evaluated with respect to the efficiency of the extractions : a microwave - assisted digestion method and an ultrasonic digestion method . although for this application 100% extraction efficiency is not required , the goal is to achieve reproducible and reliable recoveries that will enable the identification of cnt surface contamination . different cnt materials , including nist 2483 soot ( single - wall cnt ) , are digested with and without ghostwipes , as the digestion approach must be able to accommodate wipe samples which are quite bulky . the possibility that wipes may contain background contamination which can interfere with determination of the metals of interest is also considered . three swcnt products were obtained for this study : nist 2483 swcnt standard reference material ( raw soot ; national institute of standards and technology , gaithersburg , md , usa ) and two other swcnts test materials , aldrich - cnt ( sigma - aldrich co. , gillingham , uk ) and a research test sample provided by national research council ( test - cnt ) . the nist 2483-cnt is uniform and well - characterized carbon nanotube soot approx . 0.8 nm in diameter ( by tem ) produced by a chemical vapor deposition variant process . the crm was weighed straight from the bottle , as the certificate of analysis indicates that the crm does not require preparation prior to weighing . the aldrich - cnt test material was manufactured by arc discharge , with 4060 wt% carbon content and 3035% metal content . this cnt has average diameter of 1.7 0.2 nm , wall width of 0.4 0.1 nm , and bundled dimensions of 5 to 10 nm in diameter and 0.32 to 4.7 m in length . the test - cnt is a swcnt with an average diameter and wall width of 1.6 0.2 nm and 0.4 0.2 nm , respectively , narrower bundles ( diameter < 3 nm ) , and length between 0.07 and 5.2 m ( high proportion < 1 m in length ) . the test - cnt test material showed a higher proportion of amorphous material ( relative to nanotubes ) than that of aldrich - cnt . detailed characterization of test - cnt and aldrich - cnt test materials using sem and tem is provided in rasmussen et al . . nist 1633b srm ( national institute of standards and technology , gaithersburg , md , usa ) is a bituminous coal fly ash sieved through a nominal sieve opening of 90 m ( 170 mesh ) used for matrix comparisons and accuracy check . high - purity nitric and hydrofluoric acids ( seastar chemicals inc . , sidney , bc , canada ) and suprapur 30% aqueous solution of hydrogen peroxide were used for sample pretreatment . ultrapure milli - q water ( 18.2 m cm ) was used for preparation of samples and calibration standards . high - purity multielement ( mes-1107 - 01 solutions a and b , 100 g / ml ) and individual standard stock solutions ( la and gd , 1000 g / ml ) were used to prepare the calibration standards ( delta scientific laboratory products ltd . , gw procedural blanks and sample ( nist 2483 ) replicates were spiked at levels varying between 4 g / l and 250 g / l , respectively , with icp - mscs high - purity standard solution ( 10 g / ml ) . individual high - purity standards solutions of ge , in , and re ( 1000 mg / l ) were used to prepare the internal standard solution . certified reference low and high level fortified waters for trace elements materials , tm-28.4 and tmda 54.5 , respectively , were used for quality control and were purchased from environment canada ( ottawa , on ) . ghostwipes ( gw ; environmental express , charleston , south carolina , sc 4250 , premoistened with deionized water , individually sealed packets ) were used in combination with nist 2483 swcnt and nist 1633b stms to assess the effect of gw on extraction efficiency . the present study used a 3.5 cm diameter circular disk of ghostwipes material as required by the wipe sampling device used for monitoring cnt releases . composed of polyvinyl alcohol polymer , ghostwipes dissolve completely during the analytical procedure allowing the sample material / analytes to disperse in the digestion media ( http://www.envexp.com/ ) . scp science digifilters 0.45 m ( teflon membrane ) for 50 ml tubes were used for filtration of diluted extracts . mettler toledo xp205 digital analytical balance equipped with a u - shaped antistatic electrode was used to achieve accurate weighing results for the aliquots ( 2 mg ) of tested cnts used in this study . as recommended [ 1 , 11 ] , appropriate personal protective equipment was worn and care was taken to avoid inadvertent generation of aerosols when handling the cnt test materials . an ethos touch control advanced microwave labstation ( milestone microwave laboratory systems ) equipped with ethos tc built - in atc-400-ce automatic temperature control was used for all microwave digestions . digiprep ms heating block ( scp science , qc , canada ) and branson ultrasonic bath ( model b8510dth ) were used in this study . for quantification of cnt metal impurities in digested samples , a nexion 300s dual - channel universal cell icp - ms system ( perkin elmer , canada ) coupled with a sc - fast autosampler ( elemental scientific , omaha , ne ) was operated in the standard and collision mode . the instrument was equipped with a high temperature apex - st pfa microflow nebulizer , cyclonic spray chamber with a pc3x chiller ( 2c ) , and triple cone interface ( nickel - platinum skimmer and sampler cone , and aluminum hypercone ) . the following conditions were used : plasma and auxiliary argon flow rates were 18 and 1.2 l / min , respectively . the nebulizer argon gas flow rate was 1.00 ml / min for the high temperature apex - st pfa microflow concentric nebulizer and the forward rf power was 1600 w. optimization was carried out daily with a normal tuning solution . the appropriate internal standards ( ge , in , and re ) and dilution factors were selected based on a preliminary semiquantitative analysis . perkin elmer optima 5300v icp - oes equipped with radial optical system ( 163 to 782 nm range ) was used at the wavelengths recommended by manufacturer for the elements of interest . the instrument was operated at 1400 w power and flow rates of 15 l / min , 0.2 l / min , 0.8 l / min , and 1.00 ml / min for plasma , auxiliary , nebulizer , and peristaltic pump , respectively . two digestion methods were evaluated during this study to compare their efficiency in extracting elemental impurities from cnts : a microwave digestion ( md ) method using nitric acid and hydrogen peroxide and an ultrasonic digestion ( ud ) method using nitric and hydrofluoric acids . for both digestion methods three to five aliquots of 2 mg of test material were used for analysis ( to mimic the particle mass on a surface wipe sample ) . spiked samples ( 20 and 250 ppb level ) and spiked procedural blanks ( 4 and 25 ppm level ) were analyzed along with the test samples . finally , nist 2483 swcnt and nist 1633b coal fly ash were combined with ghostwipes ( gw ) in order to compare the efficiency of investigated digestion methods in the presence of gw . recoveries were calculated for each method and their advantages and disadvantages were considered . the microwave digestion ( md ) method was preceded by a digiprep sample preparation / pretreatment step . during the digiprep step , the samples were placed in 50 ml teflon digitube vessels and were treated with diluted nitric acid ( 1 : 1 , 12 ml ) and allowed to sit at room temperature for 3045 min . this step allowed the gentle and complete dissolution of gw ; otherwise the generation of effervescence during heating may affect the sample and tube integrity . after effervescence subsided , the samples were treated successively at reflux ( 85c on digiprep ms heating block ) with diluted nitric ( 12 ml , 90 min ) , concentrated nitric acid ( 10 ml , 30 min reflux ) , and 30% hydrogen peroxide ( 5 ml , 45 min ) , and evaporated to 10 ml ( 90c ) . the resultant acid - peroxide digestion solutions were then transferred quantitatively to precleaned high - pressure microwave digestion vessels ( 100 ml teflon ) and 0.5 ml 30% hydrogen peroxide . the microwave digestion step was performed at 1000 w power in the following conditions : 20 min to reach 180c temperature , 10 min to increase from 180c to 220c , and 20 min at 220c . the digested samples were evaporated to nearly dryness ( 90c ) and the resultant residues were dissolved in 1% nitric acid , vortexed , and filtered through a 0.45 m filter to remove the possible undissolved residuum ( i.e. , undissolved cnts ) that may impact the icp - ms analysis . ( based on astm method e 1644 ) to accommodate the bulk of gw samples . preliminary work showed that the digiprep method alone did not yield adequate recoveries for cnts in wipes and therefore it was necessary to add the microwave digestion step . the samples were placed in 15 ml digestion tubes with 4 ml nitric acid , 0.1 ml hydrofluoric acid , and 1.9 ml ultrapure water and allowed to stand at room temperature until gw dissolved completely and effervescence subsided ( 3045 min ) before proceeding to the next step . this step is most critical in the presence of gw because even with a small piece of gw ( 3.5 cm disc ) an exothermic reaction develops during the hot water bath stage . the capped digestion tubes were placed in the ultrasonic water bath ( 180 w , 69c ) for a total of 2 h ( 2 1 h ultrasonication with 1 min vortex in between ) . the solutions were then cooled down , diluted to 10 ml using ultrapure water , and centrifuged ( 3500 rpm , 10 min ) . a final filtration step ( 0.45 m ) was added to remove the possible undissolved residuum ( i.e. , undissolved cnts ) that may impact the icp - ms analysis . all sample digests were analyzed by icp - ms after appropriate dilution with 1% hno3 . the hno3-hf ud method is based on a method originally designed for ptfe filter - based aerosol samples by niu et al . . all sample digests were stored in the fridge in precleaned polyethylene screw - capped tubes and analyzed by icp - ms after appropriate dilution . the final digestion solutions were first screened for the presence of elements using icp - ms and isotopes such as b , al , ti , v , cr , mn , fe , co , ni , cu , as , se , sr , zn , y , mo , ba , la , gd , pb , and u were monitored . internal standard solutions of 25 ug / l ge , in , and re were used throughout the experiment for analytes in the mass ranges 87 , 95138 , and 207238 , respectively . analytical errors up to 10% for quality check standards were considered acceptable ( epa method 200.8 ) . spike recoveries of the study elements ( b , al , v , mn , co , ni , cu , mo , as , ba , la , and u ) were between 82% and 115% with md method ( except b 74% and cu 47% ; al spike not recovered ) and 86107% with ud method ( except mo 122% ) in the presence of gw ( spiked procedural blanks and nist 2483 sample ) . aluminum , boron , zinc , and copper spike recoveries were affected by the presence of gw that contain variable amounts of contaminant elements introduced during their manufacturing and/or packaging . therefore , five matrix blanks containing gw were included with each analytical batch and used for matrix blank corrections when samples were combined with gw . the lods were calculated according to the 1994 us - epa guideline for inductively coupled plasma mass spectrometry using 3 times standard deviation of 8 - 9 procedural blanks . student 's t - test and mann - whitney rank sum test were used for two sample comparisons . extraction efficiencies of two different methods , microwave digestion ( md ) and ultrasonic digestion ( ud ) , are compared for cnt impurities present at high concentrations ( > 0.5% ) in table 1 and for cnt impurities present in lower concentrations ( ppm range ) in table 2 without gw being present . nist 2483-cnt standard reference material ( srm ) , test - cnt , and aldrich - cnt were used to compare the extraction efficiency for the residual catalysts ( > 0.5% ) . nist 2483-cnt srm was used to compare the extraction efficiency for other trace metal / element impurities ( ppm range ) using the certified , informational , or reference values provided in the certificate of analysis . the catalyst residues in nist 2483-cnt are co and mo with certified mass fraction values of 9630 170 g / g and 34060 290 g / g , respectively , determined by instrumental neutron activation analysis ( inaa ) and cold neutron prompt gamma activation analysis ( pgaa ) . certified values were given also for ba , la , and gd present in smaller quantities in nist 2483-cnt , reference values for al , mn , and v , and informational values for the other metals ( b , as , and cu ) as shown in tables 2 and 3 . consequently , the efficiencies of the two investigated methods are compared using aliquots of nist 2483-cnt combined with gw ( table 3 ) . table 1 presents the high metal concentrations ( > 0.5% ) in nist 2483 , test - cnt , and aldrich - cnts obtained with both microwave and ultrasonic digestion methods . the results are presented as mean ( g / g ) and standard deviation of five independent determinations . as shown in table 1 for all investigated cnts , the md method had improved extraction efficiency for the high concentration metals ( co , ni , y , and mo ) when compared to the ud method . except for ni in test - cnt for which t - test indicated no significant difference ( p = 0.128 ) , for all other residual catalysts metals ( co , ni , y , and mo ) in all three cnts the difference between md and ud results was significant ( p < 0.05 ) . the greatest difference was observed for the nist 2483-cnt ( table 1 ) for which 25 times lower mo and co concentrations were extracted with ud method than with md method resulting in recoveries of 74 2.0% co and 82 2.5% mo with md method and just 16 1.7% co and 39 2.0% mo with ud method . although for test - cnt ( except for ni ) and aldrich - cnts the metal concentrations extracted with md method were significantly higher ( p < 0.05 ) than those obtained with ud method , the ranges were similar ( i.e. , test - cnt co : 17487 g / g md versus 14358 g / g ud ) when compared with results of nist 2483-cnt ( i.e. , co : 7100 g / g md versus 1521 g / g ud ) . overall , the results obtained with the ud method varied largely with the cnt analyzed , indicating that morphology and/or the way the residual catalyst metals were incorporated in the graphitic structure could influence the success of the sample pretreatment and consequently the recovery . this might be related to the manufacturing processes since the cnts are produced by various processes , that is , cold vapor deposition , laser ablation , and arc discharge surface mediated growth [ 3 , 4 ] , which influence the amounts and how strongly the residual catalyst metals are incorporated in the graphitic structure [ 7 , 22 ] . even though in case of ultrasonic digestion method the recoveries vary with the cnt investigated , this proves to be a quick and useful method for qualitative identification of high concentration metallic impurities in cnts . therefore , when comparing the two investigated methods , the md method appears to be the method of choice for quantification of high concentration metals from tested cnts due to improved extraction ( table 1 ) yielding high recovery of residual catalysts . the use of alternating oxidation and reflux steps with strong acids enhances the dissolution and extraction of metallic impurities from the cnt graphitic structure [ 3034 ] . 4.5 h for alternating oxidation and reflux steps plus evaporation and 4 h for microwave digestion and evaporation / filtration ) . in contrast , the ud method has a shorter sample - reagent contact time ( 2 h ultrasonication ) and does not include alternating oxidation and reflux steps . although the md method takes more time than the ud method ( and uses more reagents ) , the result is an increased extraction efficiency for high concentration metallic impurities in cnts . beside the catalyst residues present in high concentrations ( up to 3% ) , lower concentrations of other metals are present in cnts as a result of production procedures , postfabrication , and postpurification treatments . table 2 presents results obtained for lower concentration metal(loids)s in nist 2483-cnt with both md and ud methods . generally both methods extracted similar amounts for most of the lower concentration ( ppm range ) metal(loids)s investigated . even though slightly increased concentrations of al , v , mn , and ba were extracted with the ud method and of cu and la with the md method , in contrast , the md method extracted significantly higher ( p < 0.001 ) amounts of as , but significantly lower ( p < 0.05 ) amounts of b , gd , and u ( table 2 ) . figure 1 shows the percent recoveries obtained for high ( > 0.5% ) and low concentration ( ppm range ) metals in nist 2483-cnt srm obtained with ( a ) md and ( b ) ud methods with or without ghostwipes ( gw ) . percent recoveries were calculated using the certified / information / reference values available in the certificate of analysis for the investigated elements . generally the recoveries for low concentration metals ( i.e. , al , v , cu , ba , la , and gd ) ranged between 75 and 100% with md and 76 and 104% with ud . both methods resulted in low recoveries of b ( 43% md and 62% ud ) and mn ( 35% md and 51% ud ) , whereas ud gave also lower recoveries for as ( 64% ud versus 100% md ) . with similar or increased recoveries ( i.e. , 85% al , 83% v , and 84% la ) for the low concentration ( ppm range ) metals investigated ( except as ) in nist 2483-cnt , the ud method is a short and practical method that may be used to detect and measure metals present in lower amount in cnts . moreover , since nist 2483-cnt is not certified for some elements of interest ( i.e. , ni ) and other metals present in higher concentrations were not well recovered with the ultrasonic digestion method ( e.g. , 16% co ) , another srm with carbonaceous matrix ( as recommended by decker et al . ) , nist 1633b , was analyzed using ud to assess the ability of the method to extract other metals , that is , ni and co when present in lower concentrations . this srm was recommended previously for quality assurance in the absence of specific reference materials for cnt analysis . it should be noted that elements in nist 1633b are generally in lower concentration range ( i.e. , ni : 120.6 1.8 g / g ; co : 50 g / g ) with the exception of al and fe ( 150.5 mg / g al and 77.8 mg / g fe ) . the certified values along with the results obtained for nist 1633b srm with ud method are presented in table s1 ( see table s1 in the supplementary material available online at http://dx.doi.org/10.1155/2016/3834292 ) . recoveries of 7891% were obtained with the ud method for most elements in nist 1633b ( with the exception of as , zn , and gd ; table s1 ) which were better than recoveries of 2969% achieved for the same srm using us - epa method 3051 . even though the ud method showed excellent recoveries for low concentration metals in nist 2483-cnt ( i.e. , al , v , cu , ba , la , and gd ) , recoveries were poor for the catalyst impurities ( i.e. , 16% co and 39% mo , resp . ) . moreover , the extraction efficiency of the ud method for the residual catalysts / high concentration metals varied with the cnts tested as shown in table 1 with better extraction efficiency for test - cnt and aldrich - cnt than for nist 2483-cnt . compared to the md method , the ud method results varied largely with the cnt analyzed , indicating that morphology and/or the way the residual catalyst metals were incorporated in the graphitic structure could influence the success of the sample pretreatment and consequently the recovery . this variability might be related to the manufacturing processes since the cnts are produced by various processes , that is , cold vapor deposition , laser ablation , and arc discharge surface mediated growth [ 3 , 4 ] , which influence the amounts and how strongly the residual catalyst metals are incorporated in the graphitic structure [ 7 , 22 ] . based on previous concerns about signal enhancement caused by nonspectral interferences [ 20 , 36 ] , the potential for signal enhancement was investigated for the md method . this was done by analyzing the digests of all three tested cnts ( nist-2483 , nrc , and aldrich ) with both icp - oes and icp - ms techniques and comparing the results ( figure 2 ) . the elements measured were co , ni , y , mo , and fe ( 5 replicates of each ) . t - test results showed that the co , ni , y , and mo results obtained with icp - ms were not significantly higher ( p > 0.05 ) than those obtained with icp - oes for all of the three cnts tested , except for ni in aldrich - cnt ( p = 0.031 ) . figure 2(a ) presents the correlation of metal concentrations measured by icp - ms and icp - oes using all seven pairs of metal - concentration values from all cnts tested . the strong linear regression obtained ( slope = 0.9574 , r = 0.99992 , n = 7 ) indicates consistency between icp - ms and icp - oes results . also , different dilutions ( 10x and 100x ) of two spiked ( 200 ppb ) aldrich - cnt solutions obtained with md method were analyzed ( standard and/or collision mode ) and the results compared for co , ni , y , and mo ( figure 2(b ) ) . the results ( slope = 1.0002 , r = 0.999998 ) presented in figure 2(b ) showed no differences between the icp - ms results of the two dilutions . in summary , signal enhancement due to residual carbon does not occur with the md / icp - ms method and does not explain the higher recoveries observed with the md versus the ud method . wipe sampling is commonly used in occupational safety and health applications . among other sampling methods ( e.g. , filter cassettes , electrostatic precipitation ) surface wipes have been suggested as a strategy to collect samples ( including cnt ) for workplace monitoring [ 17 , 3739 ] . ghostwipes ( gw ) meet the us regulatory standard for sampling pb in house dust and were used for quantification of additional metal(loid)s ( i.e. , as , cd , cr , cu , and ni ) to provide information on typical background loadings for these metals in urban canadian homes . although gw have the advantage of dissolving completely during acid digestion , their presence in the sample matrix adds to the analytical challenges encountered in the quantification of cnt metal bound impurities , since wipes are bulky and may contain impurities . the md method includes the digiprep sample pretreatment step that controls the dissolution of the bulky gw matrix ( especially useful where the study design calls for one or two 15 15 cm gw per digestion tube ) . the present study was designed for 3.5 cm discs of gw matrix , which permitted the use of smaller test tubes ( 15 ml ) required by the ultrasonic method . the efficiencies of the two investigated methods were compared using aliquots of nist 2483-cnt combined with gw ( table 3 ) . nist 1633b srm was also analyzed with both methods with / without gw ( table s1 , supplementary material ) . results obtained in the presence of gw ( table 3 ) are significantly lower ( p < 0.05 ) for co and mo in nist 2483-cnt with the ud method than with the md method , similar to results observed without gw ( table 1 ) . regarding lower concentration ( ppm range ) elements , the md method extracted similar concentrations ( p > 0.05 ) for most elements whether gw were present or not ( except for b and as , p < 0.05 ) . in contrast , with the ud method recoveries were significantly lower ( p < 0.05 ) for almost all elements in the presence of gw than without gw ( except b , al , as , and gd ; p > 0.05 ) suggesting that the ud method is more sensitive to interferences from gw . problems are caused by high gw blanks for certain elements ( al , ti , ni , mn , zn , and fe ) , which result in nondetects after blank correction , particularly when present in cnts at lower concentrations . this is consistent with a previous study which concluded that gw blanks were so elevated for zn that residential zn loadings could not be reported . al , ti , and ni were recovered only with the ud method , and mn , fe , and zn were not recovered with either of the two methods ( table 3 ) . high blank values with poor recovery and/or nondetects after blank correction were also observed for certain elements ( e.g. , al , ti , zn , fe , and mn ) in our preliminary work with nist 2483-cnt combined with whole gw caused by uneven distribution of background contamination of these elements in the wipes used . ( however , high blanks for certain elements would not necessarily be problematic if those particular metals are not target elements . ) other factors may include interferences from organic carbon derived from gws or uncertainty arising from the small sample mass used in this study ( 2 mg versus 2540 mg recommended in the certificate of analysis ) . figure 1 shows the same information as tables 2 and 3 , expressed as percentage recoveries ( observed / certified value ) for certified elements in nist 2483 . combining the results into these histograms clarifies the overall trends : the md method ( a ) yields acceptable recoveries for most elements with or without the presence of gw , but the ud method ( b ) yields poor recoveries for the catalyst elements and is generally more sensitive to the presence of gw . the ud method does result in acceptable recoveries ( 7598% ) for certain elements ( v , la , and gd ) with or without the presence of gw . despite generally lower recoveries , the cost - effective ud method may be considered acceptable for semiquantitative purposes such as identifying cnt releases in the workplace environment using wipe samples . different responses of the cnts tested in the present study indicate that the cnt matrix may influence the success of the sample pretreatment and consequently the recovery . thus , different matrices are likely to respond differently to a given digestion method , as observed for the ud method which gave excellent results for co in nist 1633b ( table s1 , supplementary material ) but poor recovery for co in nist 2483 ( table 1 ) . poor recoveries for the ud method ( figure 1(b ) ) are not likely due to readsorption of the extracted metals on residual cnts , due to the strong acid digestion ( ph < 2 throughout ) . chen et al . previously showed that the adsorption of metals on the surface of swcnts at ph less than 2 was negligible . wipe sampling for cnt metal tracers has emerged as a useful and cost - effective qualitative technique suitable for representative identification of cnt surface contamination in the workplace ( and potential exposures ) and routine monitoring of control measures . even though wipe sampling may be considered qualitative , an analytical method is needed that includes an efficient digestion step and adequate detection limits to ensure reliable and reproducible metal determinations . for the purpose of wipe sample analysis , the microwave digestion ( md ) method yielded acceptable recoveries for catalyst elements at high concentrations ( table 1 ) as well as for elemental impurities at low concentrations ( tables 2 and 3 ) in the tested cnt reference materials . the md method involved prolonged contact time between the reagents and acid , with alternating oxidation and acid reflux steps , which permitted the elemental impurities to be efficiently extracted from the cnts . moreover , no signal enhancement due to residual carbon compounds was observed when icp - ms was used in conjunction with the md method . performance of the md extraction was not affected by the presence of ghostwipes ( gw ) in the digest matrix , with the caveat that elevated and variable gw blanks for certain elements ( such as al and zn ) must be considered . an advantage of the md method is that it eliminates the use of hf , which is avoided in many labs due to safety concerns . it is concluded that the md method is appropriate for quantification of metallic tracers in wipe samples used to identify the presence of cnt in work environments . in contrast , the ultrasonic digestion method showed incomplete extraction of catalyst metals present at high concentrations ( table 1 ) , but near - total extraction efficiencies for elemental impurities present at low concentrations ( tables 2 and 3 ) in the tested cnts . however , for nist 1633b reference material , the ud method showed equivalent or better recoveries than those reported for us - epa method 3051 , and since the ud method involves fewer steps than the md method , it may serve as a cost - effective semiquantitative approach for screening purposes . for example , the ud method was useful for identifying the presence of cnt metal impurities ( surface wipe samples ) in a preliminary mapping survey of cnt releases in a manufacturing plant . the ud method appeared to be more sensitive to interferences from gw than the md method . also , compared to the md method , the extraction efficiency of the ud method varied to a greater degree with different carbonaceous matrices ( various cnts and coal fly ash ) . nonetheless , cnt extraction efficiency is a parameter that can be evaluated using certified reference materials for cnts , unlike other sources of laboratory and sampling uncertainty which are not as easily constrained ( e.g. , weighing errors , spatial and temporal variability ) .

residual metal impurities in carbon nanotubes ( cnts ) provide a means to distinguish cnt from non - cnt sources of elemental carbon in environmental samples . a practical and cost - effective analytical approach is needed to support routine surface monitoring of cnt metal tracers using wipe sampling . wipe sampling for cnt metal tracers is considered a qualitative indicator of the presence of cnts , not a quantitative exposure metric . in this study , two digestion approaches ( microwave - assisted nitric acid / h2o2 digestion and ultrasonic nitric / hf acid digestion ) in conjunction with inductively coupled plasma mass spectrometry ( icp - ms ) determination were evaluated for their ability to extract metal impurities from cnt particles captured on wipe substrates . aliquots of different carbon nanotubes ( including nist 2483 single - wall cnt ) with and without ghostwipes ( astm e-1792 compliant ) were used to compare the performance of the digestion methods . the microwave digestion method accommodated the bulky wipe sample and also eliminated potential icp - ms signal interferences related to incomplete digestion . although quantitative recoveries requiring lengthy multistep digestion protocols may be necessary in other applications , the near - total recoveries achieved in the present study for cnt catalyst elements were adequate for identifying surface contamination of cnts in the workplace using wipe sampling .

1. Introduction 2. Experimental 3. Results and Discussion 4. Conclusion

, cold vapor deposition , laser ablation , and arc discharge surface mediated growth ) , carbon nanotubes ( cnts ) contain metal impurities ( e.g. for example , an icp - ms study of commercially important cnts showed that impurity concentrations in three multiwall cnts ( mwcnts ) were between 4738 and 51867 g / g for ni and in two commercially relevant single - wall cnts ( swcnts ) between 1798 and 4217 g / g for co and 14721672 g / g mo . the currently accepted method for determination of cnts in workplace air is thermooptical analysis ( niosh method # 5040 ) for determination of elemental carbon . a drawback of this approach is that it does not distinguish between cnt and non - cnt sources of elemental carbon such as diesel exhaust [ 11 , 12 ] . the detection of cnt - bound metal impurities in environmental samples provides a means to distinguish cnt emissions from non - cnt background sources of elemental carbon [ 1215 ] . worker exposure to cnts can occur at any stage of the manufacturing process and during transport , storage , and handling of cnts , with inhalation being the major route of exposure [ 11 , 16 ] . different sampling methods have been suggested as possible strategies to collect samples for workplace monitoring of metal impurities in cnts such as filter cassettes , electrostatic precipitation , and surface wipes [ 13 , 15 ] . wipe sampling has emerged as a useful and cost - effective qualitative technique suitable for representative mapping of spatial variations of surface metal contamination within a workplace . wipe sampling for cnt metal tracers is considered a qualitative indicator of the presence of cnts , not a quantitative exposure metric . even though wipe sampling may be considered qualitative , an analytical method is needed that includes an efficient digestion step and adequate detection limits to ensure reliable and reproducible metal determinations . the most commonly used analytical methods for determining metal impurities in cnts are neutron activation analysis ( naa ) [ 1 , 3 , 2022 ] , direct solid sampling electrothermal atomic absorption spectrometry ( dss - et aas ) , high resolution continuum source graphite furnace atomic absorption spectrometry ( hr cs gfaas ) , inductively coupled plasma optical emission spectrometry ( icp - oes ) [ 1 , 4 , 20 , 21 , 23 , 24 ] , and inductively coupled plasma mass spectrometry ( icp - ms ) [ 1 , 3 , 4 , 7 , 20 , 21 ] . out of all of these detection methods , icp - ms is a suitably sensitive approach for determination of particle - bound metals on wipe substrates , but an appropriate extraction method is needed for cnt - bound metals . the key challenge is the difficulty in quantitatively extracting cnt metal impurities during the digestion step [ 3 , 4 , 7 , 22 , 26 ] . a variety of sample pretreatments have been employed for determination of metals in cnt samples including wet digestion [ 1 , 3 , 23 ] , dry ashing combined with acid digestion [ 1 , 3 , 4 ] , carius tube digestion , microwave - assisted acid digestion [ 1 , 3 , 4 , 20 , 26 ] , and microwave - induced combustion [ 4 , 21 , 24 ] . and proposed a multistep microwave - assisted procedure that involved the repeated use of strong acids as follows : hno3 combined with h2o2 ( 1x ) , hno3 alone ( 3x ) , evaporation near dryness , hno3 and hclo4 followed by hno3 addition and evaporation to dryness ( 4x ) , and finally dissolution and dilution in 1% hno3 . , three to six heating cycles depending on cnt structure ) when using microwave digestion to measure cnt metal impurities . emphasized the importance of the digestion step as they observed positive nonspectral interferences from undigested carbon residues remaining in the sample solution which they identified by comparing icp - ms and icp - oes results for the same swcnt digest . the purpose of this study is to identify an appropriate digestion approach for icp - ms determination of metal impurities in cnts collected using wipe samples . two approaches are evaluated with respect to the efficiency of the extractions : a microwave - assisted digestion method and an ultrasonic digestion method . although for this application 100% extraction efficiency is not required , the goal is to achieve reproducible and reliable recoveries that will enable the identification of cnt surface contamination . different cnt materials , including nist 2483 soot ( single - wall cnt ) , are digested with and without ghostwipes , as the digestion approach must be able to accommodate wipe samples which are quite bulky . three swcnt products were obtained for this study : nist 2483 swcnt standard reference material ( raw soot ; national institute of standards and technology , gaithersburg , md , usa ) and two other swcnts test materials , aldrich - cnt ( sigma - aldrich co. , gillingham , uk ) and a research test sample provided by national research council ( test - cnt ) . high - purity multielement ( mes-1107 - 01 solutions a and b , 100 g / ml ) and individual standard stock solutions ( la and gd , 1000 g / ml ) were used to prepare the calibration standards ( delta scientific laboratory products ltd . , gw procedural blanks and sample ( nist 2483 ) replicates were spiked at levels varying between 4 g / l and 250 g / l , respectively , with icp - mscs high - purity standard solution ( 10 g / ml ) . individual high - purity standards solutions of ge , in , and re ( 1000 mg / l ) were used to prepare the internal standard solution . ghostwipes ( gw ; environmental express , charleston , south carolina , sc 4250 , premoistened with deionized water , individually sealed packets ) were used in combination with nist 2483 swcnt and nist 1633b stms to assess the effect of gw on extraction efficiency . the present study used a 3.5 cm diameter circular disk of ghostwipes material as required by the wipe sampling device used for monitoring cnt releases . mettler toledo xp205 digital analytical balance equipped with a u - shaped antistatic electrode was used to achieve accurate weighing results for the aliquots ( 2 mg ) of tested cnts used in this study . digiprep ms heating block ( scp science , qc , canada ) and branson ultrasonic bath ( model b8510dth ) were used in this study . for quantification of cnt metal impurities in digested samples , a nexion 300s dual - channel universal cell icp - ms system ( perkin elmer , canada ) coupled with a sc - fast autosampler ( elemental scientific , omaha , ne ) was operated in the standard and collision mode . two digestion methods were evaluated during this study to compare their efficiency in extracting elemental impurities from cnts : a microwave digestion ( md ) method using nitric acid and hydrogen peroxide and an ultrasonic digestion ( ud ) method using nitric and hydrofluoric acids . for both digestion methods three to five aliquots of 2 mg of test material were used for analysis ( to mimic the particle mass on a surface wipe sample ) . finally , nist 2483 swcnt and nist 1633b coal fly ash were combined with ghostwipes ( gw ) in order to compare the efficiency of investigated digestion methods in the presence of gw . during the digiprep step , the samples were placed in 50 ml teflon digitube vessels and were treated with diluted nitric acid ( 1 : 1 , 12 ml ) and allowed to sit at room temperature for 3045 min . after effervescence subsided , the samples were treated successively at reflux ( 85c on digiprep ms heating block ) with diluted nitric ( 12 ml , 90 min ) , concentrated nitric acid ( 10 ml , 30 min reflux ) , and 30% hydrogen peroxide ( 5 ml , 45 min ) , and evaporated to 10 ml ( 90c ) . the microwave digestion step was performed at 1000 w power in the following conditions : 20 min to reach 180c temperature , 10 min to increase from 180c to 220c , and 20 min at 220c . , undissolved cnts ) that may impact the icp - ms analysis . preliminary work showed that the digiprep method alone did not yield adequate recoveries for cnts in wipes and therefore it was necessary to add the microwave digestion step . this step is most critical in the presence of gw because even with a small piece of gw ( 3.5 cm disc ) an exothermic reaction develops during the hot water bath stage . , undissolved cnts ) that may impact the icp - ms analysis . all sample digests were analyzed by icp - ms after appropriate dilution with 1% hno3 . all sample digests were stored in the fridge in precleaned polyethylene screw - capped tubes and analyzed by icp - ms after appropriate dilution . the final digestion solutions were first screened for the presence of elements using icp - ms and isotopes such as b , al , ti , v , cr , mn , fe , co , ni , cu , as , se , sr , zn , y , mo , ba , la , gd , pb , and u were monitored . spike recoveries of the study elements ( b , al , v , mn , co , ni , cu , mo , as , ba , la , and u ) were between 82% and 115% with md method ( except b 74% and cu 47% ; al spike not recovered ) and 86107% with ud method ( except mo 122% ) in the presence of gw ( spiked procedural blanks and nist 2483 sample ) . the lods were calculated according to the 1994 us - epa guideline for inductively coupled plasma mass spectrometry using 3 times standard deviation of 8 - 9 procedural blanks . extraction efficiencies of two different methods , microwave digestion ( md ) and ultrasonic digestion ( ud ) , are compared for cnt impurities present at high concentrations ( > 0.5% ) in table 1 and for cnt impurities present in lower concentrations ( ppm range ) in table 2 without gw being present . nist 2483-cnt standard reference material ( srm ) , test - cnt , and aldrich - cnt were used to compare the extraction efficiency for the residual catalysts ( > 0.5% ) . nist 2483-cnt srm was used to compare the extraction efficiency for other trace metal / element impurities ( ppm range ) using the certified , informational , or reference values provided in the certificate of analysis . consequently , the efficiencies of the two investigated methods are compared using aliquots of nist 2483-cnt combined with gw ( table 3 ) . table 1 presents the high metal concentrations ( > 0.5% ) in nist 2483 , test - cnt , and aldrich - cnts obtained with both microwave and ultrasonic digestion methods . as shown in table 1 for all investigated cnts , the md method had improved extraction efficiency for the high concentration metals ( co , ni , y , and mo ) when compared to the ud method . except for ni in test - cnt for which t - test indicated no significant difference ( p = 0.128 ) , for all other residual catalysts metals ( co , ni , y , and mo ) in all three cnts the difference between md and ud results was significant ( p < 0.05 ) . overall , the results obtained with the ud method varied largely with the cnt analyzed , indicating that morphology and/or the way the residual catalyst metals were incorporated in the graphitic structure could influence the success of the sample pretreatment and consequently the recovery . even though in case of ultrasonic digestion method the recoveries vary with the cnt investigated , this proves to be a quick and useful method for qualitative identification of high concentration metallic impurities in cnts . 4.5 h for alternating oxidation and reflux steps plus evaporation and 4 h for microwave digestion and evaporation / filtration ) . figure 1 shows the percent recoveries obtained for high ( > 0.5% ) and low concentration ( ppm range ) metals in nist 2483-cnt srm obtained with ( a ) md and ( b ) ud methods with or without ghostwipes ( gw ) . , 85% al , 83% v , and 84% la ) for the low concentration ( ppm range ) metals investigated ( except as ) in nist 2483-cnt , the ud method is a short and practical method that may be used to detect and measure metals present in lower amount in cnts . moreover , the extraction efficiency of the ud method for the residual catalysts / high concentration metals varied with the cnts tested as shown in table 1 with better extraction efficiency for test - cnt and aldrich - cnt than for nist 2483-cnt . compared to the md method , the ud method results varied largely with the cnt analyzed , indicating that morphology and/or the way the residual catalyst metals were incorporated in the graphitic structure could influence the success of the sample pretreatment and consequently the recovery . this variability might be related to the manufacturing processes since the cnts are produced by various processes , that is , cold vapor deposition , laser ablation , and arc discharge surface mediated growth [ 3 , 4 ] , which influence the amounts and how strongly the residual catalyst metals are incorporated in the graphitic structure [ 7 , 22 ] . this was done by analyzing the digests of all three tested cnts ( nist-2483 , nrc , and aldrich ) with both icp - oes and icp - ms techniques and comparing the results ( figure 2 ) . t - test results showed that the co , ni , y , and mo results obtained with icp - ms were not significantly higher ( p > 0.05 ) than those obtained with icp - oes for all of the three cnts tested , except for ni in aldrich - cnt ( p = 0.031 ) . figure 2(a ) presents the correlation of metal concentrations measured by icp - ms and icp - oes using all seven pairs of metal - concentration values from all cnts tested . the results ( slope = 1.0002 , r = 0.999998 ) presented in figure 2(b ) showed no differences between the icp - ms results of the two dilutions . , filter cassettes , electrostatic precipitation ) surface wipes have been suggested as a strategy to collect samples ( including cnt ) for workplace monitoring [ 17 , 3739 ] . ghostwipes ( gw ) meet the us regulatory standard for sampling pb in house dust and were used for quantification of additional metal(loid)s ( i.e. although gw have the advantage of dissolving completely during acid digestion , their presence in the sample matrix adds to the analytical challenges encountered in the quantification of cnt metal bound impurities , since wipes are bulky and may contain impurities . results obtained in the presence of gw ( table 3 ) are significantly lower ( p < 0.05 ) for co and mo in nist 2483-cnt with the ud method than with the md method , similar to results observed without gw ( table 1 ) . in contrast , with the ud method recoveries were significantly lower ( p < 0.05 ) for almost all elements in the presence of gw than without gw ( except b , al , as , and gd ; p > 0.05 ) suggesting that the ud method is more sensitive to interferences from gw . , al , ti , zn , fe , and mn ) in our preliminary work with nist 2483-cnt combined with whole gw caused by uneven distribution of background contamination of these elements in the wipes used . other factors may include interferences from organic carbon derived from gws or uncertainty arising from the small sample mass used in this study ( 2 mg versus 2540 mg recommended in the certificate of analysis ) . combining the results into these histograms clarifies the overall trends : the md method ( a ) yields acceptable recoveries for most elements with or without the presence of gw , but the ud method ( b ) yields poor recoveries for the catalyst elements and is generally more sensitive to the presence of gw . the ud method does result in acceptable recoveries ( 7598% ) for certain elements ( v , la , and gd ) with or without the presence of gw . despite generally lower recoveries , the cost - effective ud method may be considered acceptable for semiquantitative purposes such as identifying cnt releases in the workplace environment using wipe samples . different responses of the cnts tested in the present study indicate that the cnt matrix may influence the success of the sample pretreatment and consequently the recovery . thus , different matrices are likely to respond differently to a given digestion method , as observed for the ud method which gave excellent results for co in nist 1633b ( table s1 , supplementary material ) but poor recovery for co in nist 2483 ( table 1 ) . poor recoveries for the ud method ( figure 1(b ) ) are not likely due to readsorption of the extracted metals on residual cnts , due to the strong acid digestion ( ph < 2 throughout ) . wipe sampling for cnt metal tracers has emerged as a useful and cost - effective qualitative technique suitable for representative identification of cnt surface contamination in the workplace ( and potential exposures ) and routine monitoring of control measures . even though wipe sampling may be considered qualitative , an analytical method is needed that includes an efficient digestion step and adequate detection limits to ensure reliable and reproducible metal determinations . for the purpose of wipe sample analysis , the microwave digestion ( md ) method yielded acceptable recoveries for catalyst elements at high concentrations ( table 1 ) as well as for elemental impurities at low concentrations ( tables 2 and 3 ) in the tested cnt reference materials . moreover , no signal enhancement due to residual carbon compounds was observed when icp - ms was used in conjunction with the md method . performance of the md extraction was not affected by the presence of ghostwipes ( gw ) in the digest matrix , with the caveat that elevated and variable gw blanks for certain elements ( such as al and zn ) must be considered . it is concluded that the md method is appropriate for quantification of metallic tracers in wipe samples used to identify the presence of cnt in work environments . in contrast , the ultrasonic digestion method showed incomplete extraction of catalyst metals present at high concentrations ( table 1 ) , but near - total extraction efficiencies for elemental impurities present at low concentrations ( tables 2 and 3 ) in the tested cnts . however , for nist 1633b reference material , the ud method showed equivalent or better recoveries than those reported for us - epa method 3051 , and since the ud method involves fewer steps than the md method , it may serve as a cost - effective semiquantitative approach for screening purposes . for example , the ud method was useful for identifying the presence of cnt metal impurities ( surface wipe samples ) in a preliminary mapping survey of cnt releases in a manufacturing plant .

this randomized , double - blind , placebo - controlled , phase iii study was conducted in 167 centers in 19 countries ( argentina , belgium , brazil , canada , czech republic , finland , germany , greece , italy , korea , mexico , the netherlands , norway , peru , russia , slovakia , spain , taiwan , and the u.s . ) . patients were eligible if they were 18 years of age with a diagnosis of type 2 diabetes , had inadequate glycemic control ( hba1c 7.0% [ 53 mmol / mol ] to 10.0% [ 86 mmol / mol ] ) , had a bmi of 45 kg / m , and were receiving treatment with basal insulin , alone or in combination with metformin and/or pioglitazone , for 12 weeks . acceptable basal insulins were insulin glargine , insulin detemir , and neutral protamine hagedorn insulin . the total prescribed insulin dose must not have changed by > 10% of the baseline value during the 12 weeks before randomization . patients were ineligible if they had uncontrolled fasting hyperglycemia ( glucose > 13.3 mmol / l during placebo run - in ) ; a myocardial infarction , stroke , or transient ischemic attack within 6 months before informed consent ; impaired hepatic function ( either alanine transaminase , aspartate transaminase , or alkaline phosphate > 3 times the upper limit of normal ) ; previous gastric bypass surgery ; or any medical history of cancer ( except basal cell carcinoma ) in the 5 years before screening . further exclusion criteria included hypersensitivity or allergy to the investigational products ; contraindications to metformin or pioglitazone ; treatment with rosiglitazone , sulfonylureas , glucagon - like peptide 1 analogs , dpp-4 inhibitors , or antiobesity drugs within the 3 months before informed consent ; a history of alcohol or drug abuse in the previous 3 months ; and current treatment with systemic steroids or change in dosage of thyroid hormones within 6 weeks before informed consent . premenopausal women who were nursing , pregnant , or not practicing an acceptable method of birth control were also ineligible . the trial protocol was approved by the independent ethics committees or institutional review boards of all participating centers . the study was carried out according to the principles of the declaration of helsinki and the international conference on harmonization guideline for good clinical practice . patients underwent a 2-week , open - label placebo run - in period to confirm their eligibility after the initial screening and to exclude those who were nonadherent . after this placebo run - in period , eligible patients were randomly assigned ( 1:1 ratio ) to receive double - blind linagliptin 5 mg once - daily or placebo in addition to continued basal insulin for at least 52 weeks . treatment assignment was determined by computer - generated random sequence with an interactive voice response system . randomization was stratified by hba1c ( < 8.5% [ 69 mmol / mol ] vs. 8.5% [ 69 mmol / mol ] ) , renal function ( estimated glomerular filtration rate [ egfr ] ) , and concomitant use of oads ( metformin only , pioglitazone only , metformin and pioglitazone , or none ) . because the patients who were randomized early in the trial were treated until the study s end , the maximal possible treatment duration was 110 weeks . during the first 24 weeks of treatment , the doses of basal insulin ( within 10% of baseline dose ) and oads were kept unchanged . after week 24 , adjustments to the dose of basal insulin ( but not oads ) were allowed according to the medical judgment of the investigator , with a treatment target for fpg of 6.1 rescue therapy could be initiated during randomized treatment if a patient met the following criteria : confirmed fpg ( after overnight fast ) > 13.3 mmol / l during the first 12 weeks , fpg > 11.1 mmol / l from weeks 12 to 24 , or fpg > 10.0 mmol / l or hba1c > 8.0% ( 64 mmol / mol ) after week 24 . for initiation of rescue medication , these criteria had to be confirmed by two measurements on separate days . patients were withdrawn from the trial if the fpg remained above this threshold despite rescue therapy . the primary efficacy end point was the change from baseline in hba1c after 24 weeks of treatment . secondary end points included changes from baseline in hba1c and fpg with time , change from baseline in fpg after 52 weeks of treatment , the proportion of patients achieving hba1c < 7% ( 53 mmol / mol ) , the proportion of patients achieving 0.5% ( 5.5 mmol / mol ) reduction in hba1c , and the change from baseline in mean basal insulin dose after 52 weeks of treatment . other end points included the use of rescue medication and mean change in body weight to the end of treatment . safety end points included the frequency and intensity of adverse events ( aes ) , including hypoglycemia and clinically relevant new or worsening findings in physical examination , 12-lead electrocardiogram , vital signs , lipid parameters , and clinical laboratory assessments . an independent external adjudication committee reviewed treatment - emergent fatal events and suspected events of stroke or cardiac ischemia ( including myocardial infarction ) , hospitalization for heart failure , stent thrombosis , and revascularization procedures . follow - up for all aes , including those persisting after a patient had completed ( or withdrawn prematurely from ) the trial , continued until the event had resolved or been sufficiently characterized . allowing for sd of change in hba1c from baseline of 1.2% ( 13.1 mmol / mol ) , 284 patients per treatment group were sufficient to achieve 93% power to detect a 0.35% ( 3.8 mmol / mol ) difference between groups in change in hba1c from baseline to week 24 . the larger sample size of 600 patients in each group allowed adequate exposure data to be collected for the regulatory requirement to detect rare cardiovascular events across the entire linagliptin program . the primary end point was evaluated with ancova at the level of = 0.025 ( one - sided ) . treatment , concomitant oads , and baseline renal function impairment category as fixed classification effects and baseline hba1c as linear covariate . this analysis was performed on the full analysis set ( fas ) , comprising all randomized patients treated with at least one dose of study medication , with a baseline hba1c measurement and at least one on - treatment hba1c measurement within the first 24 weeks of double - blind treatment . an approach of last observation carried forward ( locf ) was used to replace missing data . changes in fpg with time were analyzed for the fas ( observed case set [ oc ] ; i.e. , patients with available data ) by means of descriptive statistics . the impact of treatment on the use of rescue medication was assessed by means of logistic regression , and the time to first use of rescue therapy was evaluated by kaplan - meier analysis . the impact of treatment on the occurrence of hypoglycemia was investigated by means of logistic regression and kaplan - meier analysis . safety end points were evaluated for the treated set ( all patients who were treated with at least one dose of study medication ) by means of descriptive statistics . aes were described according to the medical dictionary for drug regulatory affairs ( version 14.0 ) . mmol / l accompanied by typical symptoms of hypoglycemia , plasma glucose <3 mmol / l accompanied by typical symptoms of hypoglycemia but without need for external assistance , and severe hypoglycemia requiring the assistance of another person to actively administer carbohydrate , glucagon , or other resuscitative actions . this randomized , double - blind , placebo - controlled , phase iii study was conducted in 167 centers in 19 countries ( argentina , belgium , brazil , canada , czech republic , finland , germany , greece , italy , korea , mexico , the netherlands , norway , peru , russia , slovakia , spain , taiwan , and the u.s . ) . patients were eligible if they were 18 years of age with a diagnosis of type 2 diabetes , had inadequate glycemic control ( hba1c 7.0% [ 53 mmol / mol ] to 10.0% [ 86 mmol / mol ] ) , had a bmi of 45 kg / m , and were receiving treatment with basal insulin , alone or in combination with metformin and/or pioglitazone , for 12 weeks . acceptable basal insulins were insulin glargine , insulin detemir , and neutral protamine hagedorn insulin . the total prescribed insulin dose must not have changed by > 10% of the baseline value during the 12 weeks before randomization . patients were ineligible if they had uncontrolled fasting hyperglycemia ( glucose > 13.3 mmol / l during placebo run - in ) ; a myocardial infarction , stroke , or transient ischemic attack within 6 months before informed consent ; impaired hepatic function ( either alanine transaminase , aspartate transaminase , or alkaline phosphate > 3 times the upper limit of normal ) ; previous gastric bypass surgery ; or any medical history of cancer ( except basal cell carcinoma ) in the 5 years before screening . further exclusion criteria included hypersensitivity or allergy to the investigational products ; contraindications to metformin or pioglitazone ; treatment with rosiglitazone , sulfonylureas , glucagon - like peptide 1 analogs , dpp-4 inhibitors , or antiobesity drugs within the 3 months before informed consent ; a history of alcohol or drug abuse in the previous 3 months ; and current treatment with systemic steroids or change in dosage of thyroid hormones within 6 weeks before informed consent . premenopausal women who were nursing , pregnant , or not practicing an acceptable method of birth control were also ineligible . the trial protocol was approved by the independent ethics committees or institutional review boards of all participating centers . the study was carried out according to the principles of the declaration of helsinki and the international conference on harmonization guideline for good clinical practice . patients underwent a 2-week , open - label placebo run - in period to confirm their eligibility after the initial screening and to exclude those who were nonadherent . after this placebo run - in period , eligible patients were randomly assigned ( 1:1 ratio ) to receive double - blind linagliptin 5 mg once - daily or placebo in addition to continued basal insulin for at least 52 weeks . treatment assignment was determined by computer - generated random sequence with an interactive voice response system . randomization was stratified by hba1c ( < 8.5% [ 69 mmol / mol ] vs. 8.5% [ 69 mmol / mol ] ) , renal function ( estimated glomerular filtration rate [ egfr ] ) , and concomitant use of oads ( metformin only , pioglitazone only , metformin and pioglitazone , or none ) . because the patients who were randomized early in the trial were treated until the study s end , the maximal possible treatment duration was 110 weeks . during the first 24 weeks of treatment , the doses of basal insulin ( within 10% of baseline dose ) and oads were kept unchanged . after week 24 , adjustments to the dose of basal insulin ( but not oads ) were allowed according to the medical judgment of the investigator , with a treatment target for fpg of 6.1 rescue therapy could be initiated during randomized treatment if a patient met the following criteria : confirmed fpg ( after overnight fast ) > 13.3 mmol / l during the first 12 weeks , fpg > 11.1 mmol / l from weeks 12 to 24 , or fpg > 10.0 mmol / l or hba1c > 8.0% ( 64 mmol / mol ) after week 24 . for initiation of rescue medication , these criteria had to be confirmed by two measurements on separate days . patients were withdrawn from the trial if the fpg remained above this threshold despite rescue therapy . the primary efficacy end point was the change from baseline in hba1c after 24 weeks of treatment . secondary end points included changes from baseline in hba1c and fpg with time , change from baseline in fpg after 52 weeks of treatment , the proportion of patients achieving hba1c < 7% ( 53 mmol / mol ) , the proportion of patients achieving 0.5% ( 5.5 mmol / mol ) reduction in hba1c , and the change from baseline in mean basal insulin dose after 52 weeks of treatment . other end points included the use of rescue medication and mean change in body weight to the end of treatment . safety end points included the frequency and intensity of adverse events ( aes ) , including hypoglycemia and clinically relevant new or worsening findings in physical examination , 12-lead electrocardiogram , vital signs , lipid parameters , and clinical laboratory assessments . an independent external adjudication committee reviewed treatment - emergent fatal events and suspected events of stroke or cardiac ischemia ( including myocardial infarction ) , hospitalization for heart failure , stent thrombosis , and revascularization procedures . follow - up for all aes , including those persisting after a patient had completed ( or withdrawn prematurely from ) the trial , continued until the event had resolved or been sufficiently characterized . allowing for sd of change in hba1c from baseline of 1.2% ( 13.1 mmol / mol ) , 284 patients per treatment group were sufficient to achieve 93% power to detect a 0.35% ( 3.8 mmol / mol ) difference between groups in change in hba1c from baseline to week 24 . the larger sample size of 600 patients in each group allowed adequate exposure data to be collected for the regulatory requirement to detect rare cardiovascular events across the entire linagliptin program . the primary end point was evaluated with ancova at the level of = 0.025 ( one - sided ) . treatment , concomitant oads , and baseline renal function impairment category as fixed classification effects and baseline hba1c as linear covariate . this analysis was performed on the full analysis set ( fas ) , comprising all randomized patients treated with at least one dose of study medication , with a baseline hba1c measurement and at least one on - treatment hba1c measurement within the first 24 weeks of double - blind treatment . an approach of last observation carried forward ( locf ) was used to replace missing data . changes in fpg with time were analyzed for the fas ( observed case set [ oc ] ; i.e. , patients with available data ) by means of descriptive statistics . the impact of treatment on the use of rescue medication was assessed by means of logistic regression , and the time to first use of rescue therapy was evaluated by kaplan - meier analysis . the impact of treatment on the occurrence of hypoglycemia was investigated by means of logistic regression and kaplan - meier analysis . safety end points were evaluated for the treated set ( all patients who were treated with at least one dose of study medication ) by means of descriptive statistics . aes were described according to the medical dictionary for drug regulatory affairs ( version 14.0 ) . hypoglycemia was analyzed by three levels of intensity : plasma glucose 4 mmol / l accompanied by typical symptoms of hypoglycemia , plasma glucose <3 mmol / l accompanied by typical symptoms of hypoglycemia but without need for external assistance , and severe hypoglycemia requiring the assistance of another person to actively administer carbohydrate , glucagon , or other resuscitative actions . a total of 1,261 patients were randomized to receive linagliptin ( n = 631 ) or placebo ( n = 630 ) once daily ( fig . 1,063 patients ( 84.3% ) completed the trial ( 543 [ 86.1% ] receiving linagliptin vs. 520 [ 82.5% ] receiving placebo ) . the main reasons for discontinuation were aes ( linagliptin 4.0% , placebo 5.2% ) and refusal to continue study medication ( linagliptin 3.3% , placebo 4.1% ) . mean exposures to study medication were 435.5 days in the linagliptin group and 422.4 days in the placebo group ( medians 444 and 448 days , respectively ) . demographic and clinical characteristics were similar between treatment groups at baseline and are presented in table 1 . baseline demographics and clinical characteristics linagliptin was superior to placebo in reducing hba1c after 24 weeks ( supplementary table 1 ) . the adjusted mean ( se ) change in hba1c from baseline at week 24 for linagliptin was 0.58% ( 0.08 ) ( 6.3 [ 0.9 ] mmol / mol ) , compared with 0.07% ( 0.08 ) ( 0.8 [ 0.9 ] mmol / mol ) for placebo , resulting in a placebo - adjusted mean change in hba1c from baseline of 0.65% ( 95% ci 0.74 to 0.55 ) ( 7.1 mmol / mol ; p < 0.0001 ) . after 52 weeks , the adjusted mean changes in hba1c from baseline were 0.48% ( 0.08 ) ( 5.2 [ 0.9 ] mmol / mol ) for linagliptin and 0.05% ( 0.08 ) ( 0.5 [ 0.9 ] mmol / mol ) for placebo ( placebo - adjusted difference 0.53% [ 95% ci 0.64 to 0.43 ] , 5.8 mmol / mol ; p < 0.0001 ) . a : mean hba1c change from baseline with time out to 76 weeks ( fas , oc ) . b : mean fpg change ( fas , oc ) and mean change in insulin dose ( fas , original results ) from baseline with time out to 76 weeks . at week 24 , mmol / l ( 95% ci 0.9 to 0.4 ; p < 0.001 ) . during the period when insulin titration was allowed and recommended , improvement in fpg was sustained in the linagliptin group , whereas fpg decreased in the placebo group in parallel with a small increase in basal insulin dose ( fig . , the mean changes in fpg from baseline were similar between the groups ( linagliptin baseline 7.8 mmol / l , 52-week 7.6 mmol / l , change from baseline 0.2 [ 0.2 ] mmol / l ; placebo baseline 7.8 mmol / l , 52-week 7.6 mmol / l , change from baseline 0.3 [ 0.2 ] mmol / l ) . mmol / l was achieved by 24.6% of patients in the linagliptin group and 19.1% of the placebo group . of these patients , 29.6% and 38.1% experienced hypoglycemia in the linagliptin and placebo groups , respectively . among patients with baseline hba1c 7.0% ( 53 mmol / mol ) , after 52 weeks an hba1c < 7.0% ( 53 mmol / mol ) was achieved by 16% and 7% of the linagliptin and placebo groups , respectively ( p < 0.001 ) . compared with the placebo group , patients in the linagliptin group were more likely to have a decrease in hba1c of 0.5% ( 5.5 mmol / mol ) after 52 weeks of treatment ( 37% linagliptin vs. 17% placebo ; p < 0.0001 ) . analysis of change in hba1c by prespecified subgroups demonstrated no significant interaction with treatment according to renal function category ( p = 0.5784 ) , type of basal insulin ( p = 0.9511 ) , age group ( p = 0.1000 ) , concomitant use of oads ( p = 0.64 ) , sex ( p = 0.98 ) , or bmi ( p = 0.99 ) , indicating that none of these factors altered the efficacy of linagliptin . subgroup variables with significant ( p < 0.10 ) interactions with treatment were baseline hba1c ( p = 0.0725 ) , geographical region ( p = 0.0548 ) , race ( p = 0.0603 ) , and time since diabetes diagnosis ( p = 0.0017 ) . placebo - adjusted mean ( sd ) changes in hba1c were greatest for patients with a baseline hba1c of 9.0% ( 75 mmol / mol ) ( 0.83% [ 0.10 ] ) , patients who were from asia ( 1.00 [ 0.15 ] ) or of asian race ( 0.93% [ 0.14 ] ) , and patients who had been diagnosed with diabetes for > 5 years ( 0.72% [ 0.05 ] ) . the placebo - adjusted mean change from baseline in hba1c after 24 weeks was 0.52% ( 5.7 mmol / mol ) for patients taking no oads at baseline ( n = 96 [ 15.50% ] ; p < 0.0001 ) , 0.67% ( 7.3 mmol / mol ) for those taking metformin only ( n = 470 [ 76.1% ] ; p < 0.0001 ) , 0.76% ( 8.3 mmol / mol ) for those taking pioglitazone only ( n = 12 [ 1.0% ] ; p = 0.129 ) , and 0.71% ( 7.8 mmol / mol ) for patients taking both metformin and pioglitazone ( n = 91 [ 7.4% ] ; p < 0.0001 ) . more patients required rescue medication in the placebo group ( 50.4% ) than in the linagliptin group ( 38.2% ) ; the odds ratio ( linagliptin vs. placebo ) for rescue medication was 0.575 ( 95% ci 0.4540.728 ; p < 0.0001 ) . the mean ( sd ) change in basal insulin dose to week 24 ( when dose was to have remained within 10% of baseline ) was 0.1 ( 0.2 ) iu for patients treated with linagliptin and 0.4 ( 0.2 ) iu for those treated with placebo . from week 24 , mean basal insulin dose increased in the linagliptin group to a lesser extent than in the placebo group ( fig . the adjusted mean ( sd ) changes from baseline in insulin dose at week 52 were 2.6 ( 0.8 ) iu for linagliptin and 4.2 ( 0.8 ) iu for placebo ( p < 0.003 ) . the overall incidence of patients with 1 reported ae was comparable between treatment groups ( linagliptin 78.4% , placebo 81.4% ) ( supplementary table 2 ) . aes were primarily of mild or moderate intensity ; aes of severe intensity occurred in 8.2% and 8.3% of patients in the linagliptin and placebo groups , respectively . the most commonly reported severe aes in both treatment groups were hypoglycemia , coronary artery disease , osteoarthritis , cardiac failure , pneumonia , diarrhea , arthralgia , gastroenteritis , acute renal failure , and subdural hematoma . aes considered to be drug - related occurred in 18.7% and 22.2% of linagliptin and placebo patients , respectively . drug - related hypoglycemia occurred in 83 ( 15.1% ) and 95 ( 13.2% ) patients in the linagliptin and placebo groups , respectively , and was the only drug - related ae with an incidence greater than 2% . aes leading to discontinuation of trial medication occurred in 21 patients ( 3.3% ) in the linagliptin group and 28 patients ( 4.4% ) in the placebo group . there were no clinically relevant changes in vital signs or laboratory parameters in either group , including no between - group imbalance in shifts in stage of renal impairment . the percentage of patients with investigator - defined hypoglycemia was not different between groups either at week 24 ( linagliptin 22.0% , placebo 23.2% ) or at the end of treatment ( linagliptin 31.4% , placebo 32.9% ) . incidence of severe hypoglycemia was also similar between groups ( week 24 linagliptin 0.3% , placebo 0.6% ; end of treatment linagliptin 1.7% , placebo 1.1% ) ( supplementary table 2 ) . there was no significant change from baseline in mean body weight at either week 24 ( linagliptin 0.16 [ 0.12 ] kg , placebo 0.12 [ 0.11 ] kg ) or at week 52 ( linagliptin 0.3 [ 0.19 ] kg , placebo 0.04 [ 0.18 ] kg ) . adjudicated cardiovascular events occurred in 18 linagliptin patients ( 2.9% ) and 11 placebo patients ( 1.7% ) . cardiovascular deaths occurred in 5 patients ( 0.8% ) in the linagliptin group and 1 ( 0.2% ) in the placebo group . total mortality , however , was comparable between the arms , with 5 deaths in each group . a total of 1,261 patients were randomized to receive linagliptin ( n = 631 ) or placebo ( n = 630 ) once daily ( fig . 1,063 patients ( 84.3% ) completed the trial ( 543 [ 86.1% ] receiving linagliptin vs. 520 [ 82.5% ] receiving placebo ) . the main reasons for discontinuation were aes ( linagliptin 4.0% , placebo 5.2% ) and refusal to continue study medication ( linagliptin 3.3% , placebo 4.1% ) . mean exposures to study medication were 435.5 days in the linagliptin group and 422.4 days in the placebo group ( medians 444 and 448 days , respectively ) . demographic and clinical characteristics were similar between treatment groups at baseline and are presented in table 1 . linagliptin was superior to placebo in reducing hba1c after 24 weeks ( supplementary table 1 ) . the adjusted mean ( se ) change in hba1c from baseline at week 24 for linagliptin was 0.58% ( 0.08 ) ( 6.3 [ 0.9 ] mmol / mol ) , compared with 0.07% ( 0.08 ) ( 0.8 [ 0.9 ] mmol / mol ) for placebo , resulting in a placebo - adjusted mean change in hba1c from baseline of 0.65% ( 95% ci 0.74 to 0.55 ) ( 7.1 mmol / mol ; p < 0.0001 ) . after 52 weeks , the adjusted mean changes in hba1c from baseline were 0.48% ( 0.08 ) ( 5.2 [ 0.9 ] mmol / mol ) for linagliptin and 0.05% ( 0.08 ) ( 0.5 [ 0.9 ] mmol / mol ) for placebo ( placebo - adjusted difference 0.53% [ 95% ci 0.64 to 0.43 ] , 5.8 mmol / mol ; p < 0.0001 ) . a : mean hba1c change from baseline with time out to 76 weeks ( fas , oc ) . b : mean fpg change ( fas , oc ) and mean change in insulin dose ( fas , original results ) from baseline with time out to 76 weeks . at week 24 , mmol / l ( 95% ci 0.9 to 0.4 ; p < 0.001 ) . during the period when insulin titration was allowed and recommended , improvement in fpg was sustained in the linagliptin group , whereas fpg decreased in the placebo group in parallel with a small increase in basal insulin dose ( fig . by week 52 , the mean changes in fpg from baseline were similar between the groups ( linagliptin baseline 7.8 mmol / l , 52-week 7.6 mmol / l , change from baseline 0.2 [ 0.2 ] mmol / l ; placebo baseline 7.8 mmol / l , 52-week 7.6 mmol / l , change from baseline 0.3 [ 0.2 ] mmol / l ) . the fpg target of < 6.1 mmol / l was achieved by 24.6% of patients in the linagliptin group and 19.1% of the placebo group . of these patients , 29.6% and 38.1% experienced hypoglycemia in the linagliptin and placebo groups , respectively . among patients with baseline 7.0% ( 53 mmol / mol ) , after 52 weeks an hba1c < 7.0% ( 53 mmol / mol ) was achieved by 16% and 7% of the linagliptin and placebo groups , respectively ( p < 0.001 ) . compared with the placebo group , patients in the linagliptin group were more likely to have a decrease in hba1c of 0.5% ( 5.5 mmol / mol ) after 52 weeks of treatment ( 37% linagliptin vs. 17% placebo ; p < 0.0001 ) . analysis of change in hba1c by prespecified subgroups demonstrated no significant interaction with treatment according to renal function category ( p = 0.5784 ) , type of basal insulin ( p = 0.9511 ) , age group ( p = 0.1000 ) , concomitant use of oads ( p = 0.64 ) , sex ( p = 0.98 ) , or bmi ( p = 0.99 ) , indicating that none of these factors altered the efficacy of linagliptin . subgroup variables with significant ( p < 0.10 ) interactions with treatment were baseline hba1c ( p = 0.0725 ) , geographical region ( p = 0.0548 ) , race ( p = 0.0603 ) , and time since diabetes diagnosis ( p = 0.0017 ) . placebo - adjusted mean ( sd ) changes in hba1c were greatest for patients with a baseline hba1c of 9.0% ( 75 mmol / mol ) ( 0.83% [ 0.10 ] ) , patients who were from asia ( 1.00 [ 0.15 ] ) or of asian race ( 0.93% [ 0.14 ] ) , and patients who had been diagnosed with diabetes for > 5 years ( 0.72% [ 0.05 ] ) . the placebo - adjusted mean change from baseline in hba1c after 24 weeks was 0.52% ( 5.7 mmol / mol ) for patients taking no oads at baseline ( n = 96 [ 15.50% ] ; p < 0.0001 ) , 0.67% ( 7.3 mmol / mol ) for those taking metformin only ( n = 470 [ 76.1% ] ; p < 0.0001 ) , 0.76% ( 8.3 mmol / mol ) for those taking pioglitazone only ( n = 12 [ 1.0% ] ; p = 0.129 ) , and 0.71% ( 7.8 mmol / mol ) for patients taking both metformin and pioglitazone ( n = 91 [ 7.4% ] ; p < 0.0001 ) . more patients required rescue medication in the placebo group ( 50.4% ) than in the linagliptin group ( 38.2% ) ; the odds ratio ( linagliptin vs. placebo ) for rescue medication was 0.575 ( 95% ci 0.4540.728 ; p < 0.0001 ) . the mean ( sd ) change in basal insulin dose to week 24 ( when dose was to have remained within 10% of baseline ) was 0.1 ( 0.2 ) iu for patients treated with linagliptin and 0.4 ( 0.2 ) iu for those treated with placebo . from week 24 , mean basal insulin dose increased in the linagliptin group to a lesser extent than in the placebo group ( fig . the adjusted mean ( sd ) changes from baseline in insulin dose at week 52 were 2.6 ( 0.8 ) iu for linagliptin and 4.2 ( 0.8 ) iu for placebo ( p < 0.003 ) . linagliptin was superior to placebo in reducing hba1c after 24 weeks ( supplementary table 1 ) . the adjusted mean ( se ) change in hba1c from baseline at week 24 for linagliptin was 0.58% ( 0.08 ) ( 6.3 [ 0.9 ] mmol / mol ) , compared with 0.07% ( 0.08 ) ( 0.8 [ 0.9 ] mmol / mol ) for placebo , resulting in a placebo - adjusted mean change in hba1c from baseline of 0.65% ( 95% ci 0.74 to 0.55 ) ( 7.1 mmol / mol ; p < 0.0001 ) . after 52 weeks , the adjusted mean changes in hba1c from baseline were 0.48% ( 0.08 ) ( 5.2 [ 0.9 ] mmol / mol ) for linagliptin and 0.05% ( 0.08 ) ( 0.5 [ 0.9 ] mmol / mol ) for placebo ( placebo - adjusted difference 0.53% [ 95% ci 0.64 to 0.43 ] , 5.8 mmol / mol ; p < 0.0001 ) . a : mean hba1c change from baseline with time out to 76 weeks ( fas , oc ) . b : mean fpg change ( fas , oc ) and mean change in insulin dose ( fas , original results ) from baseline with time out to 76 weeks . at week 24 , mmol / l ( 95% ci 0.9 to 0.4 ; p < 0.001 ) . during the period when insulin titration was allowed and recommended , improvement in fpg was sustained in the linagliptin group , whereas fpg decreased in the placebo group in parallel with a small increase in basal insulin dose ( fig . by week 52 , the mean changes in fpg from baseline were similar between the groups ( linagliptin baseline 7.8 mmol / l , 52-week 7.6 mmol / l , change from baseline 0.2 [ 0.2 ] mmol / l ; placebo baseline 7.8 mmol / l , 52-week 7.6 mmol / l , change from baseline 0.3 [ 0.2 ] mmol / l ) . the fpg target of < 6.1 mmol / l was achieved by 24.6% of patients in the linagliptin group and 19.1% of the placebo group . of these patients , 29.6% and 38.1% experienced hypoglycemia in the linagliptin and placebo groups , respectively . among patients with baseline 7.0% ( 53 mmol / mol ) , after 52 weeks an hba1c < 7.0% ( 53 mmol / mol ) was achieved by 16% and 7% of the linagliptin and placebo groups , respectively ( p < 0.001 ) . compared with the placebo group , patients in the linagliptin group were more likely to have a decrease in hba1c of 0.5% ( 5.5 mmol / mol ) after 52 weeks of treatment ( 37% linagliptin vs. 17% placebo ; p < 0.0001 ) . analysis of change in hba1c by prespecified subgroups demonstrated no significant interaction with treatment according to renal function category ( p = 0.5784 ) , type of basal insulin ( p = 0.9511 ) , age group ( p = 0.1000 ) , concomitant use of oads ( p = 0.64 ) , sex ( p = 0.98 ) , or bmi ( p = 0.99 ) , indicating that none of these factors altered the efficacy of linagliptin . subgroup variables with significant ( p < 0.10 ) interactions with treatment were baseline hba1c ( p = 0.0725 ) , geographical region ( p = 0.0548 ) , race ( p = 0.0603 ) , and time since diabetes diagnosis ( p = 0.0017 ) . placebo - adjusted mean ( sd ) changes in hba1c were greatest for patients with a baseline hba1c of 9.0% ( 75 mmol / mol ) ( 0.83% [ 0.10 ] ) , patients who were from asia ( 1.00 [ 0.15 ] ) or of asian race ( 0.93% [ 0.14 ] ) , and patients who had been diagnosed with diabetes for > 5 years ( 0.72% [ 0.05 ] ) . the placebo - adjusted mean change from baseline in hba1c after 24 weeks was 0.52% ( 5.7 mmol / mol ) for patients taking no oads at baseline ( n = 96 [ 15.50% ] ; p < 0.0001 ) , 0.67% ( 7.3 mmol / mol ) for those taking metformin only ( n = 470 [ 76.1% ] ; p < 0.0001 ) , 0.76% ( 8.3 mmol / mol ) for those taking pioglitazone only ( n = 12 [ 1.0% ] ; p = 0.129 ) , and 0.71% ( 7.8 mmol / mol ) for patients taking both metformin and pioglitazone ( n = 91 [ 7.4% ] ; p < 0.0001 ) . more patients required rescue medication in the placebo group ( 50.4% ) than in the linagliptin group ( 38.2% ) ; the odds ratio ( linagliptin vs. placebo ) for rescue medication was 0.575 ( 95% ci 0.4540.728 ; p < 0.0001 ) . the mean ( sd ) change in basal insulin dose to week 24 ( when dose was to have remained within 10% of baseline ) was 0.1 ( 0.2 ) iu for patients treated with linagliptin and 0.4 ( 0.2 ) iu for those treated with placebo . from week 24 , mean basal insulin dose increased in the linagliptin group to a lesser extent than in the placebo group ( fig . the adjusted mean ( sd ) changes from baseline in insulin dose at week 52 were 2.6 ( 0.8 ) iu for linagliptin and 4.2 ( 0.8 ) iu for placebo ( p < 0.003 ) . the overall incidence of patients with 1 reported ae was comparable between treatment groups ( linagliptin 78.4% , placebo 81.4% ) ( supplementary table 2 ) . aes were primarily of mild or moderate intensity ; aes of severe intensity occurred in 8.2% and 8.3% of patients in the linagliptin and placebo groups , respectively . the most commonly reported severe aes in both treatment groups were hypoglycemia , coronary artery disease , osteoarthritis , cardiac failure , pneumonia , diarrhea , arthralgia , gastroenteritis , acute renal failure , and subdural hematoma . aes considered to be drug - related occurred in 18.7% and 22.2% of linagliptin and placebo patients , respectively . drug - related hypoglycemia occurred in 83 ( 15.1% ) and 95 ( 13.2% ) patients in the linagliptin and placebo groups , respectively , and was the only drug - related ae with an incidence greater than 2% . aes leading to discontinuation of trial medication occurred in 21 patients ( 3.3% ) in the linagliptin group and 28 patients ( 4.4% ) in the placebo group . there were no clinically relevant changes in vital signs or laboratory parameters in either group , including no between - group imbalance in shifts in stage of renal impairment . the percentage of patients with investigator - defined hypoglycemia was not different between groups either at week 24 ( linagliptin 22.0% , placebo 23.2% ) or at the end of treatment ( linagliptin 31.4% , placebo 32.9% ) . incidence of severe hypoglycemia was also similar between groups ( week 24 linagliptin 0.3% , placebo 0.6% ; end of treatment linagliptin 1.7% , placebo 1.1% ) ( supplementary table 2 ) . there was no significant change from baseline in mean body weight at either week 24 ( linagliptin 0.16 [ 0.12 ] kg , placebo 0.12 [ 0.11 ] kg ) or at week 52 ( linagliptin 0.3 [ 0.19 ] kg , placebo 0.04 [ 0.18 ] kg ) . adjudicated cardiovascular events occurred in 18 linagliptin patients ( 2.9% ) and 11 placebo patients ( 1.7% ) . cardiovascular deaths occurred in 5 patients ( 0.8% ) in the linagliptin group and 1 ( 0.2% ) in the placebo group . total mortality , however , was comparable between the arms , with 5 deaths in each group . in patients with inadequate glycemic control despite treatment with oads , basal insulin therapy is recommended with or without additional oads ( 14 ) . this phase iii clinical trial demonstrated that , in basal - insulin treated patients with type 2 diabetes and inadequate glycemic control ( 15,16 ) , the addition of linagliptin 5 mg once - daily improved glycemic control without increasing the risk of hypoglycemia or body weight gain . these improvements were not affected by concomitant use of oads , type of basal insulin , age , or degree of renal impairment . previous studies have also shown that dpp-4 inhibitors can significantly decrease hba1c concentration when added to various insulin regimens , with the exception of sitagliptin , without an additional risk of hypoglycemia ( 813 ) . our study differs from the previous trials in specifically testing a more homogenous population of basal insulin treated patients and including both a period of stable insulin dosing , permitting robust assessment of the efficacy and safety of add - on linagliptin , and an extension period , during which patients were allowed to adjust the insulin according to the investigator criteria . addition of linagliptin to basal insulin was associated with a low frequency of hypoglycemia , implying that dose reduction of basal insulin to avoid hypoglycemia when coadministering linagliptin may not be necessary . although the extension period was intended primarily to provide long - term safety data for linagliptin , it also provided additional information on utilization in clinical practice . in the previous trials of other dpp-4 inhibitors , the insulin dose was either kept stable ( 8,9,13 ) or titrated ( 1012 ) , but not both . during the extension period , the insulin dose was increased more in the placebo group than in the linagliptin group . the magnitude of the increase in insulin dose , however , was much less than recommended in the study protocol ( i.e. , to achieve an fpg target of 6.1 mmol / l ) . because no forced titration was requested , this may have resulted in some titration inertia . of note , the proportion of elderly patients and patients with renal impairment was high in the study , and it appears likely that investigators may have been more reluctant to uptitrate basal insulin because age and renal impairment both increase risk of hypoglycemia . the mean fpg change from baseline at week 52 was similar between groups , indicating that clinicians were targeting similar fpg goals . patients themselves are also often reluctant to uptitrate insulin because of the potentially increased risk of hypoglycemia . this represents a significant consideration driving the need for additional treatments such as linagliptin to improve glycemic control . the low risk of hypoglycemia with linagliptin could potentially reduce indirect costs , such as hospitalization , and this may counterbalance the added direct expense ( 17,18 ) . in contrast to other dpp-4 inhibitors , linagliptin has a primarily nonrenal route of elimination and therefore does not require dose adjustment in patients with impaired renal function ( 6,19 ) . because many patients taking insulin have impaired renal function , this is a potential advantage of linagliptin relative to other dpp-4 inhibitors . a previous study found linagliptin to be well tolerated and efficacious in long - term use in patients with severe renal impairment ( 20 ) . in conclusion , this study shows that addition of linagliptin to basal insulin improves glycemic control without increasing hypoglycemia or inducing weight gain , with the additional advantage that the dose does not need to be altered in the elderly or in those with impaired renal function .

objectiveto evaluate the efficacy and long - term safety of linagliptin added to basal insulins in type 2 diabetes inadequately controlled on basal insulin with or without oral agents.research design and methodsa total of 1,261 patients ( hba1c 7.0% [ 53 mmol / mol ] to 10.0% [ 86 mmol / mol ] ) on basal insulin alone or combined with metformin and/or pioglitazone were randomized ( 1:1 ) to double - blind treatment with linagliptin 5 mg once daily or placebo for 52 weeks . the basal insulin dose was kept unchanged for 24 weeks but could thereafter be titrated according to fasting plasma glucose levels at the investigators discretion . the primary end point was the mean change in hba1c from baseline to week 24 . the safety analysis incorporated data up to a maximum of 110 weeks.resultsat week 24 , hba1c changed from a baseline of 8.3% ( 67 mmol / mol ) by 0.6% ( 6.6 mmol / mol ) and by 0.1% ( 1.1 mmol / mol ) with linagliptin and placebo , respectively ( treatment difference 0.65% [ 95% ci 0.74 to 0.55 ] [ 7.1 mmol / mol ] ; p < 0.0001 ) . despite the option to uptitrate basal insulin , it was adjusted only slightly upward ( week 52 , linagliptin 2.6 iu / day , placebo 4.2 iu / day ; p < 0.003 ) , resulting in no further hba1c improvements . frequencies of hypoglycemia ( week 24 , linagliptin 22.0% , placebo 23.2% ; treatment end , linagliptin 31.4% , placebo 32.9% ) and adverse events ( linagliptin 78.4% , placebo 81.4% ) were similar between groups . mean body weight remained unchanged ( week 52 , linagliptin 0.30 kg , placebo 0.04 kg).conclusionslinagliptin added to basal insulin therapy significantly improved glycemic control relative to placebo without increasing hypoglycemia or body weight .

RESEARCH DESIGN AND METHODS Study design and patients End points and assessments Statistical analyses RESULTS Patient disposition, demographics, and baseline clinical characteristics Efficacy outcomes Changes in HbA Subgroup analyses of HbA Use of rescue therapy and changes in background therapy. Safety and tolerability CONCLUSIONS

patients were eligible if they were 18 years of age with a diagnosis of type 2 diabetes , had inadequate glycemic control ( hba1c 7.0% [ 53 mmol / mol ] to 10.0% [ 86 mmol / mol ] ) , had a bmi of 45 kg / m , and were receiving treatment with basal insulin , alone or in combination with metformin and/or pioglitazone , for 12 weeks . after this placebo run - in period , eligible patients were randomly assigned ( 1:1 ratio ) to receive double - blind linagliptin 5 mg once - daily or placebo in addition to continued basal insulin for at least 52 weeks . after week 24 , adjustments to the dose of basal insulin ( but not oads ) were allowed according to the medical judgment of the investigator , with a treatment target for fpg of 6.1 rescue therapy could be initiated during randomized treatment if a patient met the following criteria : confirmed fpg ( after overnight fast ) > 13.3 mmol / l during the first 12 weeks , fpg > 11.1 mmol / l from weeks 12 to 24 , or fpg > 10.0 mmol / l or hba1c > 8.0% ( 64 mmol / mol ) after week 24 . secondary end points included changes from baseline in hba1c and fpg with time , change from baseline in fpg after 52 weeks of treatment , the proportion of patients achieving hba1c < 7% ( 53 mmol / mol ) , the proportion of patients achieving 0.5% ( 5.5 mmol / mol ) reduction in hba1c , and the change from baseline in mean basal insulin dose after 52 weeks of treatment . allowing for sd of change in hba1c from baseline of 1.2% ( 13.1 mmol / mol ) , 284 patients per treatment group were sufficient to achieve 93% power to detect a 0.35% ( 3.8 mmol / mol ) difference between groups in change in hba1c from baseline to week 24 . patients were eligible if they were 18 years of age with a diagnosis of type 2 diabetes , had inadequate glycemic control ( hba1c 7.0% [ 53 mmol / mol ] to 10.0% [ 86 mmol / mol ] ) , had a bmi of 45 kg / m , and were receiving treatment with basal insulin , alone or in combination with metformin and/or pioglitazone , for 12 weeks . after this placebo run - in period , eligible patients were randomly assigned ( 1:1 ratio ) to receive double - blind linagliptin 5 mg once - daily or placebo in addition to continued basal insulin for at least 52 weeks . after week 24 , adjustments to the dose of basal insulin ( but not oads ) were allowed according to the medical judgment of the investigator , with a treatment target for fpg of 6.1 rescue therapy could be initiated during randomized treatment if a patient met the following criteria : confirmed fpg ( after overnight fast ) > 13.3 mmol / l during the first 12 weeks , fpg > 11.1 mmol / l from weeks 12 to 24 , or fpg > 10.0 mmol / l or hba1c > 8.0% ( 64 mmol / mol ) after week 24 . secondary end points included changes from baseline in hba1c and fpg with time , change from baseline in fpg after 52 weeks of treatment , the proportion of patients achieving hba1c < 7% ( 53 mmol / mol ) , the proportion of patients achieving 0.5% ( 5.5 mmol / mol ) reduction in hba1c , and the change from baseline in mean basal insulin dose after 52 weeks of treatment . allowing for sd of change in hba1c from baseline of 1.2% ( 13.1 mmol / mol ) , 284 patients per treatment group were sufficient to achieve 93% power to detect a 0.35% ( 3.8 mmol / mol ) difference between groups in change in hba1c from baseline to week 24 . the adjusted mean ( se ) change in hba1c from baseline at week 24 for linagliptin was 0.58% ( 0.08 ) ( 6.3 [ 0.9 ] mmol / mol ) , compared with 0.07% ( 0.08 ) ( 0.8 [ 0.9 ] mmol / mol ) for placebo , resulting in a placebo - adjusted mean change in hba1c from baseline of 0.65% ( 95% ci 0.74 to 0.55 ) ( 7.1 mmol / mol ; p < 0.0001 ) . after 52 weeks , the adjusted mean changes in hba1c from baseline were 0.48% ( 0.08 ) ( 5.2 [ 0.9 ] mmol / mol ) for linagliptin and 0.05% ( 0.08 ) ( 0.5 [ 0.9 ] mmol / mol ) for placebo ( placebo - adjusted difference 0.53% [ 95% ci 0.64 to 0.43 ] , 5.8 mmol / mol ; p < 0.0001 ) . at week 24 , mmol / l ( 95% ci 0.9 to 0.4 ; p < 0.001 ) . among patients with baseline hba1c 7.0% ( 53 mmol / mol ) , after 52 weeks an hba1c < 7.0% ( 53 mmol / mol ) was achieved by 16% and 7% of the linagliptin and placebo groups , respectively ( p < 0.001 ) . compared with the placebo group , patients in the linagliptin group were more likely to have a decrease in hba1c of 0.5% ( 5.5 mmol / mol ) after 52 weeks of treatment ( 37% linagliptin vs. 17% placebo ; p < 0.0001 ) . placebo - adjusted mean ( sd ) changes in hba1c were greatest for patients with a baseline hba1c of 9.0% ( 75 mmol / mol ) ( 0.83% [ 0.10 ] ) , patients who were from asia ( 1.00 [ 0.15 ] ) or of asian race ( 0.93% [ 0.14 ] ) , and patients who had been diagnosed with diabetes for > 5 years ( 0.72% [ 0.05 ] ) . the placebo - adjusted mean change from baseline in hba1c after 24 weeks was 0.52% ( 5.7 mmol / mol ) for patients taking no oads at baseline ( n = 96 [ 15.50% ] ; p < 0.0001 ) , 0.67% ( 7.3 mmol / mol ) for those taking metformin only ( n = 470 [ 76.1% ] ; p < 0.0001 ) , 0.76% ( 8.3 mmol / mol ) for those taking pioglitazone only ( n = 12 [ 1.0% ] ; p = 0.129 ) , and 0.71% ( 7.8 mmol / mol ) for patients taking both metformin and pioglitazone ( n = 91 [ 7.4% ] ; p < 0.0001 ) . more patients required rescue medication in the placebo group ( 50.4% ) than in the linagliptin group ( 38.2% ) ; the odds ratio ( linagliptin vs. placebo ) for rescue medication was 0.575 ( 95% ci 0.4540.728 ; p < 0.0001 ) . the mean ( sd ) change in basal insulin dose to week 24 ( when dose was to have remained within 10% of baseline ) was 0.1 ( 0.2 ) iu for patients treated with linagliptin and 0.4 ( 0.2 ) iu for those treated with placebo . the adjusted mean ( sd ) changes from baseline in insulin dose at week 52 were 2.6 ( 0.8 ) iu for linagliptin and 4.2 ( 0.8 ) iu for placebo ( p < 0.003 ) . the overall incidence of patients with 1 reported ae was comparable between treatment groups ( linagliptin 78.4% , placebo 81.4% ) ( supplementary table 2 ) . the percentage of patients with investigator - defined hypoglycemia was not different between groups either at week 24 ( linagliptin 22.0% , placebo 23.2% ) or at the end of treatment ( linagliptin 31.4% , placebo 32.9% ) . incidence of severe hypoglycemia was also similar between groups ( week 24 linagliptin 0.3% , placebo 0.6% ; end of treatment linagliptin 1.7% , placebo 1.1% ) ( supplementary table 2 ) . there was no significant change from baseline in mean body weight at either week 24 ( linagliptin 0.16 [ 0.12 ] kg , placebo 0.12 [ 0.11 ] kg ) or at week 52 ( linagliptin 0.3 [ 0.19 ] kg , placebo 0.04 [ 0.18 ] kg ) . the adjusted mean ( se ) change in hba1c from baseline at week 24 for linagliptin was 0.58% ( 0.08 ) ( 6.3 [ 0.9 ] mmol / mol ) , compared with 0.07% ( 0.08 ) ( 0.8 [ 0.9 ] mmol / mol ) for placebo , resulting in a placebo - adjusted mean change in hba1c from baseline of 0.65% ( 95% ci 0.74 to 0.55 ) ( 7.1 mmol / mol ; p < 0.0001 ) . after 52 weeks , the adjusted mean changes in hba1c from baseline were 0.48% ( 0.08 ) ( 5.2 [ 0.9 ] mmol / mol ) for linagliptin and 0.05% ( 0.08 ) ( 0.5 [ 0.9 ] mmol / mol ) for placebo ( placebo - adjusted difference 0.53% [ 95% ci 0.64 to 0.43 ] , 5.8 mmol / mol ; p < 0.0001 ) . at week 24 , mmol / l ( 95% ci 0.9 to 0.4 ; p < 0.001 ) . by week 52 , the mean changes in fpg from baseline were similar between the groups ( linagliptin baseline 7.8 mmol / l , 52-week 7.6 mmol / l , change from baseline 0.2 [ 0.2 ] mmol / l ; placebo baseline 7.8 mmol / l , 52-week 7.6 mmol / l , change from baseline 0.3 [ 0.2 ] mmol / l ) . among patients with baseline 7.0% ( 53 mmol / mol ) , after 52 weeks an hba1c < 7.0% ( 53 mmol / mol ) was achieved by 16% and 7% of the linagliptin and placebo groups , respectively ( p < 0.001 ) . compared with the placebo group , patients in the linagliptin group were more likely to have a decrease in hba1c of 0.5% ( 5.5 mmol / mol ) after 52 weeks of treatment ( 37% linagliptin vs. 17% placebo ; p < 0.0001 ) . placebo - adjusted mean ( sd ) changes in hba1c were greatest for patients with a baseline hba1c of 9.0% ( 75 mmol / mol ) ( 0.83% [ 0.10 ] ) , patients who were from asia ( 1.00 [ 0.15 ] ) or of asian race ( 0.93% [ 0.14 ] ) , and patients who had been diagnosed with diabetes for > 5 years ( 0.72% [ 0.05 ] ) . the placebo - adjusted mean change from baseline in hba1c after 24 weeks was 0.52% ( 5.7 mmol / mol ) for patients taking no oads at baseline ( n = 96 [ 15.50% ] ; p < 0.0001 ) , 0.67% ( 7.3 mmol / mol ) for those taking metformin only ( n = 470 [ 76.1% ] ; p < 0.0001 ) , 0.76% ( 8.3 mmol / mol ) for those taking pioglitazone only ( n = 12 [ 1.0% ] ; p = 0.129 ) , and 0.71% ( 7.8 mmol / mol ) for patients taking both metformin and pioglitazone ( n = 91 [ 7.4% ] ; p < 0.0001 ) . more patients required rescue medication in the placebo group ( 50.4% ) than in the linagliptin group ( 38.2% ) ; the odds ratio ( linagliptin vs. placebo ) for rescue medication was 0.575 ( 95% ci 0.4540.728 ; p < 0.0001 ) . the mean ( sd ) change in basal insulin dose to week 24 ( when dose was to have remained within 10% of baseline ) was 0.1 ( 0.2 ) iu for patients treated with linagliptin and 0.4 ( 0.2 ) iu for those treated with placebo . the adjusted mean ( sd ) changes from baseline in insulin dose at week 52 were 2.6 ( 0.8 ) iu for linagliptin and 4.2 ( 0.8 ) iu for placebo ( p < 0.003 ) . the adjusted mean ( se ) change in hba1c from baseline at week 24 for linagliptin was 0.58% ( 0.08 ) ( 6.3 [ 0.9 ] mmol / mol ) , compared with 0.07% ( 0.08 ) ( 0.8 [ 0.9 ] mmol / mol ) for placebo , resulting in a placebo - adjusted mean change in hba1c from baseline of 0.65% ( 95% ci 0.74 to 0.55 ) ( 7.1 mmol / mol ; p < 0.0001 ) . after 52 weeks , the adjusted mean changes in hba1c from baseline were 0.48% ( 0.08 ) ( 5.2 [ 0.9 ] mmol / mol ) for linagliptin and 0.05% ( 0.08 ) ( 0.5 [ 0.9 ] mmol / mol ) for placebo ( placebo - adjusted difference 0.53% [ 95% ci 0.64 to 0.43 ] , 5.8 mmol / mol ; p < 0.0001 ) . at week 24 , mmol / l ( 95% ci 0.9 to 0.4 ; p < 0.001 ) . by week 52 , the mean changes in fpg from baseline were similar between the groups ( linagliptin baseline 7.8 mmol / l , 52-week 7.6 mmol / l , change from baseline 0.2 [ 0.2 ] mmol / l ; placebo baseline 7.8 mmol / l , 52-week 7.6 mmol / l , change from baseline 0.3 [ 0.2 ] mmol / l ) . among patients with baseline 7.0% ( 53 mmol / mol ) , after 52 weeks an hba1c < 7.0% ( 53 mmol / mol ) was achieved by 16% and 7% of the linagliptin and placebo groups , respectively ( p < 0.001 ) . compared with the placebo group , patients in the linagliptin group were more likely to have a decrease in hba1c of 0.5% ( 5.5 mmol / mol ) after 52 weeks of treatment ( 37% linagliptin vs. 17% placebo ; p < 0.0001 ) . analysis of change in hba1c by prespecified subgroups demonstrated no significant interaction with treatment according to renal function category ( p = 0.5784 ) , type of basal insulin ( p = 0.9511 ) , age group ( p = 0.1000 ) , concomitant use of oads ( p = 0.64 ) , sex ( p = 0.98 ) , or bmi ( p = 0.99 ) , indicating that none of these factors altered the efficacy of linagliptin . placebo - adjusted mean ( sd ) changes in hba1c were greatest for patients with a baseline hba1c of 9.0% ( 75 mmol / mol ) ( 0.83% [ 0.10 ] ) , patients who were from asia ( 1.00 [ 0.15 ] ) or of asian race ( 0.93% [ 0.14 ] ) , and patients who had been diagnosed with diabetes for > 5 years ( 0.72% [ 0.05 ] ) . the placebo - adjusted mean change from baseline in hba1c after 24 weeks was 0.52% ( 5.7 mmol / mol ) for patients taking no oads at baseline ( n = 96 [ 15.50% ] ; p < 0.0001 ) , 0.67% ( 7.3 mmol / mol ) for those taking metformin only ( n = 470 [ 76.1% ] ; p < 0.0001 ) , 0.76% ( 8.3 mmol / mol ) for those taking pioglitazone only ( n = 12 [ 1.0% ] ; p = 0.129 ) , and 0.71% ( 7.8 mmol / mol ) for patients taking both metformin and pioglitazone ( n = 91 [ 7.4% ] ; p < 0.0001 ) . more patients required rescue medication in the placebo group ( 50.4% ) than in the linagliptin group ( 38.2% ) ; the odds ratio ( linagliptin vs. placebo ) for rescue medication was 0.575 ( 95% ci 0.4540.728 ; p < 0.0001 ) . the mean ( sd ) change in basal insulin dose to week 24 ( when dose was to have remained within 10% of baseline ) was 0.1 ( 0.2 ) iu for patients treated with linagliptin and 0.4 ( 0.2 ) iu for those treated with placebo . the adjusted mean ( sd ) changes from baseline in insulin dose at week 52 were 2.6 ( 0.8 ) iu for linagliptin and 4.2 ( 0.8 ) iu for placebo ( p < 0.003 ) . the overall incidence of patients with 1 reported ae was comparable between treatment groups ( linagliptin 78.4% , placebo 81.4% ) ( supplementary table 2 ) . the percentage of patients with investigator - defined hypoglycemia was not different between groups either at week 24 ( linagliptin 22.0% , placebo 23.2% ) or at the end of treatment ( linagliptin 31.4% , placebo 32.9% ) . incidence of severe hypoglycemia was also similar between groups ( week 24 linagliptin 0.3% , placebo 0.6% ; end of treatment linagliptin 1.7% , placebo 1.1% ) ( supplementary table 2 ) . there was no significant change from baseline in mean body weight at either week 24 ( linagliptin 0.16 [ 0.12 ] kg , placebo 0.12 [ 0.11 ] kg ) or at week 52 ( linagliptin 0.3 [ 0.19 ] kg , placebo 0.04 [ 0.18 ] kg ) . this phase iii clinical trial demonstrated that , in basal - insulin treated patients with type 2 diabetes and inadequate glycemic control ( 15,16 ) , the addition of linagliptin 5 mg once - daily improved glycemic control without increasing the risk of hypoglycemia or body weight gain .

the study population was composed of members of the mutualit sociale agricole ( msa ) , a health insurance system for workers in agriculture and related services . we identified pig farmers as an exposed group and nonfarmers ( such as those working at banks or in insurance services ) as a nonexposed group . the sample size was calculated according to results on the prevalence of antimicrobial resistance in the fecal flora of french residents ( 15 ) to ensure that , for most markers measured , detection of a 10% difference in the exposed group would be found with a power of 80% and an risk of 5% . pig farmers were chosen among those working in large , exclusively pig farms ( > 84 pigs ) and contacted during the yearly msa preventive medicine visits to obtain permission for participation . one pig farmer per farm was randomly selected to fill a panel of 20 in each of the seven major french porcine production areas . one nonfarmer control , matched for sex , age , and county of residence , was selected for each pig farmer and approached similarly . nonfarmers were not living or working on a farm , in a slaughterhouse , or in the pharmaceutical industry and were not living with someone who worked on a farm . persons included in the study were judged healthy by physical examination , had no gastrointestinal symptoms or throat pain at inclusion , and reported that they had not been hospitalized or taken antimicrobial agents within the previous month . antimicrobial use in the 6 months preceding the study was retrospectively estimated from the msa reimbursement database and converted to defined daily doses , as described ( 16 ) . in cases in which methicillin - resistant staphylococcus aureus ( mrsa ) was isolated , participants were further interviewed for hospitalization and contacts with hospitalized patients and healthcare workers during the previous year , as described ( 17 ) . occurrence and type of contact with pigs and contact precautions used in farms were documented in pig farmers with a standardized questionnaire . they likely did , however , since participants were contacted during the yearly msa preventive medicine visits by the practitioner with whom they had an established confidential relationship . nasal swabs were inserted ( 1 cm ) successively in both nares and rotated three times for 10 to 15 s. pharyngeal samples were obtained by firmly pressing a swab over the tonsils and the posterior pharyngeal wall , and avoiding touching the jaws , teeth , or gingival when withdrawing the swab . all swabs were extemporaneously squeezed in sterile brain - heart infusion broth ( biomrieux , marcy - letoile , france ) with 10% glycerol , immerged in liquid nitrogen within 6 hours , and stored at 80c until processing . antimicrobial susceptibility of one isolate per participant was determined by using the disk diffusion technique ( 18 ) . for the pharyngeal samples , isolation of streptococcus pneumoniae and -hemolytic streptococci was performed on 5% sheep blood columbia agar ; isolation of haemophilus influenzae was performed on chocolate agar , staphylococcus aureus on chapman agar , and yeast on chromagar ( all biomrieux ) . isolation of antimicrobial - resistant nongroupable streptococci was performed on 5% sheep blood columbia agar supplemented with nalidixic acid and colistin . antimicrobial - resistant nongroupable streptococci were detected on the same medium , supplemented with ampicillin ( 4 mg / l ) or erythromycin ( 1 mg / l ) . for feces , aliquots were plated on chromagar , cetrimide ( bio - mrieux ) , and chapman agar for detection of yeasts , pseudomonas aeruginosa , and s. aureus , respectively . detection of enterococci of any resistance phenotype and of those resistant to erythromycin was performed on bile - esculin - agar ( bea ) ( biomrieux ) free of antimicrobial agents or supplemented with 5 mg detection of vancomycin - resistant enterococci ( vre ) was performed on bea supplemented with 10 mg vancomycin / l after an enrichment step of 18 hours in broth containing 1 mg vancomycin / l , as described ( 19,20 ) . the mechanism of vancomycin resistance was determined by polymerase chain reaction analysis , as described ( 21 ) . carriage of resistant enterobacteria was detected by using two separate procedures , as described ( 22 ) , with modifications . in the first , designed to explore the subdominant flora , 0.1 ml of broth was plated on drigalski agar supplemented with ampicillin ( 10 mg / l ) , ceftazidime ( 2 mg / l ) , streptomycin ( 20 mg / l ) , kanamycin ( 20 mg / l ) , chloramphenicol ( 20 mg / l ) , tetracycline ( 10 mg / l ) , or nalidixic acid ( 50 mg / l ) , as described ( 15 ) . one of 10 positive plates was selected for quality control , and one colony was selected for antimicrobial susceptibility testing . a study participant was defined as colonized in the subdominant fecal flora with enterobacteria resistant to a given antimicrobial agent when at least one colony grew from the plate containing the corresponding antimicrobial agent . in the second procedure , designed to explore the predominant fecal flora , drigalski agar plates without antimicrobial agents were spread with 0.1 ml of broth culture . a study participant was defined as colonized in the predominant flora by e. coli resistant to a given antimicrobial agent when at least one resistant strain was recovered from the feces by using this second procedure . the prescribed defined daily doses of an antimicrobial agent and the number of participants who had ordered antimicrobial agents within the previous 6 months were compared between pig farmers and nonfarmers by using the student t test for matched data . differences between groups for carriage of nasal , pharyngeal , and fecal microbial species were analyzed by calculating matched prevalence ratios ( pr ) ( 23 ) . for comparing antimicrobial - resistant phenotypes of s. aureus , nongroupable streptococci , e. coli , enterococci , and enterobacteria from pig farmers and nonfarmer carriers , nonmatched pr were used , since these comparisons were performed on subgroups composed of only the carriers of the species among the resistant clones for which we looked . ( for instance , rates of carriage of resistant enterobacteria were composed from subgroups of those actually carrying enterobacteria . ) because this analysis was performed only for carriers , a comparison in terms of age , sex , and location was performed to assess that pig farmers and nonfarmer carrier subgroups were comparable for these variables . frequency of co - resistance to ampicillin , streptomycin , and trimethoprim - sulfamethoxazole in predominant strains of e. coli was used as a marker for multiple resistance and compared between groups ( 23 ) . in analyzing data , we did not adjust for making multiple comparisons ( 24 ) since adjusting remains controversial ( 25,26 ) , particularly for actual observations on nature ( 27 ) . the association between isolation of resistant strains and specific farming activities and the size of farms was assessed by chi - square analysis . the study population was composed of members of the mutualit sociale agricole ( msa ) , a health insurance system for workers in agriculture and related services . we identified pig farmers as an exposed group and nonfarmers ( such as those working at banks or in insurance services ) as a nonexposed group . the sample size was calculated according to results on the prevalence of antimicrobial resistance in the fecal flora of french residents ( 15 ) to ensure that , for most markers measured , detection of a 10% difference in the exposed group would be found with a power of 80% and an risk of 5% . pig farmers were chosen among those working in large , exclusively pig farms ( > 84 pigs ) and contacted during the yearly msa preventive medicine visits to obtain permission for participation . one pig farmer per farm was randomly selected to fill a panel of 20 in each of the seven major french porcine production areas . one nonfarmer control , matched for sex , age , and county of residence , was selected for each pig farmer and approached similarly . nonfarmers were not living or working on a farm , in a slaughterhouse , or in the pharmaceutical industry and were not living with someone who worked on a farm . persons included in the study were judged healthy by physical examination , had no gastrointestinal symptoms or throat pain at inclusion , and reported that they had not been hospitalized or taken antimicrobial agents within the previous month . antimicrobial use in the 6 months preceding the study was retrospectively estimated from the msa reimbursement database and converted to defined daily doses , as described ( 16 ) . in cases in which methicillin - resistant staphylococcus aureus ( mrsa ) was isolated , participants were further interviewed for hospitalization and contacts with hospitalized patients and healthcare workers during the previous year , as described ( 17 ) . occurrence and type of contact with pigs and contact precautions used in farms were documented in pig farmers with a standardized questionnaire . they likely did , however , since participants were contacted during the yearly msa preventive medicine visits by the practitioner with whom they had an established confidential relationship . nasal swabs were inserted ( 1 cm ) successively in both nares and rotated three times for 10 to 15 s. pharyngeal samples were obtained by firmly pressing a swab over the tonsils and the posterior pharyngeal wall , and avoiding touching the jaws , teeth , or gingival when withdrawing the swab . all swabs were extemporaneously squeezed in sterile brain - heart infusion broth ( biomrieux , marcy - letoile , france ) with 10% glycerol , immerged in liquid nitrogen within 6 hours , and stored at 80c until processing . one hundred microliter aliquots of all broth samples were plated as follows . for nasal samples , antimicrobial susceptibility of one isolate per participant was determined by using the disk diffusion technique ( 18 ) . for the pharyngeal samples , isolation of streptococcus pneumoniae and -hemolytic streptococci was performed on 5% sheep blood columbia agar ; isolation of haemophilus influenzae was performed on chocolate agar , staphylococcus aureus on chapman agar , and yeast on chromagar ( all biomrieux ) . isolation of antimicrobial - resistant nongroupable streptococci was performed on 5% sheep blood columbia agar supplemented with nalidixic acid and colistin . antimicrobial - resistant nongroupable streptococci were detected on the same medium , supplemented with ampicillin ( 4 mg / l ) or erythromycin ( 1 mg , aliquots were plated on chromagar , cetrimide ( bio - mrieux ) , and chapman agar for detection of yeasts , pseudomonas aeruginosa , and s. aureus , respectively . detection of enterococci of any resistance phenotype and of those resistant to erythromycin was performed on bile - esculin - agar ( bea ) ( biomrieux ) free of antimicrobial agents or supplemented with 5 mg detection of vancomycin - resistant enterococci ( vre ) was performed on bea supplemented with 10 mg vancomycin / l after an enrichment step of 18 hours in broth containing 1 mg vancomycin / l , as described ( 19,20 ) . the mechanism of vancomycin resistance was determined by polymerase chain reaction analysis , as described ( 21 ) . carriage of resistant enterobacteria was detected by using two separate procedures , as described ( 22 ) , with modifications . in the first , designed to explore the subdominant flora , 0.1 ml of broth was plated on drigalski agar supplemented with ampicillin ( 10 mg / l ) , ceftazidime ( 2 mg / l ) , streptomycin ( 20 mg / l ) , kanamycin ( 20 mg / l ) , chloramphenicol ( 20 mg / l ) , tetracycline ( 10 mg / l ) , or nalidixic acid ( 50 mg / l ) , as described ( 15 ) . one of 10 positive plates was selected for quality control , and one colony was selected for antimicrobial susceptibility testing . a study participant was defined as colonized in the subdominant fecal flora with enterobacteria resistant to a given antimicrobial agent when at least one colony grew from the plate containing the corresponding antimicrobial agent . in the second procedure , designed to explore the predominant fecal flora , drigalski agar plates without antimicrobial agents were spread with 0.1 ml of broth culture . a study participant was defined as colonized in the predominant flora by e. coli resistant to a given antimicrobial agent when at least one resistant strain was recovered from the feces by using this second procedure . the prescribed defined daily doses of an antimicrobial agent and the number of participants who had ordered antimicrobial agents within the previous 6 months were compared between pig farmers and nonfarmers by using the student t test for matched data . differences between groups for carriage of nasal , pharyngeal , and fecal microbial species were analyzed by calculating matched prevalence ratios ( pr ) ( 23 ) . for comparing antimicrobial - resistant phenotypes of s. aureus , nongroupable streptococci , e. coli , enterococci , and enterobacteria from pig farmers and nonfarmer carriers , nonmatched pr were used , since these comparisons were performed on subgroups composed of only the carriers of the species among the resistant clones for which we looked . ( for instance , rates of carriage of resistant enterobacteria were composed from subgroups of those actually carrying enterobacteria . ) because this analysis was performed only for carriers , a comparison in terms of age , sex , and location was performed to assess that pig farmers and nonfarmer carrier subgroups were comparable for these variables . frequency of co - resistance to ampicillin , streptomycin , and trimethoprim - sulfamethoxazole in predominant strains of e. coli was used as a marker for multiple resistance and compared between groups ( 23 ) . in analyzing data , we did not adjust for making multiple comparisons ( 24 ) since adjusting remains controversial ( 25,26 ) , particularly for actual observations on nature ( 27 ) . the association between isolation of resistant strains and specific farming activities and the size of farms was assessed by chi - square analysis . the overall male - to - female ratio was 6.1 , and mean age was 37.8 years ( range 2172 ) . mean previous time in the professional position occupied at the time of the study was 9.7 + /- health insurance reimbursement data showed that antimicrobial agents had been prescribed in the month preceding the study for two pig farmers ( one with macrolide and one with broad - spectrum penicillin 24 and 28 days before participation , respectively ) and three nonfarmers ( one with oral cephalosporin , one with penicillinase - resistant penicillin , and one with tetracycline 3 , 10 , and 24 days before participation , respectively ) . however , because of the retrospective nature of this analysis , the low number of participants , the nearly even distribution between pig farmers and nonfarmers , and the fact that reimbursement data are not a formal proof that antimicrobial agents were actually taken , these five persons were included in further analysis . neither overall , nor class - specific antimicrobial prescriptions during the 6 months preceding participation in the study were significantly different between pig farmers and nonfarmers ( table 1 ) . prevalence of nasal or pharyngeal isolation of s. aureus was significantly higher in pig farmers ( pr 1.85 ; confidence intervals [ ci ] 1.26 to 2.71 ] ; p < 0.01 ) ( table 2 ) . isolation of erythromycin - resistant strains was significantly more frequent among s. aureus pig farmer carriers than among nonfarmer carriers ( pr 9.72 ; ci 2.53 to 37.30 ; p moreover , 31 ( 87% ) of 36 macrolide - resistant s. aureus isolates from pig farmers were cross - resistant to lincosamides . analysis of the antimicrobial - susceptibility profile of these strains showed that two were resistant to at least one macrolide antimicrobial agent , four were resistant to aminoglycosides , and four were resistant to pefloxacin . three of the mrsa carriers had been hospitalized within the 2 years preceding the study , including one within the previous year . the two other farmers had not been hospitalized but had visited outpatient clinics for medical problems within the year preceding the study . as determined by health insurance reimbursements to pig farmers and nonfarmers . who used any given type of antimicrobial agent . in addition of being resistant to methicillin , two strains were resistant to at least one macrolide antibiotic ( two were resistant [ r ] to erythromycin , lincomycin , and pristinamycin ; 1 susceptible [ s ] to erythromycin only ; and one susceptible to pristinamycin only ) , 4 strains were r to aminoglycosides ( 2 were rrs and 2 rrr to kanamycin , tobramycin , and gentamicin , respectively ) . prevalence of pharyngeal isolation of streptococcus pneumoniae , h. influenzae , and -hemolytic streptococci was low and did not differ significantly between groups ( table 3 ) . isolation of nongroupable streptococci was frequent and not significantly different between groups , but that of nongroupable streptococci resistant to ampicillin was significantly more frequent in pig farmers than in nonfarmers ( pr 2.02 ; ci 1.32 to 3.09 ; p < 0.01 ) . prevalence of fecal enterococci was not significantly different between groups nor was isolation of enterococci resistant to erythromycin or vancomycin ( table 4 ) . in all , 16 vre were isolated including 2 vana - type enterococcus faecium , along with 11 e. gallinarum and 3 e. casseliflavus of vanc phenotype and genotype . nearly all participants carried enterobacteria : 103 ( 94.5% ) of 109 pig farmers and 100 ( 91.7% ) of 109 nonfarmers ( pr 1.03 ; ci 0.96 to 1.10 ; not significant ) . isolation of enterobacteria resistant to nalidixic acid ( pr 7.12 ; ci 2.20 to 23.0 ; p < 0.01 ) , chloramphenicol ( pr 2.08 ; ci 1.17 to 3.68 ) ; p < 0.01 ) , tetracycline ( pr 1.65 ; ci 1.27 to 2.13 ; p < 0.01 ) , and streptomycin ( pr 1.40 ; ci 1.01 to 1.95 ; p < 0.01 ) was significantly more frequent in pig farmer carriers of enterobacteria than in nonfarmer carriers . regarding the predominant flora , the most frequent species isolated were escherichia coli ( 917/995 ; 92.2% ) followed by hafnia alvei ( 48/995 ; 4.8% ) and citrobacter freundii ( 11/995 ; 1.1% ) with no significant between - group differences . the prevalence of isolation of e. coli resistant to cotrimoxazole ( pr 3.02 ; ci 1.68 to 5.44 ; p < 0.01 ) , tetracycline ( pr 2.22 ; ci 1.48 to 3.32 ; p < 0.01 ) , streptomycin ( pr 1.40 ; ci 1.01 to 1.95 ; p = 0.04 ) , or nalidixic acid ( pr not calculable ; p < 0.01 ) was significantly higher in pig farmers carrying e. coli than in nonfarmers ( table 4 ) . in all instances in which subgroups of pig farmers and nonfarmers were compared , no significant between - group difference emerged in terms of age , sex , and county of residence . prevalence of co - resistance to ampicillin , streptomycin , and cotrimoxazole was also significantly higher in e. coli from pig farmers ( 24% , 24/100 ) than from nonfarmers ( 12.2% , 12/98 ) ( pr 1.96 ; ci 1.04 to 3.70 ; p = 0.03 ) . no strains of clostridium difficile , pseudomonas aeruginosa , or staphylococcus aureus were isolated from the feces of any study participant . prevalence of yeast was not significantly different between pig farmers and nonfarmers , and the species were evenly distributed ( table 4 ) . group a streptococcus:1 , group c : 5 , s. anginosus : 3 , s. intermedius : 1 , s. constellatus : 4 . group a streptococcus : 1 , group c : 5 , s. anginosus : 3 , s. intermedius : 1 , s. constellatus : 3 . . e. faecium : 2 , e. gallinarum : 5 , e. casseliflavus : 3 . using direct plating plating on drigalski agar without or with antimicrobial agents ( first technique , see methods ) . na , not applicable . from the predominant fecal flora ( second technique , see methods ) . candida albicans : 1 , geotrichum sp . : 15 , c. glabrata : 2 , rhodolulora sp . : 1 . c. albicans : 2 , geotrichum sp . : 14 , saccharomyces cerevesia : 2 . we found no statistical association between professional activity or use of masks and gloves and the prevalence of resistant bacteria . by contrast , prevalence of nasal isolation of s. aureus resistant to macrolides increased significantly , from 33% ( 5/15 ) in pig farmers working in farms raising 84180 swine , to 70% ( 7/10 ) , 92% ( 11/12 ) , and 100% ( 13/13 ) in those working in farms raising 181270 , 271399 , and > 400 swine , respectively ( chi - square linear slope ; p < 0.01 ) . during that activity . our results showed that the prevalence of antimicrobial drug resistance in bacteria from the nasal , pharyngeal , and fecal flora was higher in pig farmers than in nonfarmers . with a few exceptions , pig farmers and nonfarmers had not taken antimicrobial agents during the month preceding the study and had not been differentially exposed to such agents during the previous 6 months . that e. coli ( 1113 ) and enterococci ( 14 ) are significantly more resistant in persons working in farms or slaughterhouses than in urban residents had been reported , but a potential role of antimicrobial treatments in these workers could not be excluded and the increased prevalence of carriage of resistant organisms had not been quantified . the prevalence of s. aureus nasal carriage in nonfarmers was similar to that reported previously in the general population ( 28 ) , which suggests that the higher isolation rate in pig farmers was due to their work environment . this hypothesis was further supported by the increased resistance to macrolides ( still the fourth most common class of antimicrobial agents used in food production ) of s. aureus isolates from pig farmers and the link between this resistance and the size of the farm . several hypotheses , including high transfer of animal specific clones , should be raised and investigated . in the pharynx , ampicillin resistance of nongroupable streptococci in pig farmers may contribute to further transfer of -lactam resistance to streptococcus pneumoniae by transformation ( 29 ) . in the feces , antimicrobial drug resistance in enterobacteria was also greater in pig farmers for four of eight markers tested in the subdominant flora , and for four of nine markers in the predominant flora . resistance in e. coli was close to that of healthy participants from developing countries ( 22 ) . the prevalence of resistance in enterobacteria from the subdominant flora of our nonfarmers was lower than that in participants of the only study published that used the same methods ; however , that study included mostly laboratory workers ( a. andremont , pers . comm . ) , who are known to be more colonized by resistant enterobacteria than are urban and rural dwellers ( 30 ) . the rate of vre colonization that we observed differed from that reported in france ( 31 ) , which might be due to the enrichment step we used ; however , the rate of vre colonization did not differ between farmers and nonfarmers . this finding suggests that the 1997 ban ( 32 ) of avoparcin , a glycopeptide previously used as a growth promoter , was effective . although specific information on avoparcin is lacking , 145 tons of antimicrobial agents were used globally in france in 1998 in pig raising , including 70 mg of growth additive per kilogram of pork meat produced ( 33 ) . three possible explanations may explain why isolation of resistant bacteria in pig farmers was higher than in nonfarmers . first , farmers may come in contact with more antimicrobial - resistant bacteria from pigs ; these bacteria are then transferred to the farmers . second , farmers may be in frequent contact with antimicrobial agents themselves or antimicrobial residues that are given to the pigs in the workplace . the third possibility is that farmers receive more antimicrobial agents for other , i.e. , medical , reasons . the first of these possibilities appears most likely because 1 ) farmers used very few precautions during contact with animal feces , 2 ) antimicrobial exposure is a well - known risk factor for intestinal yeast colonization ( 34,35 ) , and yeast colonization in both groups was low , and 3 ) antimicrobial prescriptions were not significantly different between pig farmers and nonfarmers during the previous 6 months . we did not assess the use of antimicrobial agents for animals in each of the 113 farms where pig farmers worked . however , 1,364 tons of antimicrobial agents were sold in france in 1999 for veterinary medicinal use . of these , tetracycline , cotrimoxazole , and -lactams together accounted for 79.5% ( 8) , a finding compatible with the high resistance rates found in pig farmers . however , we could not assess the exact cause of the high antimicrobial resistance rates in farmers . determining the exact cause may not be as important as the fact that these people are colonized with a much higher rate of resistant bacteria . we minimized the risk that differences in food intake caused the higher prevalence of resistance in pig farmers by matching pig farmers with nonfarmers by age , sex , and county of residence . children can be a source of resistant bacteria in households ( 36 ) and thus might be a confounding factor if the number of children was greater in pig farmer families than in nonfarmer families . however , this factor was not documented in the study questionnaire and thus could not be investigated . some inherent limitations of cross - sectional studies invite cautious assessments of our results . the lack of preexposure data on resistance and the general design of the study preclude determining a causal relationship between exposure and acquired resistance . however , the observation we made indicates that professional pig farming is significantly associated with isolation of antimicrobial - resistant commensal species . the minimal use of contact precautions by pig farmers may have further increased this risk , but the study was not designed to assess the efficacy of contact precautions , and thus no recommendations can be drawn in this matter . pigs could be raised with considerably fewer antimicrobial agents than currently used , and many animals can be raised with little or no exposure to such drugs at all ( 37 ) . additional studies are needed to evaluate the consequences of isolating resistant bacteria in farmers and , if necessary , design appropriate preventive measures .

we assessed the quantitative contribution of pig farming to antimicrobial resistance in the commensal flora of pig farmers by comparing 113 healthy pig farmers from the major french porcine production areas to 113 nonfarmers , each matched for sex , age , and county of residence . all reported that they had not taken antiimicrobial agents within the previous month . throat , nasal , and fecal swabs were screened for resistant microorganisms on agar containing selected antimicrobial agents . nasopharyngeal carriage of staphylococcus aureus was significantly more frequent in pig farmers , as was macrolide resistance of s. aureus from carriers . nongroupable streptococci from the throat were more resistant to the penicillins in pig farmers . the intestinal isolation of enterococci resistant to erythromycin or vancomycin was not significantly higher in pig farmers in contrast to that of enterobacteria resistant to nalidixic acid , chloramphenicol , tetracycline , and streptomycin . prevalence of resistance in predominant fecal enterobacteria was also significantly higher in pig farmers for cotrimoxazole , tetracycline , streptomycin , and nalidixic acid . we determined a significant association between pig farming and isolation of resistant commensal bacteria .

Methods Participants Specimens Obtained Detection of Microbial Isolates Statistical Analysis Results Discussion

the sample size was calculated according to results on the prevalence of antimicrobial resistance in the fecal flora of french residents ( 15 ) to ensure that , for most markers measured , detection of a 10% difference in the exposed group would be found with a power of 80% and an risk of 5% . one pig farmer per farm was randomly selected to fill a panel of 20 in each of the seven major french porcine production areas . one nonfarmer control , matched for sex , age , and county of residence , was selected for each pig farmer and approached similarly . persons included in the study were judged healthy by physical examination , had no gastrointestinal symptoms or throat pain at inclusion , and reported that they had not been hospitalized or taken antimicrobial agents within the previous month . antimicrobial use in the 6 months preceding the study was retrospectively estimated from the msa reimbursement database and converted to defined daily doses , as described ( 16 ) . in cases in which methicillin - resistant staphylococcus aureus ( mrsa ) was isolated , participants were further interviewed for hospitalization and contacts with hospitalized patients and healthcare workers during the previous year , as described ( 17 ) . nasal swabs were inserted ( 1 cm ) successively in both nares and rotated three times for 10 to 15 s. pharyngeal samples were obtained by firmly pressing a swab over the tonsils and the posterior pharyngeal wall , and avoiding touching the jaws , teeth , or gingival when withdrawing the swab . for the pharyngeal samples , isolation of streptococcus pneumoniae and -hemolytic streptococci was performed on 5% sheep blood columbia agar ; isolation of haemophilus influenzae was performed on chocolate agar , staphylococcus aureus on chapman agar , and yeast on chromagar ( all biomrieux ) . isolation of antimicrobial - resistant nongroupable streptococci was performed on 5% sheep blood columbia agar supplemented with nalidixic acid and colistin . detection of enterococci of any resistance phenotype and of those resistant to erythromycin was performed on bile - esculin - agar ( bea ) ( biomrieux ) free of antimicrobial agents or supplemented with 5 mg detection of vancomycin - resistant enterococci ( vre ) was performed on bea supplemented with 10 mg vancomycin / l after an enrichment step of 18 hours in broth containing 1 mg vancomycin / l , as described ( 19,20 ) . carriage of resistant enterobacteria was detected by using two separate procedures , as described ( 22 ) , with modifications . in the first , designed to explore the subdominant flora , 0.1 ml of broth was plated on drigalski agar supplemented with ampicillin ( 10 mg / l ) , ceftazidime ( 2 mg / l ) , streptomycin ( 20 mg / l ) , kanamycin ( 20 mg / l ) , chloramphenicol ( 20 mg / l ) , tetracycline ( 10 mg / l ) , or nalidixic acid ( 50 mg / l ) , as described ( 15 ) . a study participant was defined as colonized in the subdominant fecal flora with enterobacteria resistant to a given antimicrobial agent when at least one colony grew from the plate containing the corresponding antimicrobial agent . in the second procedure , designed to explore the predominant fecal flora , drigalski agar plates without antimicrobial agents were spread with 0.1 ml of broth culture . a study participant was defined as colonized in the predominant flora by e. coli resistant to a given antimicrobial agent when at least one resistant strain was recovered from the feces by using this second procedure . the prescribed defined daily doses of an antimicrobial agent and the number of participants who had ordered antimicrobial agents within the previous 6 months were compared between pig farmers and nonfarmers by using the student t test for matched data . differences between groups for carriage of nasal , pharyngeal , and fecal microbial species were analyzed by calculating matched prevalence ratios ( pr ) ( 23 ) . for comparing antimicrobial - resistant phenotypes of s. aureus , nongroupable streptococci , e. coli , enterococci , and enterobacteria from pig farmers and nonfarmer carriers , nonmatched pr were used , since these comparisons were performed on subgroups composed of only the carriers of the species among the resistant clones for which we looked . because this analysis was performed only for carriers , a comparison in terms of age , sex , and location was performed to assess that pig farmers and nonfarmer carrier subgroups were comparable for these variables . frequency of co - resistance to ampicillin , streptomycin , and trimethoprim - sulfamethoxazole in predominant strains of e. coli was used as a marker for multiple resistance and compared between groups ( 23 ) . the association between isolation of resistant strains and specific farming activities and the size of farms was assessed by chi - square analysis . the sample size was calculated according to results on the prevalence of antimicrobial resistance in the fecal flora of french residents ( 15 ) to ensure that , for most markers measured , detection of a 10% difference in the exposed group would be found with a power of 80% and an risk of 5% . one pig farmer per farm was randomly selected to fill a panel of 20 in each of the seven major french porcine production areas . one nonfarmer control , matched for sex , age , and county of residence , was selected for each pig farmer and approached similarly . persons included in the study were judged healthy by physical examination , had no gastrointestinal symptoms or throat pain at inclusion , and reported that they had not been hospitalized or taken antimicrobial agents within the previous month . antimicrobial use in the 6 months preceding the study was retrospectively estimated from the msa reimbursement database and converted to defined daily doses , as described ( 16 ) . in cases in which methicillin - resistant staphylococcus aureus ( mrsa ) was isolated , participants were further interviewed for hospitalization and contacts with hospitalized patients and healthcare workers during the previous year , as described ( 17 ) . nasal swabs were inserted ( 1 cm ) successively in both nares and rotated three times for 10 to 15 s. pharyngeal samples were obtained by firmly pressing a swab over the tonsils and the posterior pharyngeal wall , and avoiding touching the jaws , teeth , or gingival when withdrawing the swab . for the pharyngeal samples , isolation of streptococcus pneumoniae and -hemolytic streptococci was performed on 5% sheep blood columbia agar ; isolation of haemophilus influenzae was performed on chocolate agar , staphylococcus aureus on chapman agar , and yeast on chromagar ( all biomrieux ) . isolation of antimicrobial - resistant nongroupable streptococci was performed on 5% sheep blood columbia agar supplemented with nalidixic acid and colistin . antimicrobial - resistant nongroupable streptococci were detected on the same medium , supplemented with ampicillin ( 4 mg / l ) or erythromycin ( 1 mg , aliquots were plated on chromagar , cetrimide ( bio - mrieux ) , and chapman agar for detection of yeasts , pseudomonas aeruginosa , and s. aureus , respectively . detection of enterococci of any resistance phenotype and of those resistant to erythromycin was performed on bile - esculin - agar ( bea ) ( biomrieux ) free of antimicrobial agents or supplemented with 5 mg detection of vancomycin - resistant enterococci ( vre ) was performed on bea supplemented with 10 mg vancomycin / l after an enrichment step of 18 hours in broth containing 1 mg vancomycin / l , as described ( 19,20 ) . carriage of resistant enterobacteria was detected by using two separate procedures , as described ( 22 ) , with modifications . in the first , designed to explore the subdominant flora , 0.1 ml of broth was plated on drigalski agar supplemented with ampicillin ( 10 mg / l ) , ceftazidime ( 2 mg / l ) , streptomycin ( 20 mg / l ) , kanamycin ( 20 mg / l ) , chloramphenicol ( 20 mg / l ) , tetracycline ( 10 mg / l ) , or nalidixic acid ( 50 mg / l ) , as described ( 15 ) . a study participant was defined as colonized in the subdominant fecal flora with enterobacteria resistant to a given antimicrobial agent when at least one colony grew from the plate containing the corresponding antimicrobial agent . in the second procedure , designed to explore the predominant fecal flora , drigalski agar plates without antimicrobial agents were spread with 0.1 ml of broth culture . a study participant was defined as colonized in the predominant flora by e. coli resistant to a given antimicrobial agent when at least one resistant strain was recovered from the feces by using this second procedure . the prescribed defined daily doses of an antimicrobial agent and the number of participants who had ordered antimicrobial agents within the previous 6 months were compared between pig farmers and nonfarmers by using the student t test for matched data . differences between groups for carriage of nasal , pharyngeal , and fecal microbial species were analyzed by calculating matched prevalence ratios ( pr ) ( 23 ) . for comparing antimicrobial - resistant phenotypes of s. aureus , nongroupable streptococci , e. coli , enterococci , and enterobacteria from pig farmers and nonfarmer carriers , nonmatched pr were used , since these comparisons were performed on subgroups composed of only the carriers of the species among the resistant clones for which we looked . because this analysis was performed only for carriers , a comparison in terms of age , sex , and location was performed to assess that pig farmers and nonfarmer carrier subgroups were comparable for these variables . frequency of co - resistance to ampicillin , streptomycin , and trimethoprim - sulfamethoxazole in predominant strains of e. coli was used as a marker for multiple resistance and compared between groups ( 23 ) . the association between isolation of resistant strains and specific farming activities and the size of farms was assessed by chi - square analysis . mean previous time in the professional position occupied at the time of the study was 9.7 + /- health insurance reimbursement data showed that antimicrobial agents had been prescribed in the month preceding the study for two pig farmers ( one with macrolide and one with broad - spectrum penicillin 24 and 28 days before participation , respectively ) and three nonfarmers ( one with oral cephalosporin , one with penicillinase - resistant penicillin , and one with tetracycline 3 , 10 , and 24 days before participation , respectively ) . however , because of the retrospective nature of this analysis , the low number of participants , the nearly even distribution between pig farmers and nonfarmers , and the fact that reimbursement data are not a formal proof that antimicrobial agents were actually taken , these five persons were included in further analysis . neither overall , nor class - specific antimicrobial prescriptions during the 6 months preceding participation in the study were significantly different between pig farmers and nonfarmers ( table 1 ) . prevalence of nasal or pharyngeal isolation of s. aureus was significantly higher in pig farmers ( pr 1.85 ; confidence intervals [ ci ] 1.26 to 2.71 ] ; p < 0.01 ) ( table 2 ) . isolation of erythromycin - resistant strains was significantly more frequent among s. aureus pig farmer carriers than among nonfarmer carriers ( pr 9.72 ; ci 2.53 to 37.30 ; p moreover , 31 ( 87% ) of 36 macrolide - resistant s. aureus isolates from pig farmers were cross - resistant to lincosamides . in addition of being resistant to methicillin , two strains were resistant to at least one macrolide antibiotic ( two were resistant [ r ] to erythromycin , lincomycin , and pristinamycin ; 1 susceptible [ s ] to erythromycin only ; and one susceptible to pristinamycin only ) , 4 strains were r to aminoglycosides ( 2 were rrs and 2 rrr to kanamycin , tobramycin , and gentamicin , respectively ) . prevalence of pharyngeal isolation of streptococcus pneumoniae , h. influenzae , and -hemolytic streptococci was low and did not differ significantly between groups ( table 3 ) . isolation of nongroupable streptococci was frequent and not significantly different between groups , but that of nongroupable streptococci resistant to ampicillin was significantly more frequent in pig farmers than in nonfarmers ( pr 2.02 ; ci 1.32 to 3.09 ; p < 0.01 ) . prevalence of fecal enterococci was not significantly different between groups nor was isolation of enterococci resistant to erythromycin or vancomycin ( table 4 ) . isolation of enterobacteria resistant to nalidixic acid ( pr 7.12 ; ci 2.20 to 23.0 ; p < 0.01 ) , chloramphenicol ( pr 2.08 ; ci 1.17 to 3.68 ) ; p < 0.01 ) , tetracycline ( pr 1.65 ; ci 1.27 to 2.13 ; p < 0.01 ) , and streptomycin ( pr 1.40 ; ci 1.01 to 1.95 ; p < 0.01 ) was significantly more frequent in pig farmer carriers of enterobacteria than in nonfarmer carriers . the prevalence of isolation of e. coli resistant to cotrimoxazole ( pr 3.02 ; ci 1.68 to 5.44 ; p < 0.01 ) , tetracycline ( pr 2.22 ; ci 1.48 to 3.32 ; p < 0.01 ) , streptomycin ( pr 1.40 ; ci 1.01 to 1.95 ; p = 0.04 ) , or nalidixic acid ( pr not calculable ; p < 0.01 ) was significantly higher in pig farmers carrying e. coli than in nonfarmers ( table 4 ) . in all instances in which subgroups of pig farmers and nonfarmers were compared , no significant between - group difference emerged in terms of age , sex , and county of residence . prevalence of co - resistance to ampicillin , streptomycin , and cotrimoxazole was also significantly higher in e. coli from pig farmers ( 24% , 24/100 ) than from nonfarmers ( 12.2% , 12/98 ) ( pr 1.96 ; ci 1.04 to 3.70 ; p = 0.03 ) . no strains of clostridium difficile , pseudomonas aeruginosa , or staphylococcus aureus were isolated from the feces of any study participant . prevalence of yeast was not significantly different between pig farmers and nonfarmers , and the species were evenly distributed ( table 4 ) . we found no statistical association between professional activity or use of masks and gloves and the prevalence of resistant bacteria . by contrast , prevalence of nasal isolation of s. aureus resistant to macrolides increased significantly , from 33% ( 5/15 ) in pig farmers working in farms raising 84180 swine , to 70% ( 7/10 ) , 92% ( 11/12 ) , and 100% ( 13/13 ) in those working in farms raising 181270 , 271399 , and > 400 swine , respectively ( chi - square linear slope ; p < 0.01 ) . our results showed that the prevalence of antimicrobial drug resistance in bacteria from the nasal , pharyngeal , and fecal flora was higher in pig farmers than in nonfarmers . with a few exceptions , pig farmers and nonfarmers had not taken antimicrobial agents during the month preceding the study and had not been differentially exposed to such agents during the previous 6 months . that e. coli ( 1113 ) and enterococci ( 14 ) are significantly more resistant in persons working in farms or slaughterhouses than in urban residents had been reported , but a potential role of antimicrobial treatments in these workers could not be excluded and the increased prevalence of carriage of resistant organisms had not been quantified . the prevalence of s. aureus nasal carriage in nonfarmers was similar to that reported previously in the general population ( 28 ) , which suggests that the higher isolation rate in pig farmers was due to their work environment . this hypothesis was further supported by the increased resistance to macrolides ( still the fourth most common class of antimicrobial agents used in food production ) of s. aureus isolates from pig farmers and the link between this resistance and the size of the farm . in the pharynx , ampicillin resistance of nongroupable streptococci in pig farmers may contribute to further transfer of -lactam resistance to streptococcus pneumoniae by transformation ( 29 ) . in the feces , antimicrobial drug resistance in enterobacteria was also greater in pig farmers for four of eight markers tested in the subdominant flora , and for four of nine markers in the predominant flora . resistance in e. coli was close to that of healthy participants from developing countries ( 22 ) . the prevalence of resistance in enterobacteria from the subdominant flora of our nonfarmers was lower than that in participants of the only study published that used the same methods ; however , that study included mostly laboratory workers ( a. andremont , pers . three possible explanations may explain why isolation of resistant bacteria in pig farmers was higher than in nonfarmers . second , farmers may be in frequent contact with antimicrobial agents themselves or antimicrobial residues that are given to the pigs in the workplace . the first of these possibilities appears most likely because 1 ) farmers used very few precautions during contact with animal feces , 2 ) antimicrobial exposure is a well - known risk factor for intestinal yeast colonization ( 34,35 ) , and yeast colonization in both groups was low , and 3 ) antimicrobial prescriptions were not significantly different between pig farmers and nonfarmers during the previous 6 months . of these , tetracycline , cotrimoxazole , and -lactams together accounted for 79.5% ( 8) , a finding compatible with the high resistance rates found in pig farmers . we minimized the risk that differences in food intake caused the higher prevalence of resistance in pig farmers by matching pig farmers with nonfarmers by age , sex , and county of residence . however , the observation we made indicates that professional pig farming is significantly associated with isolation of antimicrobial - resistant commensal species . the minimal use of contact precautions by pig farmers may have further increased this risk , but the study was not designed to assess the efficacy of contact precautions , and thus no recommendations can be drawn in this matter .

vitamin and mineral deficiencies adversely affect a third of the world 's population ( 49 ) , and it is estimated that one in five people worldwide lack sufficient dietary zinc ( 41 ) . adolescent girls may be more prone to zinc deficiency because physiological requirements for zinc peak during adolescence at the time of the pubertal growth spurt , which generally occurs in girls between 10 and 15 years ( 50 ) . especially , adolescents from developing countries such as india are at high risk because of low zinc intakes and poor bioavailability of zinc from vegetarian diets , which leads to low zinc status . in adolescents , a wide range of disturbances occur due to zinc deficiency , including impaired growth , defects in the immune system , dermatitis , diarrhoea , delayed sexual and bone maturation , impaired taste acuity and behavioural changes ( 41 ) . simple measures such as diet quality index are used to assess adherence to dietary guidelines . to identify micronutrient deficiencies in adolescents , in particular that of zinc , a simple tool needs to be devised , which can judge the micronutrient quality of diet . such an index would be of vital importance for identifying at - risk individuals and targeting the intervention programmes . guidelines by who have recommended food - based approaches to resolve micronutrient deficiencies ( 49 ) . moreover , zinc - rich dietary supplement has a potential to revert bad health effects of low zinc status . therefore , increasing consumption of foods with a high content of absorbable zinc can become a long - term sustainable strategy to overcome zinc deficiency . considering the enhancers and inhibitors in vegetarian diets ( 18 ) , low - cost food recipes need to be devised to provide high bio - available zinc . studies have documented positive effects of zinc supplements in infants and children for morbidity and growth ( 51 ) . however , research on the beneficial effects of improved zinc nutrition for behaviour and cognitive function in school - age children is critically needed because zinc deficiency continues to be a modern health concern in both developing and developed countries ( 46 , 52 ) . the present research aimed to study zinc status and its association with profile of other micronutrients , and to examine the effect of zinc supplementation on functional indices of zinc in adolescent girls . the specific objectives of the study were : to study zinc status and its association with the profile of other micronutrients , cognitive performance and taste acuity of the adolescent girls.to examine the diet patterns of vegetarian adolescent girls for zinc adequacy and devise recipes to improve bio - available zinc intakes.to develop and validate a nutrient quality index to assess micronutrient sufficiency of diets using statistical techniques and bioavailability of micronutrients.to examine efficacy of dietary supplementation on blood micronutrient levels and functional indices of zinc . to study zinc status and its association with the profile of other micronutrients , cognitive performance and taste acuity of the adolescent girls . to examine the diet patterns of vegetarian adolescent girls for zinc adequacy and devise recipes to improve bio - available zinc intakes . to develop and validate a nutrient quality index to assess micronutrient sufficiency of diets using statistical techniques and bioavailability of micronutrients . to examine efficacy of dietary supplementation on blood micronutrient levels and functional indices of zinc . the study was conducted in two secondary schools from pune city , maharashtra state , in western india , during 20052007 . in all , 662 girls participated in the study . a physician conducted clinical examinations to assess health status of the girls . girls who were currently suffering or those who had suffered the previous fortnight from any illness such as fever , respiratory or gastro - intestinal infections or those undergoing medical treatment or taking multi - vitamin mineral supplements as per a physician 's recommendation were excluded . thus in all , 630 apparently healthy girls aged 1016 years from the participating schools were recruited for the study . details of the procedures involved in the study were explained to the parents . after giving consent , the study protocol was reviewed and approved by the ethics committee of zensar foundation of india , pune , under the chairmanship of a retired professor of the international institute of population sciences , mumbai . all of the parents gave their written informed consent , and the girls voluntarily participated in the study . nutrient intakes were estimated using cooked foods database ( 53 ) and nutritive value tables ( 54 ) . venous blood samples ( 10 ml ) were collected at 7.30 am after an overnight fast for each girl . haemoglobin ( hb ) was estimated using automated cell counter ( actdiff ii , beckman coulter , fullerton , ca , usa ) . plasma levels of zinc , vitamin c , -carotene and retinol were measured as per the methods in the manual by national institute of nutrition , india ( 55 ) , and erythrocyte zinc was measured as per the method described by kenny et al . reference serum ( randox laboratories , india ) was used as a standard for blood estimations . cognitive performance was evaluated using a group of four tests standardised on indian children ; simple reaction time ( srt ) , recognition reaction time ( rrt ) , raven 's progressive matrices ( rpm ) and visual memory on each participant ( 5759 ) . srt and rrt measure the cognitive speed concerning perception , processing and motor response organisation for reaction tasks and visual search . srt and rrt were measured using a visual reaction time apparatus ( anand agencies , pune , india ) designed as per the procedure given by woodworth et al . rpm is a nonverbal test of performance that measures a person 's ability to form perceptual relations and reason by analogy in research settings and form comparisons ( 58 ) . a short - term visual memory test was carried out for measuring the ability of the girls to retain and reproduce information within a short time span . this test was adopted from the subtest of pgi memory scale developed by the post graduate institute of medical education and research , chandigarh , india , for children and adolescents ( 59 ) . taste acuity was assessed by identifying recognition threshold for salt ( rts ) using 10 different salt solutions ranging from 0 to 25 more details of the measurements and methods used in the study are presented in five research papers ( 6064 ) . the study was conducted in two secondary schools from pune city , maharashtra state , in western india , during 20052007 . in all , 662 girls participated in the study . a physician conducted clinical examinations to assess health status of the girls . girls who were currently suffering or those who had suffered the previous fortnight from any illness such as fever , respiratory or gastro - intestinal infections or those undergoing medical treatment or taking multi - vitamin mineral supplements as per a physician 's recommendation were excluded . thus in all , 630 apparently healthy girls aged 1016 years from the participating schools were recruited for the study . details of the procedures involved in the study were explained to the parents . after giving consent , the study protocol was reviewed and approved by the ethics committee of zensar foundation of india , pune , under the chairmanship of a retired professor of the international institute of population sciences , mumbai . all of the parents gave their written informed consent , and the girls voluntarily participated in the study . girls who were currently suffering or those who had suffered the previous fortnight from any illness such as fever , respiratory or gastro - intestinal infections or those undergoing medical treatment or taking multi - vitamin mineral supplements as per a physician 's recommendation were excluded . thus in all , 630 apparently healthy girls aged 1016 years from the participating schools were recruited for the study . details of the procedures involved in the study were explained to the parents . after giving consent , the study protocol was reviewed and approved by the ethics committee of zensar foundation of india , pune , under the chairmanship of a retired professor of the international institute of population sciences , mumbai . all of the parents gave their written informed consent , and the girls voluntarily participated in the study . nutrient intakes were estimated using cooked foods database ( 53 ) and nutritive value tables ( 54 ) . venous blood samples ( 10 ml ) were collected at 7.30 am after an overnight fast for each girl . haemoglobin ( hb ) was estimated using automated cell counter ( actdiff ii , beckman coulter , fullerton , ca , usa ) . plasma levels of zinc , vitamin c , -carotene and retinol were measured as per the methods in the manual by national institute of nutrition , india ( 55 ) , and erythrocyte zinc was measured as per the method described by kenny et al . reference serum ( randox laboratories , india ) was used as a standard for blood estimations . cognitive performance was evaluated using a group of four tests standardised on indian children ; simple reaction time ( srt ) , recognition reaction time ( rrt ) , raven 's progressive matrices ( rpm ) and visual memory on each participant ( 5759 ) . srt and rrt measure the cognitive speed concerning perception , processing and motor response organisation for reaction tasks and visual search . srt and rrt were measured using a visual reaction time apparatus ( anand agencies , pune , india ) designed as per the procedure given by woodworth et al . rpm is a nonverbal test of performance that measures a person 's ability to form perceptual relations and reason by analogy in research settings and form comparisons ( 58 ) . a short - term visual memory test was carried out for measuring the ability of the girls to retain and reproduce information within a short time span . this test was adopted from the subtest of pgi memory scale developed by the post graduate institute of medical education and research , chandigarh , india , for children and adolescents ( 59 ) . taste acuity was assessed by identifying recognition threshold for salt ( rts ) using 10 different salt solutions ranging from 0 to 25 mmol / l ( 40 ) . more details of the measurements and methods used in the study are presented in five research papers ( 6064 ) . average age , weight , height and body mass index of the girls were 12.21 1.11 years , 33.05 7.22 kg , 143.74 8.24 cm and 15.81 2.62 kg / m , respectively . twenty - four percent of the girls were below the 5th percentile and 2.1% were above the 85th percentile of the indian reference bmi for age ( 65 ) . family size of the girls ranged from 2 to 11 persons in the household , with an average of 5 2 persons . amongst fathers , parents educational status indicated that 13.8% of the fathers and 9.0% of the mothers completed high school , and 18.7% of the fathers and 13.2% of the mothers completed a college education . the mean intake of energy and protein was 1373 381 kcal / d and 30.4 9.6 g / d , respectively . comparison with age sex - specific reference intake indicated that these intakes were 68.7 and 60% of recommendation ( 24 ) . average calcium , iron , -carotene and vitamin c intakes were 289 146 , 6.9 2.3 , 619 564 and 25.6 14.5 mg / d , respectively . thus , average intakes of micronutrients were 3060% of the respective rdi , indicating dietary deficiencies of these minerals and vitamins in the girls . the present study has offered estimates of prevalence of dietary micronutrient deficiencies in indian urban adolescent girls , which are in agreement with previous findings on rural indian adolescents ( 13 , 16 ) and available studies on urban slum adolescents ( 14 , 15 ) . however , data on zinc intake are missing in these studies and the current literature . we have observed that average zinc intake ( 3.6 1.2 mg / d ) of the girls was low , around 40% of the indian rdi for zinc ( 66 ) , with almost all girls having their zinc intake below rdi . plasma zinc levels were low ( < 0.7 mg / l ) ( 41 ) in 72.4% , and erythrocyte zinc levels were low ( < 8 g / g of packed cells ) ( 56 ) in 23.6% of the girls . prevalence of anemia ( hb < 12 g / dl ) was 27.2% , and 26.6% girls had low serum ferritin lpar ; ( < 12 g / l ) ( 67 ) . plasma retinol was below the normal cutoff of 200 g / l ( 68 ) in 65.4% girls . plasma vitamin c level of 10.8% girls was below the normal cut - off of 0.2 mg / dl ( 69 p. 534 ) , and 67.6% of the girls had low plasma vitamin c status . this high prevalence of micronutrient deficiencies confirmed inadequate dietary intakes of micronutrients by the girls from the study group . paper i described the cognitive performance and assessed its relation with status of zinc and other micronutrients ( 60 ) . the data on cognitive scores in our girls indicated that 60% of the girls were slow in simple reaction tasks , 71.5% of the girls had their rpm scores below 50th percentile and memory deficits were seen in 44% girls ( 60 ) . reaction time and rpm percentiles of majority of the girls were below the normal range , indicating deficits in the cognitive performance of the girls . to explore the relative significance of dietary nutrient intakes with cognitive performance , weighted multiple regression adjusted for energy intake the analysis revealed that intakes of -carotene , iron and zinc were significant predictors for srt ( p<0.01 ) . further , our study also examines association of biomarkers of iron and zinc with cognitive scores of the girls . we have noted that plasma zinc and erythrocyte zinc were negatively correlated with srt and rrt and positively with memory and rpm ( p<0.05 ) , whereas hb was significantly correlated with rrt ( p<0.05 ) in girls with iron deficiency anaemia . taste rts was found to be high in 45.2% girls , indicating impaired taste function . rts showed significant negative correlation with erythrocyte zinc ( r = 0.25 , p<0.05 ) , implying that lower the zinc status higher would be the salt concentration for taste perception . thus , our data indicated high prevalence of micronutrient deficiencies including zinc in adolescent girls . moreover , we observed improvement in cognitive performance and taste perception with increased blood zinc levels in the girls . therefore , an attempt has been made to develop zinc - rich recipes to improve total and bio - available zinc intake of the girls . in paper ii ( 61 ) , dietary food intakes on three random days by 24-hour recall were analysed for investigating prevailing diet patterns in the girls . it was observed that the girls were consuming rice - legumes meals , wheat - legumes / vegetables , mixed meals or bakery items . in all , the consumption of 86 different food items was reported by the girls during the 3 days diet recall . of these , 19 foods were different cereals preparations comprising rice , wheat or millets , ; 16 were legume preparations ; seven different bakery products ; five leafy vegetables ; 14 other vegetables ; 13 fried foods ; six fruits ; and six milk and milk products . these foods were categorised into 12 food groups based on their major ingredients , such as rice , whole wheat , pearl millet , sorghum , bakery products , fried foods , sprouts , un - sprouted legumes , leafy vegetables , other vegetables , milk and milk products and fruits . for example , plain rice , rice flakes and puffed rice were categorised under the rice food group , or foods such as unleavened pancakes ( chapatti , paratha , roti ) were categorised under the wheat group . food intake data from 12 food subgroups were used to identify diet patterns of the girls . principal component analysis was performed to identify diet patterns using orthogonal rotation method ( varimax ) . food subgroups with factor loading > 0.3 , eigenvalues and percentage of variance explained by each of the major dietary patterns identified in adolescent girls is given in table 1 . the factor loading indicated the degree to which the girls diet conformed to one of the dietary patterns identified . five patterns with eigenvalues greater than one were identified , which explained about 55.1% of the variance ( table 1 ) . the patterns were characterised by typical combination of major cereal with food subgroups , namely ( 1 ) rice meal , ( 2 ) snack type meal , ( 3 ) wheat meal , ( 4 ) pearl millet meal and ( 5 ) sorghum meal ( 61 ) . food subgroups , eigenvalues and percentage of variance explained by major dietary patterns identified adolescent girls extraction method : principle component analysis . the results indicated that consumption of healthy foods mainly whole grains , sprouts and vegetables were high in wheat , pearl millet or sorghum - based patterns than the rice meal and the snacks type meal ( p<0.05 ) . in general , girls with the rice meal and the snacks type meal had low micronutrient intakes and low intake of bio - available zinc and iron than girls from other diet patterns ( p<0.05 ) . however , all diet patterns were deficient in micronutrients including zinc when compared with the rdi ( 24 , 66 ) ; mainly due to low intakes of healthy foods by the girls . recipes were devised considering the dietary habits of the girls and representing each of the five diet patterns . recipes were formulated ( 1 ) using foods that have high zinc contents such as pearl millet , chick peas , soybean , rajma , green gram , amaranthus seeds and onion stalks ( 54 ) . amongst the zinc - rich foods , selection was made on the basis of higher bio - availability ( 32 ) and local availability ; ( 2 ) zinc density of the recipes was increased by using small amounts of sesame , amaranthus and pumpkin seeds ; and ( 3 ) using methods like sprouting / fermentation , which increase the bio - availability of recipes . dialysability of the recipes was determined by simulating gastrointestinal conditions and atomic absorption spectrometry ( model 3110 , perkin elmer , norwalk , ct , usa ) . contents of -carotene and vitamin c were measured by spectrophotometry ( beckman coulter , inc . energy , protein , calcium and iron contents were computed using the indian national database of nutrient composition of raw foods ( 54 ) and adjusting for observed moisture contents of cooked recipes . average energy and protein content per 100 g of cooked weight of the recipes was 205 kcal and 2.5 mg , respectively . average content of zinc was 1.5 mg/100 g of cooked weight , which was three times more than the observed average daily intake ( 0.47 mg/ 100 g cooked weight ) of the girls . average zinc dialysability of the recipes was 18.3% , which was observed to be higher than 11% dialysability of the diets consumed by the girls . the zinc - rich snacks containing meat developed for kenyan schoolchildren provided 2.89 mg/225 g of zinc ( 37 ) , which was similar to the zinc content of proposed study ( 3.3 mg/225 g ) . thus , consumption of these new recipes as a supplement to the existing diet has potential to improve zinc intake of the vegetarian girls . contents of iron , calcium , -carotene and vitamin c were 6.2 g , 105 mg , 716 g and 4.4 mg , respectively . wallis test indicated that the mean content of micronutrients such as iron , -carotene , vitamin c of new recipes were significantly higher than the observed intakes of the girls ( p<0.01 ) . therefore , modification in the existing diet using these recipes can increase micronutrient intakes of the girls . for assessing dietary inadequacies of zinc and other micronutrients , especially in vegetarian populations , a simple tool such as micronutrient quality index is required to identify at - risk individuals . it is important to consider the content as well as bio - availability of micronutrients for evaluation of dietary adequacy . we have developed adolescent micronutrient quality index ( amqi ) to classify an individual with nutritional deficiencies ( paper iii ) ( 62 ) using the indian dietary guidelines ( 24 ) and the recent us dietary guidelines for choice of healthy foods and habits ( 23 ) . food intake data collected through 24-hour recall method for 3 days were utilised to develop the amqi . information about frequency of consuming sprouts , fermented foods and habit of taking tea with meals was utilised to judge bio - available intakes . efficacy of the amqi was assessed by studying its associations with nutrient intakes , micronutrient adequacy ratio , body mass index and plasma biomarkers of -carotene , vitamin c and zinc . the amqi is divided into 13 components , where each component represents a specific grouping of foods . seven components measured the degree of compliance , with recommendations for the major food groups such as cereals and millets , legumes , milk and milk products , vegetables , fruits , sugar and visible fats / oils . six components relate to healthy eating and micronutrient bio - availability such as adherence to at least 50% consumption of whole grains and micronutrient - dense legumes ( i.e. chick pea , moth beans , lentil ) , food variety ( 23 , 26 , 70 , 71 ) , use of practices enhancing bio - availability and component to evaluate excess fried food intake ( 71 ) . each component was scored from zero indicating lack of adherence to its maximum score for full adherence , with intermediate scores calculated to indicate the degree of adherence with dietary recommendations . the mean amqi score was 41.59.4 , and 86.8% girls had the amqi score lower than 50 , indicating inadequate dietary nutrient intakes by the girls . the majority of the girls showed adherence to three of the healthy choice components , i.e. at least 50% grains as whole grains , low frequency of consuming fried foods and greater food variety . adherence by the girls to the consumption of micronutrient dense pulses , sprouts , salad and fermented foods and not consuming tea with meals was poor . association of socio - demographic variables with amqi was evaluated using the data on family size , mother 's and father 's education and occupation . in our data , mother 's education and occupation were significantly associated with amqi . a logistic regression analysis of the amqi with socio - demographic factors revealed that age , mother 's education and occupation were significantly and positively associated with the amqi ( r = 0.07 , p<0.01 ) . partial correlations of the micronutrient intakes with the amqi scores after adjusting for energy intake and socio - demographic factors were at the range of 0.40.5 and were statistically significant ( p<0.01 ) , which demonstrated the concurrent validity of the new index . higher amqi scores were associated with higher concentrations of plasma vitamin c ( r=0.26 ) , -carotene ( r=0.34 ) and zinc ( r=0.12 ) after controlling for socio - demographic factors ( p<0.05 ) . thus , the amqi provides an updated measure to evaluate micronutrient quality of diets of lacto - vegetarian indian adolescent girls . our index is the first of its kind to consider bio - availability aspects of diet in the evaluation of diet quality . we anticipate that such concept of emphasising adequate contents and bio - availability of micronutrients while constructing the diet quality index can be widely applicable to different lacto - vegetarian population groups , although some of the components of the index may require modification with respect to nutrient requirements of different age sex groups and food habits . dietary intervention strategies have been identified as the principal approach to tackle nutritional problems in the population . we have developed diet supplements in the form of zinc - rich recipes to improve intake of zinc and other micronutrients . an intervention trial was conducted to determine the efficacy of proposed recipes in improving zinc and other micronutrient status ( paper iv ) ( 63 ) and functional indices of zinc ( paper v ) ( 64 ) . effect of the diet supplementation was viewed in light of supplementing ayurvedic zinc ( jasad bhasma ) tablet . diet and jasad supplementation both showed a significant increase in plasma zinc ( 9.9 and 61.3% , respectively ) ( p<0.01 ) ( 63 ) . further , the diet supplement showed significant increase in blood status of -carotene and vitamin c as compared to control group ( p<0.01 ) . thus , devising such recipes would prove a better sustainable strategy for promoting micronutrient status of adolescents . our results demonstrated the beneficial effects of zinc supplementation on cognitive scores of the girls by reducing reaction time ( 613% ) and improving in memory ( 25% ) and rpm scores ( 15% ) than the control group ( 64 ) . median recognition threshold for salt taste reduced significantly from 5 to 2.5 mmol / l after both diet and zinc supplementation ( p<0.01 ) ; however , it remained same in the control group . this suggests a positive effect of supplementation on cognitive performance and taste acuity of the girls . as observed in our study , decrease in reaction time has been reported by pennland et al . furthermore , our study demonstrated that zinc supplementation improved memory and non - verbal intelligence in adolescent girls as well as established positive effect of zinc supplementation on taste perception . thus , zinc supplementation through diet or ayurvedic jasad tablet not only improves zinc status of the girls but also was a useful strategy for improving cognitive performance and taste acuity function in adolescent girls . the mean intake of energy and protein was 1373 381 kcal / d and 30.4 9.6 g / d , respectively . comparison with age sex - specific reference intake indicated that these intakes were 68.7 and 60% of recommendation ( 24 ) . average calcium , iron , -carotene and vitamin c intakes were 289 146 , 6.9 2.3 , 619 564 and 25.6 14.5 mg / d , respectively . thus , average intakes of micronutrients were 3060% of the respective rdi , indicating dietary deficiencies of these minerals and vitamins in the girls . the present study has offered estimates of prevalence of dietary micronutrient deficiencies in indian urban adolescent girls , which are in agreement with previous findings on rural indian adolescents ( 13 , 16 ) and available studies on urban slum adolescents ( 14 , 15 ) . however , data on zinc intake are missing in these studies and the current literature . we have observed that average zinc intake ( 3.6 1.2 mg / d ) of the girls was low , around 40% of the indian rdi for zinc ( 66 ) , with almost all girls having their zinc intake below rdi . plasma zinc levels were low ( < 0.7 mg / l ) ( 41 ) in 72.4% , and erythrocyte zinc levels were low ( < 8 g / g of packed cells ) ( 56 ) in 23.6% of the girls . prevalence of anemia ( hb < 12 g / dl ) was 27.2% , and 26.6% girls had low serum ferritin lpar ; ( < 12 g / l ) ( 67 ) . plasma retinol was below the normal cutoff of 200 g / l ( 68 ) in 65.4% girls . plasma vitamin c level of 10.8% girls was below the normal cut - off of 0.2 mg / dl ( 69 p. 534 ) , and 67.6% of the girls had low plasma vitamin c status . this high prevalence of micronutrient deficiencies confirmed inadequate dietary intakes of micronutrients by the girls from the study group . paper i described the cognitive performance and assessed its relation with status of zinc and other micronutrients ( 60 ) . the data on cognitive scores in our girls indicated that 60% of the girls were slow in simple reaction tasks , 71.5% of the girls had their rpm scores below 50th percentile and memory deficits were seen in 44% girls ( 60 ) . reaction time and rpm percentiles of majority of the girls were below the normal range , indicating deficits in the cognitive performance of the girls . to explore the relative significance of dietary nutrient intakes with cognitive performance , weighted multiple regression adjusted for energy intake the analysis revealed that intakes of -carotene , iron and zinc were significant predictors for srt ( p<0.01 ) . further , our study also examines association of biomarkers of iron and zinc with cognitive scores of the girls . we have noted that plasma zinc and erythrocyte zinc were negatively correlated with srt and rrt and positively with memory and rpm ( p<0.05 ) , whereas hb was significantly correlated with rrt ( p<0.05 ) in girls with iron deficiency anaemia . taste rts was found to be high in 45.2% girls , indicating impaired taste function . rts showed significant negative correlation with erythrocyte zinc ( r = 0.25 , p<0.05 ) , implying that lower the zinc status higher would be the salt concentration for taste perception . thus , our data indicated high prevalence of micronutrient deficiencies including zinc in adolescent girls . moreover , we observed improvement in cognitive performance and taste perception with increased blood zinc levels in the girls . therefore , an attempt has been made to develop zinc - rich recipes to improve total and bio - available zinc intake of the girls . in paper ii ( 61 ) , dietary food intakes on three random days by 24-hour recall were analysed for investigating prevailing diet patterns in the girls . it was observed that the girls were consuming rice - legumes meals , wheat - legumes / vegetables , mixed meals or bakery items . in all , the consumption of 86 different food items was reported by the girls during the 3 days diet recall . of these , 19 foods were different cereals preparations comprising rice , wheat or millets , ; 16 were legume preparations ; seven different bakery products ; five leafy vegetables ; 14 other vegetables ; 13 fried foods ; six fruits ; and six milk and milk products . these foods were categorised into 12 food groups based on their major ingredients , such as rice , whole wheat , pearl millet , sorghum , bakery products , fried foods , sprouts , un - sprouted legumes , leafy vegetables , other vegetables , milk and milk products and fruits . for example , plain rice , rice flakes and puffed rice were categorised under the rice food group , or foods such as unleavened pancakes ( chapatti , paratha , roti ) were categorised under the wheat group . food intake data from 12 food subgroups were used to identify diet patterns of the girls . principal component analysis was performed to identify diet patterns using orthogonal rotation method ( varimax ) . food subgroups with factor loading > 0.3 , eigenvalues and percentage of variance explained by each of the major dietary patterns identified in adolescent girls is given in table 1 . the factor loading indicated the degree to which the girls diet conformed to one of the dietary patterns identified . five patterns with eigenvalues greater than one were identified , which explained about 55.1% of the variance ( table 1 ) . the patterns were characterised by typical combination of major cereal with food subgroups , namely ( 1 ) rice meal , ( 2 ) snack type meal , ( 3 ) wheat meal , ( 4 ) pearl millet meal and ( 5 ) sorghum meal ( 61 ) . food subgroups , eigenvalues and percentage of variance explained by major dietary patterns identified adolescent girls extraction method : principle component analysis . the results indicated that consumption of healthy foods mainly whole grains , sprouts and vegetables were high in wheat , pearl millet or sorghum - based patterns than the rice meal and the snacks type meal ( p<0.05 ) . in general , girls with the rice meal and the snacks type meal had low micronutrient intakes and low intake of bio - available zinc and iron than girls from other diet patterns ( p<0.05 ) however , all diet patterns were deficient in micronutrients including zinc when compared with the rdi ( 24 , 66 ) ; mainly due to low intakes of healthy foods by the girls . in paper ii ( 61 ) , dietary food intakes on three random days by 24-hour recall were analysed for investigating prevailing diet patterns in the girls . it was observed that the girls were consuming rice - legumes meals , wheat - legumes / vegetables , mixed meals or bakery items . in all , the consumption of 86 different food items was reported by the girls during the 3 days diet recall . of these , 19 foods were different cereals preparations comprising rice , wheat or millets , ; 16 were legume preparations ; seven different bakery products ; five leafy vegetables ; 14 other vegetables ; 13 fried foods ; six fruits ; and six milk and milk products . these foods were categorised into 12 food groups based on their major ingredients , such as rice , whole wheat , pearl millet , sorghum , bakery products , fried foods , sprouts , un - sprouted legumes , leafy vegetables , other vegetables , milk and milk products and fruits . for example , plain rice , rice flakes and puffed rice were categorised under the rice food group , or foods such as unleavened pancakes ( chapatti , paratha , roti ) were categorised under the wheat group . food intake data from 12 food subgroups were used to identify diet patterns of the girls . principal component analysis was performed to identify diet patterns using orthogonal rotation method ( varimax ) . food subgroups with factor loading > 0.3 , eigenvalues and percentage of variance explained by each of the major dietary patterns identified in adolescent girls is given in table 1 . the factor loading indicated the degree to which the girls diet conformed to one of the dietary patterns identified . five patterns with eigenvalues greater than one were identified , which explained about 55.1% of the variance ( table 1 ) . the patterns were characterised by typical combination of major cereal with food subgroups , namely ( 1 ) rice meal , ( 2 ) snack type meal , ( 3 ) wheat meal , ( 4 ) pearl millet meal and ( 5 ) sorghum meal ( 61 ) . food subgroups , eigenvalues and percentage of variance explained by major dietary patterns identified adolescent girls extraction method : principle component analysis . the results indicated that consumption of healthy foods mainly whole grains , sprouts and vegetables were high in wheat , pearl millet or sorghum - based patterns than the rice meal and the snacks type meal ( p<0.05 ) . in general , girls with the rice meal and the snacks type meal had low micronutrient intakes and low intake of bio - available zinc and iron than girls from other diet patterns ( p<0.05 ) however , all diet patterns were deficient in micronutrients including zinc when compared with the rdi ( 24 , 66 ) ; mainly due to low intakes of healthy foods by the girls . recipes were devised considering the dietary habits of the girls and representing each of the five diet patterns . recipes were formulated ( 1 ) using foods that have high zinc contents such as pearl millet , chick peas , soybean , rajma , green gram , amaranthus seeds and onion stalks ( 54 ) . amongst the zinc - rich foods , selection was made on the basis of higher bio - availability ( 32 ) and local availability ; ( 2 ) zinc density of the recipes was increased by using small amounts of sesame , amaranthus and pumpkin seeds ; and ( 3 ) using methods like sprouting / fermentation , which increase the bio - availability of recipes . dialysability of the recipes was determined by simulating gastrointestinal conditions and atomic absorption spectrometry ( model 3110 , perkin elmer , norwalk , ct , usa ) . contents of -carotene and vitamin c were measured by spectrophotometry ( beckman coulter , inc . energy , protein , calcium and iron contents were computed using the indian national database of nutrient composition of raw foods ( 54 ) and adjusting for observed moisture contents of cooked recipes . average energy and protein content per 100 g of cooked weight of the recipes was 205 kcal and 2.5 mg , respectively . average content of zinc was 1.5 mg/100 g of cooked weight , which was three times more than the observed average daily intake ( 0.47 mg/ 100 g cooked weight ) of the girls . average zinc dialysability of the recipes was 18.3% , which was observed to be higher than 11% dialysability of the diets consumed by the girls . the zinc - rich snacks containing meat developed for kenyan schoolchildren provided 2.89 mg/225 g of zinc ( 37 ) , which was similar to the zinc content of proposed study ( 3.3 mg/225 g ) . thus , consumption of these new recipes as a supplement to the existing diet has potential to improve zinc intake of the vegetarian girls . contents of iron , calcium , -carotene and vitamin c were 6.2 g , 105 mg , 716 g and 4.4 mg , respectively . wallis test indicated that the mean content of micronutrients such as iron , -carotene , vitamin c of new recipes were significantly higher than the observed intakes of the girls ( p<0.01 ) . therefore , modification in the existing diet using these recipes can increase micronutrient intakes of the girls . for assessing dietary inadequacies of zinc and other micronutrients , especially in vegetarian populations , a simple tool such as micronutrient quality index is required to identify at - risk individuals . it is important to consider the content as well as bio - availability of micronutrients for evaluation of dietary adequacy . we have developed adolescent micronutrient quality index ( amqi ) to classify an individual with nutritional deficiencies ( paper iii ) ( 62 ) using the indian dietary guidelines ( 24 ) and the recent us dietary guidelines for choice of healthy foods and habits ( 23 ) . food intake data collected through 24-hour recall method for 3 days were utilised to develop the amqi . information about frequency of consuming sprouts , fermented foods and habit of taking tea with meals was utilised to judge bio - available intakes . efficacy of the amqi was assessed by studying its associations with nutrient intakes , micronutrient adequacy ratio , body mass index and plasma biomarkers of -carotene , vitamin c and zinc . the amqi is divided into 13 components , where each component represents a specific grouping of foods . seven components measured the degree of compliance , with recommendations for the major food groups such as cereals and millets , legumes , milk and milk products , vegetables , fruits , sugar and visible fats / oils . six components relate to healthy eating and micronutrient bio - availability such as adherence to at least 50% consumption of whole grains and micronutrient - dense legumes ( i.e. chick pea , moth beans , lentil ) , food variety ( 23 , 26 , 70 , 71 ) , use of practices enhancing bio - availability and component to evaluate excess fried food intake ( 71 ) . each component was scored from zero indicating lack of adherence to its maximum score for full adherence , with intermediate scores calculated to indicate the degree of adherence with dietary recommendations . the mean amqi score was 41.59.4 , and 86.8% girls had the amqi score lower than 50 , indicating inadequate dietary nutrient intakes by the girls . the majority of the girls showed adherence to three of the healthy choice components , i.e. at least 50% grains as whole grains , low frequency of consuming fried foods and greater food variety . adherence by the girls to the consumption of micronutrient dense pulses , sprouts , salad and fermented foods and not consuming tea with meals was poor . association of socio - demographic variables with amqi was evaluated using the data on family size , mother 's and father 's education and occupation . in our data , mother 's education and occupation were significantly associated with amqi . a logistic regression analysis of the amqi with socio - demographic factors revealed that age , mother 's education and occupation were significantly and positively associated with the amqi ( r = 0.07 , p<0.01 ) . partial correlations of the micronutrient intakes with the amqi scores after adjusting for energy intake and socio - demographic factors were at the range of 0.40.5 and were statistically significant ( p<0.01 ) , which demonstrated the concurrent validity of the new index . higher amqi scores were associated with higher concentrations of plasma vitamin c ( r=0.26 ) , -carotene ( r=0.34 ) and zinc ( r=0.12 ) after controlling for socio - demographic factors ( p<0.05 ) . thus , the amqi provides an updated measure to evaluate micronutrient quality of diets of lacto - vegetarian indian adolescent girls . our index is the first of its kind to consider bio - availability aspects of diet in the evaluation of diet quality . we anticipate that such concept of emphasising adequate contents and bio - availability of micronutrients while constructing the diet quality index can be widely applicable to different lacto - vegetarian population groups , although some of the components of the index may require modification with respect to nutrient requirements of different age sex groups and food habits . dietary intervention strategies have been identified as the principal approach to tackle nutritional problems in the population . we have developed diet supplements in the form of zinc - rich recipes to improve intake of zinc and other micronutrients . an intervention trial was conducted to determine the efficacy of proposed recipes in improving zinc and other micronutrient status ( paper iv ) ( 63 ) and functional indices of zinc ( paper v ) ( 64 ) . effect of the diet supplementation was viewed in light of supplementing ayurvedic zinc ( jasad bhasma ) tablet . diet and jasad supplementation both showed a significant increase in plasma zinc ( 9.9 and 61.3% , respectively ) ( p<0.01 ) ( 63 ) . further , the diet supplement showed significant increase in blood status of -carotene and vitamin c as compared to control group ( p<0.01 ) . thus , devising such recipes would prove a better sustainable strategy for promoting micronutrient status of adolescents . our results demonstrated the beneficial effects of zinc supplementation on cognitive scores of the girls by reducing reaction time ( 613% ) and improving in memory ( 25% ) and rpm scores ( 15% ) than the control group ( 64 ) . median recognition threshold for salt taste reduced significantly from 5 to 2.5 mmol / l after both diet and zinc supplementation ( p<0.01 ) ; however , it remained same in the control group . this suggests a positive effect of supplementation on cognitive performance and taste acuity of the girls . as observed in our study , decrease in reaction time has been reported by pennland et al . furthermore , our study demonstrated that zinc supplementation improved memory and non - verbal intelligence in adolescent girls as well as established positive effect of zinc supplementation on taste perception . thus , zinc supplementation through diet or ayurvedic jasad tablet not only improves zinc status of the girls but also was a useful strategy for improving cognitive performance and taste acuity function in adolescent girls . the present research provided first - hand estimates of micronutrient deficiencies in a cohort of urban indian adolescent girls . our data demonstrated that the diets of the girls were deficient in zinc and other micronutrients . blood biomarkers of zinc and vitamin a were below the normal range in majority of the girls . dietary zinc deficiency and low blood zinc status lead to poor cognition and taste perception amongst adolescent girls . therefore , there is a concurrent requirement of a simple tool to identify dietary micronutrient deficiencies and a diet intervention strategy to improve zinc and other micronutrient status of the girls . the new diet quality index i.e. amqi has successfully assessed micronutrient deficiency risk among study population and thus provided an updated measure to assess micronutrient quality of diets of lacto - vegetarian adolescent girls . an intervention trial on a group of 180 girls has demonstrated effectiveness of the proposed recipes in improving zinc status . large - scale studies are needed to confirm its potential as a diet diversification strategy for vegetarian population . this study is the only one so far to demonstrate a positive effect of zinc supplementation in improving cognitive performance and taste acuity of adolescent girls . effect of zinc supplementation on other aspects of health such as bone development , infections and obesity may be studied in adolescents . thus , the findings of the research demonstrate the necessity of adopting healthy micronutrient - rich diets for positive health building in adolescents . further , from a global perspective , the methodologies and approaches used to assess dietary inadequacies in the form of amqi and diet diversification strategies could be useful to explore and alleviate problem of micronutrient deficiencies in population . this study was funded as a part of the project by zensar foundation , india .

backgroundzinc is important in adolescence because of its role in growth and sexual maturation . adolescents from developing countries such as india may be at high risk of zinc deficiency because of unwholesome food habits and poor bioavailability of zinc from plant - based diets.objective(1 ) to study zinc status and its association with profile of other micronutrients , ( 2 ) to construct a simple tool in the form of adolescent micronutrient quality index ( amqi ) to assess quality of diets of the girls and ( 3 ) to examine the effect of zinc supplement on health of adolescent girls.methodsgirls ( 1016 years ) from two secondary schools of pune , maharashtra state , in western india were enrolled in a cross - sectional study ( n = 630 ) . data were collected on dietary intake , cognitive performance , taste acuity , haemoglobin , erythrocyte zinc and plasma levels of zinc , vitamin c , -carotene and retinol . amqi was developed using age sex - specific indian dietary guidelines and healthy foods and habits described in the recent us dietary guidelines . zinc - rich recipes were developed considering habitual diets of the girls and vegetarian sources of zinc . an intervention trial ( n = 180 ) was conducted to assess the effect of zinc - rich dietary supplements and ayurvedic zinc ( jasad ) supplementation.resultsprevalence of micronutrient deficiencies was high in these girls . poor cognitive performance was seen in half of the girls , and salt taste perception was affected in 45% . amqi was correlated with nutrient intakes and blood micronutrient levels ( p < 0.01 ) , indicating the potential of amqi to measure micronutrient quality of diets of adolescent girls . results of the intervention trial indicated that supplementation of zinc - rich recipes vis - a - vis ayurvedic jasad zinc has the potential to improve plasma zinc status , cognitive performance and taste acuity in adolescent girls.conclusionsreview of the studies on indian adolescent girls demonstrates the necessity of adopting zinc and micronutrient - rich diets for positive health building in adolescents .

Rationale and significance of the study Study objectives Material and methods Participants Exclusion criteria Outcome measures Results and discussion Nutrient profile and functional indices of zinc in adolescent girls Diet patterns of adolescent girls and food-based approach for improving zinc intake Description of diet patterns Development of recipes to increase total and dialysable zinc content Development of a diet quality index with special reference to micronutrient adequacy for adolescent girls Efficacy of dietary supplementation in improving blood micronutrient status and functional indices of zinc in adolescent girls Conclusions Conflict of interest and funding

vitamin and mineral deficiencies adversely affect a third of the world 's population ( 49 ) , and it is estimated that one in five people worldwide lack sufficient dietary zinc ( 41 ) . adolescent girls may be more prone to zinc deficiency because physiological requirements for zinc peak during adolescence at the time of the pubertal growth spurt , which generally occurs in girls between 10 and 15 years ( 50 ) . especially , adolescents from developing countries such as india are at high risk because of low zinc intakes and poor bioavailability of zinc from vegetarian diets , which leads to low zinc status . in adolescents , a wide range of disturbances occur due to zinc deficiency , including impaired growth , defects in the immune system , dermatitis , diarrhoea , delayed sexual and bone maturation , impaired taste acuity and behavioural changes ( 41 ) . simple measures such as diet quality index are used to assess adherence to dietary guidelines . to identify micronutrient deficiencies in adolescents , in particular that of zinc , a simple tool needs to be devised , which can judge the micronutrient quality of diet . moreover , zinc - rich dietary supplement has a potential to revert bad health effects of low zinc status . the present research aimed to study zinc status and its association with profile of other micronutrients , and to examine the effect of zinc supplementation on functional indices of zinc in adolescent girls . the specific objectives of the study were : to study zinc status and its association with the profile of other micronutrients , cognitive performance and taste acuity of the adolescent girls.to examine the diet patterns of vegetarian adolescent girls for zinc adequacy and devise recipes to improve bio - available zinc intakes.to develop and validate a nutrient quality index to assess micronutrient sufficiency of diets using statistical techniques and bioavailability of micronutrients.to examine efficacy of dietary supplementation on blood micronutrient levels and functional indices of zinc . to study zinc status and its association with the profile of other micronutrients , cognitive performance and taste acuity of the adolescent girls . to examine the diet patterns of vegetarian adolescent girls for zinc adequacy and devise recipes to improve bio - available zinc intakes . to develop and validate a nutrient quality index to assess micronutrient sufficiency of diets using statistical techniques and bioavailability of micronutrients . to examine efficacy of dietary supplementation on blood micronutrient levels and functional indices of zinc . the study was conducted in two secondary schools from pune city , maharashtra state , in western india , during 20052007 . all of the parents gave their written informed consent , and the girls voluntarily participated in the study . plasma levels of zinc , vitamin c , -carotene and retinol were measured as per the methods in the manual by national institute of nutrition , india ( 55 ) , and erythrocyte zinc was measured as per the method described by kenny et al . cognitive performance was evaluated using a group of four tests standardised on indian children ; simple reaction time ( srt ) , recognition reaction time ( rrt ) , raven 's progressive matrices ( rpm ) and visual memory on each participant ( 5759 ) . the study was conducted in two secondary schools from pune city , maharashtra state , in western india , during 20052007 . all of the parents gave their written informed consent , and the girls voluntarily participated in the study . all of the parents gave their written informed consent , and the girls voluntarily participated in the study . plasma levels of zinc , vitamin c , -carotene and retinol were measured as per the methods in the manual by national institute of nutrition , india ( 55 ) , and erythrocyte zinc was measured as per the method described by kenny et al . cognitive performance was evaluated using a group of four tests standardised on indian children ; simple reaction time ( srt ) , recognition reaction time ( rrt ) , raven 's progressive matrices ( rpm ) and visual memory on each participant ( 5759 ) . comparison with age sex - specific reference intake indicated that these intakes were 68.7 and 60% of recommendation ( 24 ) . thus , average intakes of micronutrients were 3060% of the respective rdi , indicating dietary deficiencies of these minerals and vitamins in the girls . the present study has offered estimates of prevalence of dietary micronutrient deficiencies in indian urban adolescent girls , which are in agreement with previous findings on rural indian adolescents ( 13 , 16 ) and available studies on urban slum adolescents ( 14 , 15 ) . we have observed that average zinc intake ( 3.6 1.2 mg / d ) of the girls was low , around 40% of the indian rdi for zinc ( 66 ) , with almost all girls having their zinc intake below rdi . plasma zinc levels were low ( < 0.7 mg / l ) ( 41 ) in 72.4% , and erythrocyte zinc levels were low ( < 8 g / g of packed cells ) ( 56 ) in 23.6% of the girls . plasma vitamin c level of 10.8% girls was below the normal cut - off of 0.2 mg / dl ( 69 p. 534 ) , and 67.6% of the girls had low plasma vitamin c status . paper i described the cognitive performance and assessed its relation with status of zinc and other micronutrients ( 60 ) . the data on cognitive scores in our girls indicated that 60% of the girls were slow in simple reaction tasks , 71.5% of the girls had their rpm scores below 50th percentile and memory deficits were seen in 44% girls ( 60 ) . reaction time and rpm percentiles of majority of the girls were below the normal range , indicating deficits in the cognitive performance of the girls . to explore the relative significance of dietary nutrient intakes with cognitive performance , weighted multiple regression adjusted for energy intake the analysis revealed that intakes of -carotene , iron and zinc were significant predictors for srt ( p<0.01 ) . we have noted that plasma zinc and erythrocyte zinc were negatively correlated with srt and rrt and positively with memory and rpm ( p<0.05 ) , whereas hb was significantly correlated with rrt ( p<0.05 ) in girls with iron deficiency anaemia . rts showed significant negative correlation with erythrocyte zinc ( r = 0.25 , p<0.05 ) , implying that lower the zinc status higher would be the salt concentration for taste perception . thus , our data indicated high prevalence of micronutrient deficiencies including zinc in adolescent girls . moreover , we observed improvement in cognitive performance and taste perception with increased blood zinc levels in the girls . therefore , an attempt has been made to develop zinc - rich recipes to improve total and bio - available zinc intake of the girls . the patterns were characterised by typical combination of major cereal with food subgroups , namely ( 1 ) rice meal , ( 2 ) snack type meal , ( 3 ) wheat meal , ( 4 ) pearl millet meal and ( 5 ) sorghum meal ( 61 ) . the results indicated that consumption of healthy foods mainly whole grains , sprouts and vegetables were high in wheat , pearl millet or sorghum - based patterns than the rice meal and the snacks type meal ( p<0.05 ) . recipes were devised considering the dietary habits of the girls and representing each of the five diet patterns . amongst the zinc - rich foods , selection was made on the basis of higher bio - availability ( 32 ) and local availability ; ( 2 ) zinc density of the recipes was increased by using small amounts of sesame , amaranthus and pumpkin seeds ; and ( 3 ) using methods like sprouting / fermentation , which increase the bio - availability of recipes . the zinc - rich snacks containing meat developed for kenyan schoolchildren provided 2.89 mg/225 g of zinc ( 37 ) , which was similar to the zinc content of proposed study ( 3.3 mg/225 g ) . wallis test indicated that the mean content of micronutrients such as iron , -carotene , vitamin c of new recipes were significantly higher than the observed intakes of the girls ( p<0.01 ) . for assessing dietary inadequacies of zinc and other micronutrients , especially in vegetarian populations , a simple tool such as micronutrient quality index is required to identify at - risk individuals . we have developed adolescent micronutrient quality index ( amqi ) to classify an individual with nutritional deficiencies ( paper iii ) ( 62 ) using the indian dietary guidelines ( 24 ) and the recent us dietary guidelines for choice of healthy foods and habits ( 23 ) . efficacy of the amqi was assessed by studying its associations with nutrient intakes , micronutrient adequacy ratio , body mass index and plasma biomarkers of -carotene , vitamin c and zinc . the mean amqi score was 41.59.4 , and 86.8% girls had the amqi score lower than 50 , indicating inadequate dietary nutrient intakes by the girls . higher amqi scores were associated with higher concentrations of plasma vitamin c ( r=0.26 ) , -carotene ( r=0.34 ) and zinc ( r=0.12 ) after controlling for socio - demographic factors ( p<0.05 ) . thus , the amqi provides an updated measure to evaluate micronutrient quality of diets of lacto - vegetarian indian adolescent girls . we anticipate that such concept of emphasising adequate contents and bio - availability of micronutrients while constructing the diet quality index can be widely applicable to different lacto - vegetarian population groups , although some of the components of the index may require modification with respect to nutrient requirements of different age sex groups and food habits . we have developed diet supplements in the form of zinc - rich recipes to improve intake of zinc and other micronutrients . an intervention trial was conducted to determine the efficacy of proposed recipes in improving zinc and other micronutrient status ( paper iv ) ( 63 ) and functional indices of zinc ( paper v ) ( 64 ) . effect of the diet supplementation was viewed in light of supplementing ayurvedic zinc ( jasad bhasma ) tablet . this suggests a positive effect of supplementation on cognitive performance and taste acuity of the girls . furthermore , our study demonstrated that zinc supplementation improved memory and non - verbal intelligence in adolescent girls as well as established positive effect of zinc supplementation on taste perception . thus , zinc supplementation through diet or ayurvedic jasad tablet not only improves zinc status of the girls but also was a useful strategy for improving cognitive performance and taste acuity function in adolescent girls . comparison with age sex - specific reference intake indicated that these intakes were 68.7 and 60% of recommendation ( 24 ) . thus , average intakes of micronutrients were 3060% of the respective rdi , indicating dietary deficiencies of these minerals and vitamins in the girls . the present study has offered estimates of prevalence of dietary micronutrient deficiencies in indian urban adolescent girls , which are in agreement with previous findings on rural indian adolescents ( 13 , 16 ) and available studies on urban slum adolescents ( 14 , 15 ) . we have observed that average zinc intake ( 3.6 1.2 mg / d ) of the girls was low , around 40% of the indian rdi for zinc ( 66 ) , with almost all girls having their zinc intake below rdi . plasma zinc levels were low ( < 0.7 mg / l ) ( 41 ) in 72.4% , and erythrocyte zinc levels were low ( < 8 g / g of packed cells ) ( 56 ) in 23.6% of the girls . plasma vitamin c level of 10.8% girls was below the normal cut - off of 0.2 mg / dl ( 69 p. 534 ) , and 67.6% of the girls had low plasma vitamin c status . paper i described the cognitive performance and assessed its relation with status of zinc and other micronutrients ( 60 ) . the data on cognitive scores in our girls indicated that 60% of the girls were slow in simple reaction tasks , 71.5% of the girls had their rpm scores below 50th percentile and memory deficits were seen in 44% girls ( 60 ) . reaction time and rpm percentiles of majority of the girls were below the normal range , indicating deficits in the cognitive performance of the girls . to explore the relative significance of dietary nutrient intakes with cognitive performance , weighted multiple regression adjusted for energy intake the analysis revealed that intakes of -carotene , iron and zinc were significant predictors for srt ( p<0.01 ) . we have noted that plasma zinc and erythrocyte zinc were negatively correlated with srt and rrt and positively with memory and rpm ( p<0.05 ) , whereas hb was significantly correlated with rrt ( p<0.05 ) in girls with iron deficiency anaemia . rts showed significant negative correlation with erythrocyte zinc ( r = 0.25 , p<0.05 ) , implying that lower the zinc status higher would be the salt concentration for taste perception . thus , our data indicated high prevalence of micronutrient deficiencies including zinc in adolescent girls . moreover , we observed improvement in cognitive performance and taste perception with increased blood zinc levels in the girls . therefore , an attempt has been made to develop zinc - rich recipes to improve total and bio - available zinc intake of the girls . the patterns were characterised by typical combination of major cereal with food subgroups , namely ( 1 ) rice meal , ( 2 ) snack type meal , ( 3 ) wheat meal , ( 4 ) pearl millet meal and ( 5 ) sorghum meal ( 61 ) . the results indicated that consumption of healthy foods mainly whole grains , sprouts and vegetables were high in wheat , pearl millet or sorghum - based patterns than the rice meal and the snacks type meal ( p<0.05 ) . in general , girls with the rice meal and the snacks type meal had low micronutrient intakes and low intake of bio - available zinc and iron than girls from other diet patterns ( p<0.05 ) however , all diet patterns were deficient in micronutrients including zinc when compared with the rdi ( 24 , 66 ) ; mainly due to low intakes of healthy foods by the girls . the patterns were characterised by typical combination of major cereal with food subgroups , namely ( 1 ) rice meal , ( 2 ) snack type meal , ( 3 ) wheat meal , ( 4 ) pearl millet meal and ( 5 ) sorghum meal ( 61 ) . the results indicated that consumption of healthy foods mainly whole grains , sprouts and vegetables were high in wheat , pearl millet or sorghum - based patterns than the rice meal and the snacks type meal ( p<0.05 ) . in general , girls with the rice meal and the snacks type meal had low micronutrient intakes and low intake of bio - available zinc and iron than girls from other diet patterns ( p<0.05 ) however , all diet patterns were deficient in micronutrients including zinc when compared with the rdi ( 24 , 66 ) ; mainly due to low intakes of healthy foods by the girls . recipes were devised considering the dietary habits of the girls and representing each of the five diet patterns . amongst the zinc - rich foods , selection was made on the basis of higher bio - availability ( 32 ) and local availability ; ( 2 ) zinc density of the recipes was increased by using small amounts of sesame , amaranthus and pumpkin seeds ; and ( 3 ) using methods like sprouting / fermentation , which increase the bio - availability of recipes . the zinc - rich snacks containing meat developed for kenyan schoolchildren provided 2.89 mg/225 g of zinc ( 37 ) , which was similar to the zinc content of proposed study ( 3.3 mg/225 g ) . wallis test indicated that the mean content of micronutrients such as iron , -carotene , vitamin c of new recipes were significantly higher than the observed intakes of the girls ( p<0.01 ) . for assessing dietary inadequacies of zinc and other micronutrients , especially in vegetarian populations , a simple tool such as micronutrient quality index is required to identify at - risk individuals . we have developed adolescent micronutrient quality index ( amqi ) to classify an individual with nutritional deficiencies ( paper iii ) ( 62 ) using the indian dietary guidelines ( 24 ) and the recent us dietary guidelines for choice of healthy foods and habits ( 23 ) . efficacy of the amqi was assessed by studying its associations with nutrient intakes , micronutrient adequacy ratio , body mass index and plasma biomarkers of -carotene , vitamin c and zinc . the mean amqi score was 41.59.4 , and 86.8% girls had the amqi score lower than 50 , indicating inadequate dietary nutrient intakes by the girls . thus , the amqi provides an updated measure to evaluate micronutrient quality of diets of lacto - vegetarian indian adolescent girls . we anticipate that such concept of emphasising adequate contents and bio - availability of micronutrients while constructing the diet quality index can be widely applicable to different lacto - vegetarian population groups , although some of the components of the index may require modification with respect to nutrient requirements of different age sex groups and food habits . we have developed diet supplements in the form of zinc - rich recipes to improve intake of zinc and other micronutrients . an intervention trial was conducted to determine the efficacy of proposed recipes in improving zinc and other micronutrient status ( paper iv ) ( 63 ) and functional indices of zinc ( paper v ) ( 64 ) . effect of the diet supplementation was viewed in light of supplementing ayurvedic zinc ( jasad bhasma ) tablet . this suggests a positive effect of supplementation on cognitive performance and taste acuity of the girls . furthermore , our study demonstrated that zinc supplementation improved memory and non - verbal intelligence in adolescent girls as well as established positive effect of zinc supplementation on taste perception . thus , zinc supplementation through diet or ayurvedic jasad tablet not only improves zinc status of the girls but also was a useful strategy for improving cognitive performance and taste acuity function in adolescent girls . the present research provided first - hand estimates of micronutrient deficiencies in a cohort of urban indian adolescent girls . our data demonstrated that the diets of the girls were deficient in zinc and other micronutrients . blood biomarkers of zinc and vitamin a were below the normal range in majority of the girls . dietary zinc deficiency and low blood zinc status lead to poor cognition and taste perception amongst adolescent girls . therefore , there is a concurrent requirement of a simple tool to identify dietary micronutrient deficiencies and a diet intervention strategy to improve zinc and other micronutrient status of the girls . amqi has successfully assessed micronutrient deficiency risk among study population and thus provided an updated measure to assess micronutrient quality of diets of lacto - vegetarian adolescent girls . an intervention trial on a group of 180 girls has demonstrated effectiveness of the proposed recipes in improving zinc status . this study is the only one so far to demonstrate a positive effect of zinc supplementation in improving cognitive performance and taste acuity of adolescent girls . effect of zinc supplementation on other aspects of health such as bone development , infections and obesity may be studied in adolescents . thus , the findings of the research demonstrate the necessity of adopting healthy micronutrient - rich diets for positive health building in adolescents . further , from a global perspective , the methodologies and approaches used to assess dietary inadequacies in the form of amqi and diet diversification strategies could be useful to explore and alleviate problem of micronutrient deficiencies in population .